Produ c t ion of Re c om bin ant
Prote in s of B r uc e lla a b or t us and
U s e in th e S eroepide m iolog ic al
S tu die s
Hy ung - Jin E oh
B rain K ore a 2 1 Proje ct f or M e dic al S c ie nc e s
T h e Gradu ate S ch ool of Y on s e i U niv ers ity
Produ c t ion of Re c om bin ant
Prote in s of B r uc e lla a b or t us and
U s e in th e S eroepide m iolog ic al
S tu die s
D irec ted by A s s oc iate d Profe s s or S an g - N ae Cho
A dis s ert at ion s ub m itte d t o th e F ac ulty of
th e Gradu ate S ch ool of the Y on s e i U niv ers ity
D e c e m b er 2000
by
Hy u ng - Jin E oh
B rain K ore a 2 1 Proje ct f or M e dic al S c ie nc e s
T h e Gradu ate S ch ool of Y on s e i U niv ers ity
A dis s ert at ion f or th e D e g re e of M as te r of
S c ie nc e by Hy ung - Jin E oh h as be e n
approv e d by
T h e Gradu ate S ch ool of Y on s e i U niv ers ity
D e c e m b er 2000
(Supervisory Committee, Chairman )
(Supervisory Committee)
(Supervisory Committee)
Content s
List of F igures and T ables
Abstract s 1
Ⅰ . Introdu ction 4
Ⅱ . M aterial s an d M ethods 11
1. Bacterial strain s and v ectors 11
2. Isolation of genomic DNA 12
3. Cloning 12
4. Expression and purificat ion of recombinant protein s 15
5. Charact erizat ion of recombinant proteins 16
(1) Sodium dodecyl sulfat e- polyacrylamide gel electrophoresis 16
(2) W estern blot hybridizat ion 17
6. Preparation of rabbit antiserum to B rucella abortus 18
7. Preparation of m ou se antisera to recombinant proteins 19
8. Serum samples from cattle infected w ith B . abortus 19
9. Serum samples from resident s in the Cheju province 20
10. Enzym e- linked immunosorbent assay (ELISA ) 20
Ⅲ . Re s ult s 22
1. Charact erizat ion of recombinant proteins of B . abortus 22
2. W estern blot analy sis of recombinant proteins 24
3. Seroreactivity to recombinant prot ein s in sera from cat tle 27
4. Prevalence of ant ibodies to recombinant protein s am ong resident s
in the Cheju prov ince 34
Ⅳ . D is cu s s ion 37
Ⅴ . Con clu s ion 41
Ⅵ . Ref erenc e s 43
Abstract s (in Korean ) 48
Lis t of F ig ure s
Figure 1. SDS - PA GE analy sis of recombinant prot ein antigen s of
B . abortus 23
Figure 2. W estern blot analy sis of recombinant proteins u sing sera
from mice immunized with each protein 25
Figure 3. W estern blot analy sis of recombinant proteins u sing sera
from rabbit s immunized w ith B . abortus w hole cells 26
Figure 4. Scat t er g r am of Ig G s er or ea ct iv it y t o r 16 .5 kD a in s er a
fr om cattle 29
Figure 5. Sc a t t er g r am of Ig G s e r or e a ct iv it y t o r 26 k D a in s er a
fr om cat tle 30
Figure 6. Sc a t t er g r am of Ig G s e r or e a ct iv it y t o r 39 k D a in s er a
fr om cattle 31
Figure 7. Scat tergram of IgG seroreactiv ity to rERC protein in sera
from cattle 32
Figure 8. Scat tergram of IgG seroreactiv ity of B . abortus w hole cell
ly sates in sera from cattle 33
Figure 9. Scat tergram of IgG seroreactiv ity to recombinant prot eins
and B . abortus ly sates in sera from resident s in the Cheju
province (N =500) 35
Lis t of T able s
T able 1. S ources of bacterial st rains and vector s 11
T able 2. Oligonucleot ide prim er s for cloning each gene 14
T able 3. Prevalence of ant ibodies to recombinant protein s among
resident s in the Cheju province (N =500) 36
T able 4. Prevalence of ant ibodies to r39 kDa am ong resident s in
the Cheju prov ince by sex and age groups 36
A c kn ow le dg e m e nt s
지난 2년 간 몸소 연구라는 것이 무엇인가를 보여주시고, 때로는 꾸지람으
로 때로는 칭찬으로 이끌어 주신 조상래 교수님께 진심으로 감사드립니다. 그
리고 이 논문이 완성되기 까지 조언을 아끼지 않으셨던 김세종 교수님과 김준
명 교수님께도 깊은 감사를 드립니다. 그리고 박전한 교수님과 김주덕 교수님,
이원영 교수님, 이봉기 교수님, 김종선 교수님 신전수 교수님, 박현주 선생님께
도 곁에서 묵묵히 가르침 주신 것에 감사드립니다. 2년 전 아무것도 모르는 나
를 친구처럼 지도해준 이혜영 선생님과 명한정 선생님 그리고 친 누나처럼 많
은 것을 가르쳐준 이혜존 선생님, 어려울 때 도움을 준 방혜은 선생님, 그리고
형처럼 타일러 주신 신유철 선생님과 김승철 선생님 또한 감사의 마음을 전합
니다. 겉으로 드러나지 않게 많은 것을 타일러 주신 전보영 선생님과 박성도
선생님께도 감사의 마음을 전합니다. 김지수 선생님, 김근희 선생님, 조장은 선
생님, 노인순 선생님을 비롯한 미생물학 교실 여러 선생님들께도 감사하다는
말을 전하고 싶습니다.
가끔 술잔을 기울이며 나에게 용기를 준 정말 아끼는 친구인 박영기 군과
최휘복 군에게 심심한 감사의 마음을 전합니다.
그리고 마지막으로 힘들 때나 괴로울 때나 아플 때나 슬플 때나 기쁠 때
나 항상 옆에서 바라봐 주고 이해로 다독여 준 집사람 김영미 님과 장인, 장모
님께도 감사의 마음을 전하고, 대견하게 성장해서 자기 앞가림 훌륭하게 하고
있는 동생 광진과 지금까지 이렇게 장성하게 키워주시고, 어려움 없이 공부할
수 있게 도와주신 큰사랑을 베풀어주신 부모님께 이 논문을 바칩니다.
Abs t rac t
Pro duc tio n o f Re c o m binant Pro te ins o f Bruc e lla abo rtus a nd
Us e in the S e ro e pide mio lo g ic a l S tud ie s
Brucellosis is one of the major zoonoses infecting both domest ic
animals and human s . Recent increase of B rucella cases in catt le in Korea
has raised concern s about infect ion among farm ers , w orkers at slaughter
houses and vet erinarian s . Due to difficulty in isolat ion of B rucella abortus ,
the main cau sative organism of brucellosis , from patient s suspected having
brucellosis , diagnosis of the disease relies m ainly on serological test s .
How ev er , the current methods of serological test s are far from optimum
mainly due to presence of common antigen s betw een B rucella and other
gram - negat ive organism s and to difficulty in distinguishing active from
inactive infections . T his study w as thus designed to develop serological
test s using recombinant protein s , w hich are specific to brucella , and to
evaluate the protein - based serological t est s for use in serodiagnosis of
brucellosis . In addition , prev alence of antibodies to the proteins am ong
resident s in the Cheju province w as determined in this study .
Among B . abortus prot eins in the literature, the 16.5 kDa
outermembrane protein Omp 16, the 26 kDa periplasmic prot ein , and the 39
kDa cytoplasmic prot ein w ere chosen in this study based on their
antigenicity and specificity . In addition , the erythritol cat abolism C (ERC)
1
prot ein , w hich is involved in the metabolism of erythrit ol- the m ajor carbon
source of brucella , w as included . T he genes encoding the proteins w ere
cloned and expressed in E scher ichia coli u sing the pQE30 expression
vector . Purified recombinant protein s , r 16.5 kDa, r26 kDa , r39 kDa and
rERC, w ere then ex amined for their m olecular charact eristics by
SDS - PAGE and immunogenicity by W estern blot ting . All recombinant
prot ein s w ere immunogenic in mice in generat ing strong antibody responses
w hich w ere determined by W estern blot analy sis . In addit ion ,
hyper - immune rabbit ant ibodies to B . abortus reacted with the r16.5 kDa ,
r26 kDa, and r39 kDa, indicat ing that the recombinant protein s are
antigenic in detecting antibodies to the nat ive proteins of B . abortus . T he
recombinant protein s , r 16.5 kDa, r26 kDa, r39 kDa, and rERC, w ere also
effectiv e in detect ing ant ibodies in tube agglutination test (T AT ) posit ive
sera from cattle naturally infect ed w ith B . abortus by enzym e- linked
immunosorbent assay (ELISA ). With the serological test condit ion s using
recombinant proteins , serum samples from resident s representing
populations in the Cheju province w ere ex amined for presence of ant ibodies .
Of 500 sera ex amined, seven (1.4% ) sera had elev ated antibodies to r 16.5
kDa and r26 kDa protein s , and 11 (2.2% ) to the r39 kDa proteins .
Int erest ingly , four (0.8% ) sera show ed seroreactivity to the rERC protein
and to the B . abortus w hole cell ly sate antigen s .
T he result s thus indicate that the recombinant protein s above are
effectiv e in detecting antibodies to their nativ e protein s of B . abortus and
2
thus can be used in supplem ental serodiagnost ic test s to the current
agglutinat ion test s . In addition , this study provides a serological ev idence
that a portion of resident s in the Cheju prov ince hav e been exposed to B .
abortus . F urther studies on indiv iduals at high risk of contract ing
brucellosis will w arrant s usefulness of the recombinant protein s for
serodiagnosis of the disease.
Key W ords : brucellosis , recombinant protein, B rucella abortus ,
serological test
3
Pro duc tio n o f Re c o m binant Pro te ins o f Bruc e lla abo rtus a nd
Us e in the S e ro e pide mio lo g ic a l S tud ie s
Hyu ng -J in Eo h
Brain Korea 21 Project for Medical Sciences
T he Graduate School, Yon sei Univer sity
< directed by A ssociated Professor S ang -Nae Cho >
Ⅰ . Introdu ct ion
B ruce lla spp . are facult ativ e, intracellular gram - negative bacteria w hich
develop m ainly in the reticuloendothelial sy stem and occasionally in other
target organ s , such as joint s and placent a, and can cau se brucellosis in
animals and human s 1 . Brucellosis has been a frequent public health and
safety problem in dev eloping countries , such as Latin Am erica, w ith the
highest numbers of cases occurring in Mexico, Argent ina and Peru since
the discov ery of B rucella m elitens is in 18872 .
B ruce lla have been classified to six species according to virulence and
their host s - B . abortus , B . s uis , B . ovis , B . m elitens is , B . can is and B .
neotom ae . B . abortus cau ses abort ion s and reduces fertility in cattle and
4
chronic zoonotic infect ion s in human s resulting in undulant fev er ,
endocarditis , arthritis , and osteomyelit is3 . Besides B . abortus , B . s uis , B .
m e litens is and B . can is also can cau ses brucellosis in hum an s .
Con sumption of contaminated foods and occupational contact t o infect ed
tis sues or fluids , is the major sources of infection . Ex amples of
human - to- hum an tran smission by tissue transplantation or sexual contact
are occasionally report ed but in significant . T here also have been case
report s of transmission via blood tran sfu sion , bone m arrow tran splantat ion ,
and breast milk of m other with brucellosis4 .
In Korea, B ruce lla sp . w as isolated from a patient su spect ed having
brucellosis for the fir st tim e in 1939, and then it had not been reported
until 19865 . In 1986, five patient s with fever and nine patient s in the
contact w ith anim als w ith brucellosis w ere discovered as serologically
positive, thus indicating that Korea becam e an endemic country for
brucellosis 6 . On the other hand, there w as a report of being clinically
su spiciou s animals w ith brucellosis in 1944, and of a seropositiv e catt le in
1947, but it w as v ery rare at that tim e7 , 8 . In 1955, thirty cattle imported
from U .S .A w ere found seropositiv e9 , 1 0 . B rucella s uis w as also isolated
from a dog in 19721 1 , 1 2 . T herefore, it w as proven intermit tently that the
bacteria ex isted in anim als , especially in cat tle in Korea, but the number of
animals m arkedly increased last 20 years . In early 1990s , ov er 400 cat tle
w ere infected by B rucella in the Cheju province per year 1 3 . Although the
number of infected catt le in farm has increased and disseminated to other
5
animal hu sbandry areas , hum an brucellosis has not been reported yet in the
Cheju province6 .
In a recent study , how ev er , there seem ed to be subclinical hum an
infect ion s in the Cheju province. In the prev iou s report , a total of 2,372
serum samples from resident s in the Cheju island had been ex amined by
the tube agglut ination test , and the seropositiv e rat e w as reported to be
0.59% . No significant differences in the positive rates betw een males and
fem ales w ere found in the study . How ev er , older age groups had slightly
higher seroreactiv ity . T he report s also indicated that the differences in
positive rat e betw een the resident ial areas and betw een their occupations
w ere not noted . Consequently , the report suggest ed the need of surveillance
on brucellosis 6 .
One of the m ost difficult ob stacles for diagnosis of human brucellosis
originates in the ex ist ence of serologically posit ive individuals w ho hav e
low titer s of both agglut ination and complem ent fix ation , and are either
asymptomatic or oligosymptom atic . T his group includes serologically
positive individuals w ith no act ive brucella infection or those having an
epidemiological probability of exposure to brucella infection but no know n
history of disease1 4 .
T he cell w all envelope of B rucella spp . is a three- layered structure in
w hich an inner or cytopla smic membrane, a peripla smic space, and an outer
membrane can be differentiated, similar to other gram - negative bacteria 1 5 , 1 6 ,
1 7 , 1 8 . T he outer m embrane contain s the lipopoly saccharide (LP S ), various
6
prot ein s , the phospholipids , etc . LP S or the ex tract containing LPS is the
major antigen for diagnosis of brucellosis by detecting anti- LPS antibodies ,
but there are som e hindrances . B rucella S 19 vaccines induce production of
antibodies to LP S that are detectable in the serological t est u sing whole
cells . Con sequently , it becom es difficult to interpret result s of the test s in
vaccinated animals , because positive test results may indicate the occurrence
of antibodies by vaccines1 9 , 2 0 . On the other hand, test s preformed w ith
either LP S or LPS - rich ex tract s lead to ex ten siv e cross - reaction s with
other gram - negat ive bacteria , such as Y ers inia en terocolitica O:9 and
E schr ichia coli O:157 LPS . T his phenom ena result s in the loss of specificity
at the early and later phase of brucellosis2 1 , 2 2 .
T he diagnosis of brucellosis depends mainly upon the serological t est
because it is difficult t o culture B rucella organism s in clinical samples . T he
routine serological test s for the brucellosis with LPS containing w hole cells
include standard tube agglutination t est (T AT ), rapid slide agglutinat ion
test (RAT ), rosebengal test , milk ring t est (MRT ), Coombs test , and
complem ent fix at ion test (CF T )2 3 , 2 4 , 2 5 . T AT has become the st andard
method for serodiagnosis of brucellosis . T his t est also gives quant itat ive
inform ation about the immune respon se to brucellosis . Although T AT is a
standard m ethod, it is laboriou s , reagent consuming , and tim e inten sive and,
therefore, not suit able a s a primary t est for laboratories w ith large
specimen w orkloads or for conducting serologic survey s epidemiologic
invest igations . Several rapid screening test s have been proposed, but
7
variat ion s in ant igen s , incubat ion tim es , and interpretat ion of signigicant
reactiv ity compared w ith that of T AT m ake it difficult to evaluate their
suitability for screening hum an sera .
Other serological m ethods for the different iation of v accinated cat tle
from cat tle infect ed w ith field strain of B . abortus have been proposed. It
is inevitable to find other diagnostic method with antigen s not containing
LP S , because vaccine strains also contains the LPS antigen 2 6 , 2 7 , 2 8 . It m ay
be assumed that the det ermination of the humoral response again st
LP S - free protein s could help to av oid those undesirable react ivit ies seen in
LP S - based test s .
Little is know n about the relevance of brucella prot eins in the immune
response in naturally infected host s . A s m entioned previously , B . abortus
is an intracellular parasite . T hus immune respon se may be direct ed to
both surface and internal protein s , depending on the processing of the
bacteria by macrophages . T he outermembrane protein s of v ariou s m olecular
masses do not react w ith antibodies to other gram - negativ e bact eria LPS .
It w as report ed that there are three m ajor and four minor outermembrane
prot ein s 1 8 . T hese three m ajor outer membrane prot eins have been found to
be tight ly as sociated w ith pepetidoglycan and 16.5 kDa protein is a
represent ativ e of the outerm embrane prot ein s3 1 . But antibodies to outer
membrane prot ein s have been found to be ineffect ive or less efficaciou s
than LPS - ant ibodies for the prev ent ion again st brucella infection s 1 8 , 2 9 .
On the other hand, the delay - type hyper sensitiv ity (DT H ) test has
8
been w idely u sed for the diagnosis of brucellosis in ruminant s , and various
allergen s , namely protein ant igens from different brucella species have been
prepared for this purpose3 0 , 3 1 , 3 2 . Allergic diagnosis is u sed in a number of
countries , but the type and specificity of the methods are variable . Indeed,
the nature of the allergen s differ s depending on the strain s used, the
culture techniques , and the m ethods of production and purificat ion . Am ong
these allergen s , the brucellin consist s of a mix ture of 20∼30 cytopla smic
prot ein s and is prepared from B rucella m eliten sis , and it w as prov ed
valuable in identifying B rucella - infected catt le . Becau se the 39 kDa protein
in the cytoplasmic proteins has been show n to be act ive in the DT H test ,
it s efficiency and specificity w ere compared w ith those of brucellergen in
DT H 3 0 , 3 1 , 3 2 , 3 3 . In this study , therefore, the 39 kDa prot ein w as included as
one of diagnostic ant igens . Other B ruce lla structural protein , periplasmic
prot ein , 26 kDa prot ein w as also u sed as LP S - free protein antigen in this
study 3 4 , 3 5 .
It has been report ed that the erythritol is a carbon source in B rucella
growth , and activates the grow th rate, but the B . abortus S 19 v accine
strain w as at tenuat ed to this m aterial. On the contrary , therefore, the
growth rate of S 19 v accine strain is inhibit ed by erythritol. T he metabolism
of erythrit ol m ay thus play a major role in expressing the bact erial
virulence. Erythrit ol catabolism genes consist of three open reading
fram es (ORF s ), called ORF a, ORF b, and ORF c. Particularly , the ORF c w as
reported playing a major role in erythritol m etabolism 3 6 , and it s protein ,
9
ERC, w as also included in this study to know it s seroreact ivity .
T he m ain purpose of this study w as to develop serological test based
on the protein antigen s above and to ev aluat e the prot ein - based test s for
use in seroepidemiological studies . Since it is not easy to obtain nativ e
prot ein s from B . abortus culture, recombinant protein s w ere prepared based
or DNA sequence of the genes encoding the prot eins . the recombinant
prot ein s w ere then characterized for their m olecular characteristics and
immunogenicity . Subsequently , the recombinant prot eins w ere evaluated for
their efficiency in detecting antibodies to the nat ive proteins of B . abortus .
F inally , the serological test s u sing the recombinant protein s w ere used in
determining prevalence of antibodies among resident s in the Cheju province,
in which brucellosis outbreak s among cat tle occurred last tw o decades .
10
Ⅱ . M aterials and m ethods
1. Bac te ria l s tra ins and ve cto rs
T he sources of the bacterial st rains and v ectors w ere listed in T able 1.
B rucella abortus 2308 strain u sed in the present study w as obtained from
the National Veterinary Research In stitut e, Anyang , Korea . W orking
cultures of B rucella strains w ere grow n and maint ained on tryptic soy
broth (T SB ) supplemented w ith 5 % bovine serum albumin (BSA ) at 37℃
shaking incubator , and E . coli strain s w ere cultured on Luria Bert ani(LB )
media (DIF CO Laboratories , Inc ., Detroit , U .S .A ).
T able 1. S ources of bacterial st rains and vector s
Strain/ Vector Name Source
B ruce lla strain B rucella abortus 2308National Veterinary
Reseach In st itute
E . coli cloning host T op 10 Invitrogen , CA , U .S .A
E . coli expression
hostM 15 Qiagen , CA , U .S .A
cloning vector pT 7 Blue vectorNov agen , Darm stedt ,
Germany
expression vector pQE30 Qiagen , CA , U .S .A
11
2 . Is o latio n o f g e no mic DNA
B . abortus genomic DNA w as isolated by the method described by
Silhavy et al.3 7 . Briefly , B . abortus 2308 w ere grow n in T SB supplemented
w ith 5 % BSA , at 37℃ on an orbital shaker for 3∼4 day s . T he cells w ere
then harvest ed by centrifugation at 4,500 rpm for 10 min at 4℃. T he cells
w ere resuspended in 567 ㎕ of T EN buffer (50 mM T ris , 50 mM EDT A ,
100 mM NaCl, at pH 8.0) and mixed w ith 30 ㎕ of 10% (w/ v ) sodium
dodecyl sulfate (SDS ) solution and 3 ㎕ of 2% (w/ v ) prot einase K solut ion ,
and then incubat ed at 37℃ for 1 h . T he mix ture w as centrifuged at the
sam e condition , and the supernatant w as collected . T o precipitate genomic
DNA , the cold ethanol w as added to the collected supernatant , and the
mixture w as placed at - 70℃ for 30 min . Genomic DNA w as recov ered by
centrifugation at 13,000 rpm for 15 min at 4℃. T he genomic DNA w as
then dissolved in 100 ㎕ of T E buffer (10 mM T ris , 1 mM EDT A , at pH
8.0) and analyzed by electrophoresis on 0.8 % agarose gel.
3 . Clo ning
T he nucleotide sequences of the genes coding for the 16.5 kDa
outermembrane protein , 26 kDa peripla smic protein , 39 kDa cytoplasmic
prot ein , and fram e C of erythritol catabolism protein , ERC of B . abortus
w ere obtained from the GenBank database located at the National Center
12
for Biotechnology Information of the National Library of Medicine (Bethesda,
Md.). Prim er set s based on the nucleot ide sequences w ere designed and
w ere synthesized by Bioneer Co.(T aejeon , Korea ).
T o clone the DNA fragm ent coding for each protein into the
expression vector , pQE30, each oligonucleot ide prim er set w as designed by
adding the restriction enzym e sites underlined (T able 2) at the end of
forw ard primer (B am HI) and the end of rever se prim er (H indⅢ) in case
of 16.5 kDa and 26 kDa protein s . How ever , since the 39 kDa and ERC
protein ORF s have the H indⅢ site, the H indⅢ sit e w as replaced w ith
P s tⅠsite for the rever se end of 39 kDa and Sp hⅠ site for the rever se end
of ERC protein , respectiv ely . T he sequences of oligonucleotides used for
cloning are listed in T able 2.
T o amplify the gene coding each prot ein , 50 ㎕ P CR reaction mix ture
w as prepared w ith 1 ㎍ of genomic DNA , 5 ㎕ of 2.5 mM dNT P , 1 ㎕ of
10 pmol each primer s , 5 ㎕ of PCR buffer (10 mM T ris - Cl pH 8.3, 50 mM
KCl, 1.5 mM MgCl2 ), 1 U of T aq DNA polym erase (Prekin - Elm er Cetu s ,
Norw alk , Conn .), and up to 50 ㎕ of d .w . PCR react ion w as perform ed by
follow ing cycling condition s ; initial denaturat ion at 94℃ for 10 min follow ed
by 94℃ for 1 min , annealing at 58℃ for 1 min and 72℃ for 2 min (for 35
cycles ) follow ed by a final ex ten sion of 72℃ for 10 min . P CR product s
w ere analyzed on a 0.8% agarose gel and w ere cloned into the T 7 Blue
vector (Nov agen , Darm stadt , Germ any ).
13
T able 2. Oligonucleot ide prim er s for cloning each gene
T arget
prot ein sPrimer sequences
16.5 kDaforw ard 5 ' - GGAT CCAT GCGCCGT AT CCA GT CGAT T GCA - 3 '
rever se 5 ' - AAGCT T A CCGT CCGGCCCCGT T GAGAA C- 3 '
26 kDaforw ard 5 ' - GGAT CCAT GAA CACT CGT GCT A GC- 3 '
rever se 5 ' - AAGCT T CAT A CCCCGGCT AT T T A C- 3 '
39 kDaforw ard 5 ' - GGAT CCGT GA CGT CCGGCGGCGAA G- 3 '
rever se 5 ' - CT GCA GT T AT T T T GCGGCT T CAA CCGCCAC- 3 '
ERCforw ard 5 ' - GGAT CCA CAA CGCCAAT CT AGT T C- 3 '
rever se 5 ' - GCAT GCAT CT GCCAT T GAA CGCGG- 3 '
F or ligation of PCR product s w ith cloning v ector , reaction mix ture w as
prepared w ith 5 ㎕ of P CR product s , 1 ㎕ of 10× buffer , 1 ㎕ of 10 mM
AT P and 4 unit s of liga se and 2 ㎕ of H 2 O, and the reaction mix ture w as
incubated at 16℃ for 16 h .
T o con struct ant igen expression vector , B am HI- H indⅢ fragm ent s (16.5
kDa and 26 kDa), B am HI- P s tⅠ fragment s (39 kDa), and B am HI- Sp hⅠ
fragment s (ERC protein ) w ere obtained from the T 7 Blue vector containing
each DNA fragment by digest ing with corresponding restrict ion enzymes
and purified by Geneclean Ⅲ kit (Bio 101, Vista , CA , U .S .A ). T he
fragment s w ere subcloned into the corresponding sit es of pQE30 (Quiagen ,
14
CA , U .S .A ). After t ran sforming into T op 10 competent cells (Inv itrogen ),
the expression vector w as extracted by QIAprep Spin Miniprep Kit
(Qiagen ) and electroporated into the expression competent cells , M 15, for
tran sform ation . T he M 15 cells transform ed with the ligat ed plasmids w ere
select ed on an LB agar plat es containing 100 ㎍/ ㎖ ampicillin and 25 ㎍/ ㎖
kanamycin . T he cloned genes into expression v ector w ere confirmed by
sequencing (Yonsei University Medical Research Center ).
4 . Ex pre s s io n a nd pu rific atio n of re c o m bina nt pro te ins
T he optim al conditions of expressing each target protein w ere
determined by pilot t est s . Since the pQE30 vector has the six hist idine t ag
at the 5 ' end of the multicloning sit es , each recombinant protein prepared
by the pQE expression sy stem contains hist idine t ags . Becau se the histidine
has the potential binding ability to Ni heavy m etal, the expressed protein
w as purified through the Ni- NT A column . Briefly , E . coli M 15 containing
each expres sion vector w ere propagated in 5 ㎖ of LB media supplem ented
w ith 100 ㎍/ ㎖ of ampicillin and 25 ㎍/ ㎖ of kanamycin at 37℃ for 18 h
w ith agit ation , and the culture w as poured to 1 ℓ of LB broth w ith
antibiot ics and incubated until the O.D6 0 0 of culture reached from 0.7 to 0.9.
T hen , optimal concentration of isoprophyl- β- D - thiogalactopyranoside
15
(IPT G) w as added to the culture and incubat ed for 4 h w ith agitat ion . T he
inductive M 15 cells with clone w ere recov ered by centrifugation at 5,000
rpm for 10 min , and the recovered bacteria w ere frozen at - 20℃. T he 5 ㎖
of guanidine HCl- phosphate buffer A (6 M guanidine HCl, 0.1 M
Na- phosphate, 0.01 M T ris - HCl, pH 8.0) w as add per 1 g of the m elted
bacteria , and the mix ture w as incubat ed slow ly in a shaking incubator for
1 h at room temperature. T he mix ture w as then centrifuged at 13,000 rpm
for 30 min , and the supernatant w as collected and mix ed with 2 ㎖ of
Ni- NT A resin for 1 h at room temperature by the sam e method. T he
resin - bounded protein w as loaded into column and w ashed with 10 v olumes
of urea - phosphate buffer B (8 M Urea, 100 mM Na - phosphate, 10 mM
T ris - HCl, pH 8.0) and 50 volum es of urea - phosphat e buffer C ( 8 M
Urea , 100 mM Na- phosphat e, 10 mM T ris - HCl, pH 6.3). F inally , the t arget
prot ein w as eluted with 250 mM imidazole in buffer C.
5 . Cha rac te rizatio n o f re c o mbina nt prote ins
(1) Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS - PA GE )
SDS - PA GE w as performed according to the method of Laemmli3 8 on
12% polyacrylamide gels . Briefly , the purified protein mixed w ith the
SDS - PAGE sample buffer (60 mM T ris , 2% SDS , 2% β- m ercaptoethanol,
16
and 10% glycerol) w as boiled for about 5 min and centrifuged at 12,000
rpm for 5 min . T he supernatant of the mix ture w as collect ed and loaded
into the SDS - PA GE gel w hich w as m ade of 3% stacking gel and 12%
separat ing gel. T he gels w ere stained w ith coomas sie brilliant blue R - 250
(Sigma, St . Louis Mo., U.S .A ), and the molecular weight and overproduction
rate of recombinant proteins w ere estim ated .
(2) W est ern blot hybridization 3 9
After electrophoresis , the proteins w ere tran sferred to nit rocellulose
membrane by using a tran sblot apparatus (Hoefer Scient ific In strum ent ,
San francisco, U .S .A ) at 30 V for 16 h at 4℃ in a tran sfer buffer (25 mM
T ris - HCl, 194 mM glycine, and 20% methanol). F rom the posit ive to
negat ive pole, the sponge, three 3MM paper , NC paper , electrophoresed gel,
three 3MM paper and sponge w ere put in order . After tran sblott ing ,
Unoccupied sites on the nit rocellulose membranes w ere blocked by
incubation in the phosphate buffered saline- 0.05% T w een 20- 5% norm al
goat serum (PBST - NGS ) at room temperature w ith v igorou s agit ation for
1 h . T he m embranes w ere then incubated for 1 h w ith serum from mice
immunized with each recombinant prot ein antigen diluted 1/ 100 in the
PBST - NGS , follow ed by incubat ion with rabbit ant i- mouse immunoglobulin
G conjugated with perox idase diluted 1/ 5000 in PBST - NGS . Betw een
17
incubation periods , the membranes w ere w ashed with PBST three tim es for
10 min w ith agitation . After the last three w ashing s , the enzyme activ ity
w as det ected by ECL kit (Am ersham Pharm acia Biot ech , Piscataw ay ,
U .S .A ), and the film s w ere exposed to the membranes and developed for
visualizat ion .
6 . Pre pa ratio n of ra bbit antis e ru m to Bruc e lla abo rtus
B rucella abortus cells w ere cultured in T SB supplemented with BSA for
3∼4 days and harvested by centrifugation at 3,500 rpm for 10 min . T o
eliminate the residual media, the recovered bacteria w ere w ashed with PBS
(phosphate buffered saline, pH 7.4), and divided into tw o groups. One group
w as autoclaved at 120℃ for 15 min after adjusting the number of bacteria at
Macfarland #3 (9×108/ ㎖), and the other group w as inactivated by incubating
the bacteria with 1.5% formalin at 37℃ for 1 h . A volume of 500 ㎕ of each
bacterial suspension w as mixed w ell with the same volume of Freund ' s
incomplete adjuvant and injected into the New Zealand white rabbit s
intramuscularly on the hind legs . T he rabbit s w ere immunized again with the
same mixtures after 3 w eeks. T hree w eeks after the 2nd immunization , the
large volume of blood w as withdrawn from the heart by cardiac puncture.
18
7 . Pre pa ratio n of mo us e antis e ra to re c o m binant prote ins
F ive m ale BALB/ c mice of four w eeks old w ere immunized with each
purified recombinant protein for obtaining ant ibodies to each antigen . F ir st ,
each recombinant prot ein denatured w ith 8 M urea in buffer C and 250
mM immidazole w as dialy sed again st PBS (pH 7.4) for 48 h to remove
urea and other chemicals . After adju sting each protein concentration at 30
㎍/ ㎖, the recombinant protein w as mix ed w ith the sam e v olume of
Freund ' s incomplet e adjuvant (Sigma ) and inject ed to the mice
subcutaneou sly . About three w eeks aft er the 2nd immunizat ion , blood
samples w ere obtained from heart at least 1 ㎖ per a m ou se.
8 . S e rum s am ple s fro m c attle infe c te d w ith B. abo rtus
A total of 100 serum samples from catt le, w hich w ere positive by tube
agglutinat ion t est (T AT ), w ere provided by the Kyunggi- do Veterinary
Inst itute, Suw on , Korea . In addition , 200 serum samples from catt le, w hich
w ere negat ive in the agglut ination test , w ere also obtained from the
in stitut e .
19
9 . S e rum s am ple s fro m re s ide nts in the Cheju pro v inc e
A set of over 1,000 serum samples from resident s in Cheju island,
w here brucellosis outbreak s occurred among catt le population for a long
period, w ere prov ided kindly Dr . J .M . Kim at the Yon sei Univ ersity college
of Medicine, Seoul, Korea . Based on demographic information of the Cheju
island, 500 serum samples represent ing sex and age groups of the island
w ere randomly select ed for this study .
10. Enzy me -linke d im mu no s o rbe nt as s ay (ELIS A)4 0
All serum samples w ere assayed for antibody act ivity again st each
recombinant prot ein antigen w ith optim al dilution in 0.05 M carbonate
buffer (pH 9.6) by ELISA . T he volum e of antigen s coated in the each w ell
w as 100 ㎕, and the coat ed ELISA plates (Cost ar , NY, U .S .A ) w ere
incubated at 4℃ for overnight . Aft er w ells of the plat es w ere saturat ed
w ith PBST - NGS at 37℃ for 1 h and emptied, opt imally dilut ed serum in
PBST - NGS w ere added and incubat ed for 1 h at 37℃. Binding of the
prim ary ant ibody to ant igen w as rev ealed by incubation for 1 h at 37℃
w ith a secondary IgG peroxidase conjugat e diluted 1/ 20,000 in PBST - NGS .
Reagent s in ex ces s w ere removed betw een each step by at least four
20
w ashing s with PBST . ο- Phenylenediamine (OPD ) (Sigm a) with 2 mM
H 2 O2 in a citrat e- phosphate buffer (0.05 M Na2 HP O4 , 0.025 M citric acid[pH
5.0]) w as used to develop the assay . T he signal w as read at 490 nm and
630 nm , and the difference w as recorded w ith a BIO Kinet ics Reader
EL - 340 (Bio- tek In strum ent s , Inc., Winooski, Vt ). Each serum w as test ed
in duplicat e, and the absorbance at w ells w ithout test antigen w as
subtract ed from the absorbance at w ells with test antigen before analy sis .
21
Ⅲ . Re sult s
1. Cha rac te rizatio n o f re c o mbina nt prote ins of B. abo rtus
T hree structural protein s , 16.5 kDa , 26 kDa, 39 kDa and one cat abolism
prot ein , ERC w ere expressed by the E . coli expression sy stem s . Fig . 1
show s the SDS - PA GE profiles of the purified recombinant protein s of B .
abortus . A s shown in the figure, there w ere some discrepancy in molecular
size of recombinant prot ein s . F or ex amples , the recombinant (r ) 26 kDa and
r39 kDa protein s moved fast er than expected, w hile the r 16.5 kDa protein
show ed the right size. A ddit ion of six hist idine tag to each recombinant
prot ein might cau se the m obility change of the prot eins in electrophoresis .
Int erest ingly , there w ere tw o clear protein bands in the lane w ith the rERC
protein , which w as expect ed to giv e a 34 kDa in molecular w eight . T he
sm aller m olecules w ere con sidered as a degradation product of the rERC
protein .
22
Fig . 1. S D S - PA GE an aly s i s of re c om bin ant prot ein antig e n s of B .
ab or t us Lane 1, low m olecular w eight m arker (kilodaltons );
lane 2, r 16.5 kDa ; lane 3, r26 kDa ; lane 4, rERC; lane 5 r39 kDa,
T he recombinant proteins expressed in E . coli were purified
using Ni- agarose columns .
23
2 . We s te rn blo t ana lys is o f re c o mbina nt prote ins
Immunogenicity of recombinant protein s w as ex amined by W estern blot
analy sis . F ir st , mouse ant iserum w as prepared by immunizing mice w ith
each recombinant protein . T he m ou se antiserum w as then u sed in W estern
blotting for reactiv ity w ith the corresponding recombinant protein . A s
show n in Fig . 2, mouse antiserum to r 16.5 kDa protein gave a strong band
in the 16 kDa region and a minor but significant band in the 30 kDa
region . T his indicat es that the r 16.5 kDa protein is immunogenic. Mouse
anti- r26 kDa protein also gav e a strong band in the 26 kDa region and
several minor bands in the smaller molecular w eight region . Likew ise,
mouse antiserum to r39 kDa and to rERC protein reacted to the r39 kDa
protein and the rERC protein in the Western blot , respectively (Fig . 2, c & d).
A s seen the SDS - PAGE analy sis , the rERC protein gave tw o strong bands
in the W estern blott ing , indicat ing that both prot eins are immunogenic .
Rabbit antiserum w as also prepared by immunizing rabbit s w ith B .
abortus whole cells and u sed in the W estern blot analy sis . A s shown in
Fig . 3, rabbit ant iserum also show ed strong reactiv ity w ith r 16.5 kDa (Lane
a ), r26 kDa (Lane b ) and r39 kDa (Lane c) prot eins . Unlike in m ou se
antiserum to the prot eins , there w as only single band in each recombinant
prot ein . How ever , there w as w eak reacit ivity betw een rabbit serum and
upper protein of rERC protein (Lane d), indicating that B . abortus culture did
not contain enough upper ERC protein to produce antibodies in the rabbit s .
24
Fig . 2. W e s t ern blot an aly s i s of re c om bin ant prot ein s u s in g s era
from mice immunized w ith e ach protein . Lane a, r 16.5 kDa ;
lane b , r26 kDa; lane c, r39 kDa; lane d, rERC protein .
Molecular mass standards (in kDa) are indicated on the left , and
the arrow s on the right of each lane indicate the corresponding
recombinant protein . A 12% acrylamide gel and 100 fold dilution
of m ouse serum and 5,000 fold dilution of anti m ouse IgG
HRP - conjugated were used.
25
Fig . 3. W e s tern blot an aly s i s of re c om bin ant protein s u s in g s era
from rabbit s im m uniz e d w ith B . ab or t us w h ole c ell s . Lane
a, r 16.5 kDa ; lane b, r26 kDa ; lane c, r39 kDa ; lane d, rERC
protein . Molecular mass standards (in kDa) are indicated on the
left , and the arrow s on the right of each lane indicate the
corresponding recombinant protein . A 12% acrylamide gel and
50 fold dilution of mouse serum and 5,000 fold dilution of
anti- mouse IgG HRP - conjugated were used.
26
3 . S e ro re ac tiv ity to re c o mbina nt prote ins in s e ra fro m c attle
S erum samples from cat tle, w hich w ere con sidered infected w ith B .
abortus by tube agglutinat ion test (T AT ), w ere also included in this study
in order to determine ant igenicity of the recombinant protein s . ELISA w as
employed to det ect antibodies to each r ecom bin ant prot ein , an d dilut ed
serum sam ples w ere prein cub ated w ith E . coli ly sates to reduce reactiv ity
of residual E . coli component s in purified recombinant protein s . IgG
seroreact ivity to the r16.5 kDa prot ein w as detectable in the sub stantial
port ion of T AT seroposit ive cat tle sera and also in sev eral T AT negative
sera from catt le in the sam e geographic areas (F ig . 4). T his indicat es
clearly that cat tle infected w ith B . abortus elicited ant ibodies to 16.5 kDa
prot ein , and the r 16.5 kDa prot ein w as u seful in detect ing ant ibodies to the
nativ e 16.5 kDa outerm embrane protein Omp 16
IgG seroreactiv ity to r26 kDa w as more prominent than other
recombinant protein s (F ig . 5). More than 50% of T AT positiv e serum
samples gave a strong reactivity to the r26 kDa protein , w hile litt le
seroreact ivity to the prot ein w as found in T AT negative catt le sera . T his
also indicates that the r26 kDa prot ein is effective in detect ing antibodies
to the native 26 kDa periplasmic prot ein of B . abortus .
Unexpectedly , seroreactiv ity to the r39 kDa protein w as v ery w eak in
general and w as det ectable only in several T AT positiv e sera (F ig . 6).
None of the T AT negativ e sera w as reactiv e to the r39 kDa protein . In
27
contrast , IgG seroreactivity of the rERC protein w as detect able in almost
50% of T AT positive catt le sera, thu s indicating that cattle infected w ith
B . abortus elicited antibodies to the native ERC protein (Fig . 7).
T he w hole cell ly sates of B . abortus w ere also included in this study
to verify the T AT result s . A s show n in Fig . 8, the m ajority of the T AT
positive cat tle sera w ere react ive to the whole cell ly sate antigen s in
ELISA . T his supported that the T AT positiv e cat tle w ere likely infected
w ith B . abortus . T herefore, it seem s clear that all r ecombinant protein s are
compatible w ith their native protein s in detect ing antibodies , although there
is a m arked difference in antibody lev el among B . abortus infected catt le .
28
Fig . 4. S c atte rg ram of Ig G s erore activ ity t o r 16 .5 kD a in s era from
c att le . T he r 16.5 kDa prot ein w as used at 2 ㎍/ ㎖ concentrat ion
for coating for ELISA ; 300 fold dilution of bovine serum ,
20,000 fold dilution of anti- bovine IgG conjugated with HRP , and
OPD w ere used in ELISA , and the absorbance w as read at 490
nm . Each dot represent s an individual serum .
29
Fig . 5. S c att erg ram of Ig G s erore act iv ity t o r26 kD a in s era from
c att le . T he r26 kDa protein was used at 2 ㎍/ ㎖ concentration
for coating for ELISA ; 300 fold dilution of bovine serum ,
20,000 fold dilution of anti- bovine IgG conjugated with HRP , and
OPD w ere used in ELISA , and the absorbance w as read at 490
nm . Each dot represent s an individual serum .
30
Fig . 6. S c att erg ram of Ig G s erore act iv ity t o r39 kD a in s era from
c att le . T he r39 kDa protein was used at 2 ㎍/ ㎖ concentration
for coating for ELISA ; 300 fold dilution of bovine serum ,
20,000 fold dilution of anti- bovine IgG conjugated with HRP , and
OPD w ere used in ELISA , and the absorbance w as read at 490
nm . Each dot represent s an individual serum .
31
Fig . 7. S c atte rg ram of Ig G s erore activ ity t o rE RC protein in s era
from c attle . T he rERC protein was used at 2 ㎍/㎖
concentration for coating for ELISA ; 300 fold dilution of bovine
serum , 20,000 fold dilution of anti- bovine IgG conjugated
with HRP , and OPD were used in ELISA, and the absorbance
was read at 490 nm . Each dot represent s an individual serum .
32
Fig . 8. S c atte rg ram of Ig G s erore ac tiv ity t o B . ab or t us w h ole c e ll
ly s at e s in s e ra from c at tle . T he B rucella whole cell ly sates
were used at 0.2 ㎍/ ㎖ concentration for coat ing for ELISA ;
300 fold dilution of bovine serum , 20,000 fold dilution of
anti- bovine IgG conjugated with HRP , and OPD were used in
ELISA , and the absorbance was read at 490 nm . Each dot
represent s an individual serum .
33
4 . Pre va le nc e of a ntibo d ie s to re c o m binant prote ins amo ng
re s ide nts in the Cheju pro v inc e
Since there were continuous outbreaks among cattle population in the
Cheju province, it was of interest to know the degree of exposure of
resident s to B . abortus . In order to determine the prevalence of antibodies to
B . abortus , serum samples w ere selected randomly to represent general
populations in the Cheju province. A total of 500 serum samples w ere chosen
based on sex and age groups which are representative the population . Serum
samples w ere then examined for the presence of antibodies to recombinant
proteins and the B . abortus lysate antigens by ELISA . Although the
seroreactivity to each recombinant varied markedly , it was obvious that a
small portion of the resident s in the Cheju province had circulating antibodies
to recombinant proteins and B . abortus lysate antigens (Fig . 9). More than
90% of serum samples examined in this study had seroactivity of O.D. 0.2 or
less . When O.D. 0.4 in all recombinant proteins and O.D. 0.5 in r39 kDa
protein w ere used as criteria for seropositivity in this study , prevalence of
antibodies varied from 0.8% against the rERC and B . abortus lysate antigens
to 2.2% to the r39 kDa protein (T able 3). T he r26 kDa protein , which was
very effective in detecting T AT positive serum , gave a positive rate of 1.4%.
When the prevalence of antibodies to r39 kDa was further analyzed, there
was no signigicant difference betw een age groups (T able 4). How ever ,
prevalence of anti- r39 kDa antibodies w as significantly greater in female than
in male (p<0.05, Chi- square test ). T herefore, this study indicates clearly that
some resident s in the Cheju province had chances to expose to B . abortus .
34
Fig . 9. S c at terg ram of Ig G s e rore ac tiv ity t o re c om bin ant prote in s
an d B . ab or t us ly s ate s in s era from re s ide nt s in th e Ch eju
prov in c e (N =500 ) . 300 fold dilution of human serum ,
20,000 fold dilut ion of anti- hum an IgG conjugat ed with HRP ,
and OPD were used in ELISA , and the absorbance w as read at
490 nm . Each dot represent s an individual serum , and the
number s in the box indicate the number of sera with O.D . 490
nm les s than 0.20.
35
T able 3. Prevalence of ant ibodies to recombinant proteins among resident s
in the Cheju province (N =500)
Antigen sSeropositiv e *
No. %
r16.5 kDa 7 (1.4)
r26 kDa 7 (1.4)
r39 kDa 11 (2.2)
rERC 4 (0.8)
w hole cell ly sates 4 (0.8)
* Criteria for seropositivity : IgG O.D. at 490 nm ≥0.40 recombinant
protein s , r16.5 kDa, r26 kDa , rERC, and whole cell ly sates ,
and IgG O.D. at 490 nm ≥0.50 for r39 kDa prot ein .
T able 4. Prevalence of ant ibodies to r39 kDa am ong resident s in the Cheju
province by sex and age groups .
A geMale F emale
No.
ex amined
positiv e
No. (% )
No.
ex amined
posit ive
No. (% )
≤10 38 0(0.0) 36 3(8.3)
11- 20 39 0(0.0) 36 1(2.8)
21- 30 46 1(2.2) 45 1(2.2)
31- 40 46 0(0.0) 47 0(0.0)
41- 50 34 0(0.0) 33 3(9.1)
51- 60 22 0(0.0) 25 1(4.0)
≥61 22 0(0.0) 41 1(2.4)
247 1(0.4)) 253 10(4.0)
36
Ⅳ . D is cu s s ion
T his study dem on strat es that recombinant protein s , r 16.5 kDa, r26 kDa,
r39 kDa and rERC of B . abortus are immunogenic and are effect ive in
detecting ant ibodies to the nat ive prot eins in sera from cat tle and humans .
Although these recombinant proteins were reported in the literature3 1 , 3 4 , 3 6 , 4 0 ,
the proteins have not been u sed in serodiagnosis of brucellosis . T herefore,
it w as not possible to compare the result s obt ained in this study directly
w ith others .
In this study , the 16.5 kDa, Omp 16, protein w as chosen becau se it s
presence in the surface of B ruce lla4 0 . T he native 16.5 kDa prot ein w as
reported being acylated, ie , lipoprotein 4 1 . It w as not known w hether or not
the r 16.5 kDa produced in this study are acylated . But the r 16.5 kDa
prot ein w as immunogenic in mice, and rabbit anti- B . abortus ant iserum
recognized the recombinant 16.5 kDa prot ein in the W estern blot analy sis .
T he r 16.5 kDa protein w as thu s fully compatible w ith the nativ e Omp 16
prot ein in term s of antigenicity 4 0 .
T he peripla smic 26 kDa protein w as also expressed in this study based
on the report that sera from catt le naturally infected w ith B . abortus w ere
reactiv e with this protein in the W estern blot analy sis3 4 . T he molecular
w eight of the r26 kDa protein prepared in this study w as somew hat
sm aller at about 23 kDa . T his might due to conform ational change by the
presence of six hist idine tag s or due to cleavage of signal peptides . Despit e
37
the change in mobility in SDS - PAGE analy sis , the r26 kDa w as both
immunogenic in mice and reactiv e with rabbit antisera again st B . abortus .
T he cytoplasmic 39 kDa prot ein w as reported to elicit strong DT H
response in cattle naturally infected w ith B . abortus and w as specific to
B rucella species3 1 . T he recombinant 39 kDa protein prepared in this study
w as effectiv e in producing antibodies in mice. But rabbit antibodies to B .
abortus gav e a very faint band in W estern blot ting , maybe due to small
am ount of the 39 kDa protein in B . abortus whole cells which w ere used
in immunization of rabbit .
Recombinant ERC protein has not been reported in the literature yet ,
but w as prepared in this study mainly based on scientific interest w ith
postulation that cattle naturally infect ed w ith B rucella , part icularly aborted
cow s w ould have antibodies to the protein . Interestingly , the rERC protein
gave tw o protein bands , 34 kDa and 20 kDa, in SDS - PAGE analy sis with
no clear explanation . It m ay be pos sible to postulat e that the smaller size
w ere deriv ed by degradation of the full size ERC protein . Both protein s
w ere apparent ly immunogenic in mice and rabbit s . How ever rabbit
antiserum to B . abortus show s a w eak upper band and relativ e strong
low er band in W estern blot analy sis . T his might be due to less amount of
nativ e upper ERC protein fraction in the B . abortus antigen s w hich w ere
used in immunization of rabbit s .
In order to support the finding s above, serum samples from catt le
naturally infected w ith B . abortus w ere used in ELISA for detection of
38
antibodies to the native prot eins . A s expected, there w as a m arked
difference in seroreact ivity to recombinant prot eins in sera from T AT
positive and negative catt le . F or ex ample, only a sm all portion , about 20%
or less , of T AT positiv e serum samples gave a significant level of
antibodies to r 16.5 kDa, r39 kDa and rERC protein s . Interestingly , how ever ,
the r26 kDa protein w as react ive with m ore than 50% of T AT positiv e
sera, supporting the prev iou s report in w hich sera from cattle naturally
infect ed w ith B rucella gave a reactivity w ith 26 kDa in W estern blotting 3 4 .
Presence of positive react ivity of recombinant protein s in sera from T AT
negat ive cattle might be due to asymptomatic carrier s because the catt le
w ere from the sam e farm s or geographic areas w here T AT posit ive
animals w ere ident ified .
It was also interesting to note that B . abortus whole cell lysate antigen s
w ere very effectiv e in detect ing antibodies to B rucella in ELISA . Most of
T AT positiv e sera w as also positiv e to the w hole cell ly sat e in ELISA .
T his indicates that ELISA using the B . abortus ly sate antigen s can be
used as a screening test just like milk ring test or slide agglutinat ion test .
Presence of seroreactiv ity in several T AT negative sera m ay indicate the
greater sensitivity of ELISA over the agglutination test employed in this study .
S erological t est result s using recombinant proteins in sera from
resident s in the Cheju province w ere som ewhat ambiguou s . In general,
majority of serum samples gave a low seroreactiv ity to recombinant
prot ein s . Interestingly , how ever , four serum samples had an elev ated level
39
of seroreactiv ity to the B . abortus ly sat e ant igen s . If these sera are
considered as seropositiv e, the prevalence of ant ibodies to the B . abortus
ly sat e ant igen becomes 0.8% . T his w as similar to the previous report by
Yeom e t al., in w hich 0.6% of resident s in the Cheju prov ince w ere
seroposit ive by tube agglut ination test 6 .
Among recombinant protein antigen s , the r39 kDa prot ein gave the
strongest seroreactiv ity . T his w as in contrast w ith the finding s w ith the
T AT posit ive cat tle serum , in w hich the r39 kDa protein gav e the low est
seroreact ivity . Only possible explanation for such difference w ould be due
to host variation in humoral immune respon ses to exposure to B . abortus .
About 1.4% of sera ex amined in this study gave an elevated ant ibody
response to r26 kDa, which w as strongly reactiv e w ith the m ajority of
T AT posit ive cat tle sera .
In summary , the result s presented in this study emphasize the diversity
of humoral immune responses in infected host s to B . abortus proteins. In
addition , this study show ed a strong evidence that a small portion of
resident s in the Cheju province had exposed to B . abortus . T he recombinant
proteins , r 16.5 kDa, r26 kDa, r39 kDa, and rERC, could be useful in
serological test s for brucellosis as a supplement to the current agglutination
test s . Particularly , rERC can be used in animals vaccinated with B19 strain .
Further studies , how ever , need to be conducted to determine sensitivity and
specificity of the recombinant protein - based serological test s before field
application .
40
Ⅴ . Conc lu s ion
Brucellosis is one of the m ajor zoonoses infect ing dom estic animals and
humans . Recent increase in B rucella cases in cat tle in Korea has brought
concern s about human infection . Because the current diagnostic method
based on LPS - containing whole cells have been far from the optimal tool.
T his study w as initiat ed to explore protein - based t est s for serodiagnosis of
brucellosis . Among B . abortus prot ein s in the literature, the outermembrane
prot ein Omp 16, 16.5 kDa, a peripla smic prot ein , 26 kDa, and a cytoplasmic
prot ein , 39 kDa, w ere chosen in this study based on their ant igenicity and
specificity . In addition , the ERC protein , which is involved in the
metabolism of erythrit ol- the major carbon source of B rucella , w as included .
All the recombinant protein s prepared in this study w ere immunogenic in
mice and rabbit s in generating strong antibody respon ses w hich w ere
determined by W estern blot analy sis . T he expressed recombinant protein s ,
r 16.5 kDa, r26 kDa, r39 kDa, and rERC, w ere also effect ive in detecting
antibodies by ELISA in sera from catt le naturally infect ed w ith B . abortus .
In addition , serum samples from resident s in the Cheju province w ere
ex amined . Of 500 sera test ed, sev en (1.4% ) serum samples had elev ated
antibodies to r 16.5 kDa and r26 kDa protein s , and 11 (2.2% ) to the r39 kDa
prot ein s . Interest ingly , four (0.8% ) sera show ed seroreactiv ity to the rERC
protein and to the B . abortus whole cell ly sate antigens .
T he result s thus indicate that the recombinant protein s above are
41
effectiv e in detecting antibodies to their nativ e protein s of B . abortus and
thus can be used in supplem ental serodiagnost ic test s to the current
agglutinat ion test s . In addition , this study provides a serological ev idence
that a portion of resident s in the Cheju prov ince hav e been exposed to B .
abortus . F urther studies on indiv iduals at high risk of contract ing
brucellosis will w arrant s usefulness of the recombinant protein s for
serodiagnosis of the disease.
42
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47
국문요약
Bruce lla abo rtus의 유전자 재조합 항원을 이용한 브루셀라병의
혈청학적 진단과 항체분포조사
어 형 진
의과학사업단
연세대학교 대학원
(지도교수 조 상 래 부교수)
브루셀라병은 사람과 가축에 감염되는 대표적인 인수공통전염병이다. 최근
한국에서 증가되고 있는 브루셀라병은 가축과 접촉이 잦은 직업의 사람에서
감염에 대한 관심이 증가되고 있다. 사람에서 주요 감염세균인 B rucella
abortus는 분리, 동정이 난해해서 브루셀라병의 감염이 의심되는 사람에서의
진단에는 혈청학적 진단에 의존하고 있다. 그러나 B rucella 균체를 이용하는
현재의 혈청학적 방법은 다른 그람 음성세균과의 교차반응이 존재하고, 현재
활동성 감염자와 과거병력자를 감별하는데 적절하지 못하다는 평가를 받고 있
다. 본 연구에서는 B rucella 세균에 특이한 단백질 항원을 유전자재조합 기법
으로 생산하여 혈청학적 진단에 이용하고, 단백질을 사용한 혈청학적 진단을
평가하고자 하였다. 그리고 제주지역의 주민을 상대로 유전자재조합 단백질에
대한 항체 분포를 파악하고 혈청학적 조사에 유용성을 조사하고자 하였다.
기존에 발표되었던 B . abortus 단백질 중에서 항원성과 특이성을 고려하
여 세포외막단백 Omp16 인 16.5 kDa, 세포막주 단백인 26 kDa, 그리고 세포
48
막 단백인 39 kDa을 선택하였다. 추가로 브루셀라균이 성장하는데 필요한 탄
소를 얻는 주요 물질인 ERC 단백질을 포함하여 실험을 하였다. 각 각의 단백
질 유전자를 pQE30 발현벡터에 클로닝하고 E . coli에서 발현을 시켰다. 정제
된 유전자재조합 단백질인 r 16.5 kDa , r26 kDa, r39 kDa , 그리고 rERC 단백질
의 분자생물학적 특성을 SDS - PA GE 분석을 통해 규명하였고, W est ern
blotting 분석을 통하여 면역원성을 확인하였다. 위의 유전자재조합 단백질들
모두 W estern blot ting 분석을 통하여 보면 mice에서 생성된 항체에 강한 면
역반응을 보이는 것을 확인하였다. 더불어, 토끼에서 얻어진 항체에 대해서는
r 16.5 kDa, r26 kDa, 그리고 r39 kDa의 단백질과 반응하는 것을 확인하였고
이 사실로 이들 유전자재조합 단백질들이 B . abortus이 단백질과 반응하는 항
원으로서 역할을 한다는 사실을 간접적으로 증명하였다.
이들 유전자재조합 단백질들은 또한 ELISA를 통한 실험으로 시험관 응집
반응에서 양성판정으로 브루셀라병에 자연감염판정을 받은 100두의 젖소들로
부터 얻어진 혈청 내 항체를 찾는데도 효과적이었다. 위와 같은 유전자재조합
항원을 이용한 혈청학적 진단으로 제주지역 주민을 대표하는 거주자들의 혈청
내의 항체존재 여부를 조사하였다. 모두 500명의 검사 혈청 중에서 r 16.5 kDa
단백질과 r26 kDa 단백질에 대한 양성율은 7 검체로 1.4%였고, r39 kDa 단백
질에 대해서는 11 검체로 2.2%였다. 흥미로운 사실은 4 검체에 해당하는 0.8%
가 rERC 단백질과 B rucella 융해체에 대해 혈청학적인 양성을 보였다.
본 연구 결과는 위에서 제시되어진 4가지 유전자재조합 단백질이 B .
abortus 의 단백질 항원에 대한 항체를 찾아내는데 유용하다는 것을 제시하고
있으며, 현재 사용되고 있는 여러 가지 응집 진단 방법과 함께 사용되어 질 수
있는 가능성을 시사하고 있다. 또한 이 연구에서는 제주지방에 거주하는 주민
49
들 중 일부가 B . abortus에 노출되어 있음을 시사하는 여러 가지 결과를 제시
하고 있다. 앞으로의 연구 진행방향은 브루셀라병이 확실한 개체에 대한 연구
를 바탕으로 이 질병의 혈청학적 진단에 유전자재조합 단백질의 유용성을 증
명해 내는 일이 절실히 요구될 것이다.
핵심되는말 : 브루셀라병, 유전자재조합 단백질, B rucella abortus ,
혈청학적 진단
50
