Wus Research, 26 (1992) 127-139 0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1702/92/$05.00 VIRUS 00833 Expression of influenza neuraminidase in baculovirus-infected cells * Karen A. Mather a, Jacinta F. White b, Peter J. Hudson b and Jennifer L. McKimm-Breschkin a a Biomolecular Research Institute, and b CSIRO, Diuision of Biomolecular Engineering, Parkoille, Australia (Received 28 October 1991; revision received 22 July 1992; accepted 27 July 1992) 127 Summary Recombinant influenza neuraminidase (NA, subtype 2, A/NT/60/68) was produced by recombinant baculovirus-infected insect cells. The recombinant NA retained enzyme activity and was located on the cell surface. Enzyme activity was both cell-associated and in the cell free supernatant; maximal NA activity was found in the supernatant. Recombinant NA was recognised by polyclonal antisera and by three monoclonal antibodies specific for NA (subtype 2). Enzyme activity was also neutralised by polyclonal antisera. Recombinant NA thus retains most of the immunological and activity properties of authentic influenza NA. Immunopre- cipitation of [ 35S]Methionine-labelled cells and supernatant and partial purifica- tion of NA indicated that a N 50-kDa form of NA was present in the supernatant, whilst the expected size ( N 67-kDa) was cell-associated. Purified recombinant extracellular virus was also enzymatically active, and contained the 67-kDa NA which was located on the membrane capsule of the virus. This suggests that the virus had acquired the cell-associated form of recombinant NA during the budding process from the cell. Neuraminidase; Influenza; Baculovirus; Expression Correspondence to: K.A. Mather, Biomolecular Research Institute, 343 Royal Parade, Parkville, Australia, 3052. * Presented in part at the 8th International Conference on Negative Strand Viruses. Charleston, S.C. Abbreuiations: AcNPV, Autographica californica nuclear polyhedrosis virus; ECV, extracellular virus; FITC, fluorescein isothiocyanate; MUNANA, 2’-(4-methylumbelliferyl)-a-D-N-acetyl neuraminic acid; TMB, 3,3’,5,5’-tetramethylbenzidine; ABTS, 2,2’-azino-di-[3-athyl-benzthiazolinsulfonate].
Transcript
Wus Research, 26 (1992) 127-139
0 1992 Elsevier Science Publishers B.V. All rights reserved 0168-1702/92/$05.00
VIRUS 00833
Expression of influenza neuraminidase in baculovirus-infected cells *
Karen A. Mather a, Jacinta F. White b, Peter J. Hudson b and Jennifer L. McKimm-Breschkin a
a Biomolecular Research Institute, and b CSIRO, Diuision of Biomolecular Engineering, Parkoille, Australia
(Received 28 October 1991; revision received 22 July 1992; accepted 27 July 1992)
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Summary
Recombinant influenza neuraminidase (NA, subtype 2, A/NT/60/68) was produced by recombinant baculovirus-infected insect cells. The recombinant NA retained enzyme activity and was located on the cell surface. Enzyme activity was both cell-associated and in the cell free supernatant; maximal NA activity was found in the supernatant. Recombinant NA was recognised by polyclonal antisera and by three monoclonal antibodies specific for NA (subtype 2). Enzyme activity was also neutralised by polyclonal antisera. Recombinant NA thus retains most of the immunological and activity properties of authentic influenza NA. Immunopre- cipitation of [ 35S]Methionine-labelled cells and supernatant and partial purifica- tion of NA indicated that a N 50-kDa form of NA was present in the supernatant, whilst the expected size ( N 67-kDa) was cell-associated. Purified recombinant extracellular virus was also enzymatically active, and contained the 67-kDa NA which was located on the membrane capsule of the virus. This suggests that the virus had acquired the cell-associated form of recombinant NA during the budding process from the cell.
Neuraminidase; Influenza; Baculovirus; Expression
Correspondence to: K.A. Mather, Biomolecular Research Institute, 343 Royal Parade, Parkville,
Australia, 3052.
* Presented in part at the 8th International Conference on Negative Strand Viruses. Charleston, S.C.
The influenza genome has 8 negative-sense single-stranded RNA segments. Neuraminidase (NA) is a type II integral membrane glycoprotein which is encoded by a single RNA segment. NA plays an important role in the infectivity and immunogenicity of the influenza virus, a major human pathogen. Cloned NA has been expressed at low levels in E. coZi whilst high levels of NA have been expressed in whole insects using baculovirus methods (Price et al., 1989). Weyer and Possee (1991) expressed NA in the baculovirus system but no detailed studies of the locality of NA were made.
The baculovirus system for expression of recombinant proteins is capable of producing high yields of foreign proteins, which can undergo a number of post- translational modifications (For review, see Luckow and Summers, 1988). Here we describe the expression of the neuraminidase gene, subtype 2 (A/NT/60/68), in insect cells using the baculovirus system and the analysis of the location of recombinant products.
Materials and methods
Cells and viruses AcNPV and recombinant AcNPV were grown in Spodoptera frugiperda (Sf) cells
in Grace’s medium (Hazelton, USA) supplemented with 10% foetal bovine serum, 0.3% yeastolate and 0.5% tryptose broth. The production of recombinant bac- ulovirus stock was carried out according to the methods of Summers and Smith (1987). The recombinant virus was purified by limiting dilution (Harley et al., 1990), screened with DNA probes, and non-occluded plaques were selected (Summers and Smith, 1987).
Insertion of neuraminidase into pvL941 shuttle vector The neuraminidase gene, sub-type 2, (A/NT/60/68) was excised from a
plasmid, gel-purified and blunt-end cloned into the BamHl site of the shuttle vector, pVL941 (Luckow and Summers, 1989). Transformed bacterial cells were screened with nick-translated alpha-“*P or digoxigenin-labelled DNA probes. Restriction enzyme analysis was used to analyse clones.
Neuraminidase assays Neuraminidase activity was assayed by the use of the artificial substrate,
2’-(4-methylumbelliferyl)-a-o-N-acetyl neuraminic acid (MUNANA) as described by Myers et al. (1980). At the appropriate times (see figures) cells were harvested and centrifuged in a bench top centrifuge for 1200 rpm/5 min. The cell pellet was washed 2 x with phosphate-buffered saline (PBS). The cell pellet was lysed in 0.5% NP40 at 4°C for 10 min and sonicated for 3 min. Lysed cell pellets and supernatants were analysed for activity.
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Antibodies Rabbit polyclonal antisera was generated against purified pronased released
NA heads (M&mm-Breschkin et al., 1991) of NWS/Tokyo (virus kindly supplied by Dr. R. Webster, St. Jude Children’s Hospital, Memphis, TN). This antisera was specific for NA, subtype 2. Monoclonal antibodies specific for NA, subtype 2, were provided by D. Marshall (this laboratory). Secondary antibodies conjugated to horseradish peroxidase were purchased from Silenus (Australia) or Bio-Rad (Australia).
NA inhibition test Various dilutions of polyclonal or monoclonal antibodies specific for NA sub-
type 2 were mixed with a volume of recombinant baculovirus-infected cell super- natant or native influenza virus (A/NWS/Tokyo, NA subtype 2). The reactions were incubated at room temperature for 30 min. The samples were then tested for NA activity as per standard protocol (see above).
Recognition of recombinant NA by antibodies Various dilutions of non-denatured baculovirus supernatant and partly purified
supernatant and cell NA forms were applied to a nitrocellulose membrane. Denatured proteins were transferred from SDS-PAGE slab gels (Laemmli, 1970) to nitrocellulose (BA-85 0.45 PM, Schleicher and Schuell, Germany) by a Bio-Rad transblot apparatus under the conditions described by the manufacturer (Western transfer). The membranes were blocked with PBS-l% casein and then reacted with polyclonal and/or monoclonal antisera. Secondary conjugated antibodies were then reacted with the nitrocellulose and bound antibody was detected with TMB (Boehringer-Mannheim, Germany), as per McKimm-Breschkin, 1990.
Immunofluorescence Sf cells were grown on microscope slides until confluent and were then infected
with recombinant, wild-type or mock infected at a multiplicity of 0.1 for 1 h/27”C. The inoculum was replaced with fresh media. At 72 h post-infection cells were fixed by either of the following two methods. (1) In acetone at 4°C for 5 min and then ethanol at 4°C for a further 5 min. (2) In 2% formaldehyde for 30 min and then washed in phosphate-buffered saline (PBS). Non-fixed cells were washed in PBS 3 X . Each sample was incubated with either non-specific antisera or rabbit anti-neuraminidase sera for one hour at room temperature. The antisera was then removed and the slide washed 3 times with PBS. The cells were then reacted with sheep anti-rabbit FITC (Silenus, Australia) for one hour at room temperature. The antisera was removed and after three PBS washes and a water wash (for fixed cells only) the cells were examined for fluorescence.
Immunoprecipitation Sf cells were grown in 35 mm Petri dishes to confluency and infected with either
recombinant or wild-type virus (m.o.i. N 5). Before labelling cells were starved in media lacking methionine for one hour. At 48 h p.i. cells were labelled with 100
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PCi of [35S]Methionine (Amersham, U.K.) in methionine-free medium for 8 h, replaced with fresh medium, and samples were harvested at 72 h post-infection. Cell pellets were washed 2 x with PBS, lysed in 150 mM NaCl, 50 mM Tris pH 8 and 1% NP40, freeze thawed 3 times and sonicated for 3 min. Lysed pellets and supernatants were precleared by reacting with non-specific rabbit anti-sera conju- gated to Protein A-Sepharose beads (CL-4B, Pharmacia, Sweden) for 5 h at 4°C with gentle agitation and then centrifuged at 650 rpm/2 min. The supernatant was then added to highly specific rabbit anti-neuraminidase antisera conjugated to Protein A-sepharose beads and reacted overnight at 4°C. The reaction was then centrifuged at 650 rpm/3 min and the beads washed 3 X with immunoprecipita- tion buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8, 0.1% NP40). SDS-gel loading buffer (0.0625 M Tris-Cl pH 6.8, 10% glycerol, 2% SDS, 0.05% P-mercaptoethanol, 0.00125% bromophenol blue) was then added to the sample, heated to lOo”C/5 min and analysed by SDS-PAGE.
ELBA Monoclonal antibody 17 (anti NA, subtype 2) was bound to an ELISA plate
(Dynatech, C a. a a concentration of 10 pg/ml in 0.05 M carbonate buffer pH 9.3 > t overnight at 4°C. All further steps were performed at room temperature. The plate was blocked with PBS/l% casein for 1 h. Dilutions of purified Tokyo NA heads of known concentration, allantoic fluid (A/NWS/Tokyo), recombinant baculovirus- infected supernatant or purified NA recombinant baculovirus in PBS were added to the plate for 1 h. The plates were washed 3 X with PBS and were then incubated with polyclonal anti-NA serum conjugated to biotin at room tempera- ture for 1 h. The plates were then washed 3 x with PBS and incubated with streptavidin conjugated to horseradish peroxidase for 1 h, washed with PBS 3 x ,
rinsed once with H,O, and then incubated with the substrate, ABTS (Boehringer Mannheim, Germany).
Partial purification of NA Lysed cell pellet (see above) or concentrated extracellular medium (Amicon
filtration) were partly purified by sucrose step gradients. Samples were loaded onto a lo-25% sucrose gradient and centrifuged for 18 h/30 K/4”C in a SW41 rotor. Fractions containing NA activity were pooled, pelleted by ultracentrifugation (TLA 100.3/90 K/16 h/4”c) and resuspended in PBS. The samples were then analysed by SDS-PAGE and Western-blotted.
Purification of extracellular virus (ECV) One hundred ml of culture medium from - 1 X 10’ cells infected with NA
recombinant virus (m.o.i. - 5) was harvested 4 days post-infection. The ECV was then purified by the method of Summers and Smith (1987) except that the viral band was removed from the sucrose gradient and stored at 4°C until further analysis.
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Electron microscopy A 1: 5 dilution of the ECV preparation in PBS was allowed to settle onto a
celloidin carbon-coated grid (400 mesh) for more than 30 min and then the excess removed. The virus-coated grid was incubated with various dilutions of polyclonal anti-NA sera in filtered PBS/O.l% BSA for 45 min at room temperature. The antisera was then removed and washed 3 X with filtered PBS/O.l% BSA. The grid was incubated with a 1: 20 dilution of goat anti-rabbit antibody conjugated with 10 nm gold particles (Amersham, U.K.) in filtered 20 mM Tris, pH 8.2, 0.1% BSA for 45 min at room temperature. The grid was washed 2 x with PBS, 1 x with distilled water and was negatively stained with 2% uranyl acetate (OA) for 3 s and allowed to air-dry before examination by an Transmission JEOL 1OOB electron microscope.
Results
Construction of recombinant AcNPV The baculovirus shuttle vector containing NA was constructed as described in
the Methods section. Restriction enzyme analysis confirmed that the NA gene was present as only one copy and in the correct orientation for expression under the control of the polyhedrin promoter.
Recombinant virus was produced by the co-transfection of Sf cells with wild-type AcNPV DNA and purified pVL941-NA DNA. Limit dilution of the transfection supernatant and screening of possible recombinants by DNA probes yielded three positives. The partially purified recombinants were analysed for NA activity and all were found to be positive. The ‘recombinant’ with the highest activity was further purified by three rounds of plaque purification. High-titred stocks of the plaque- purified recombinant virus were grown.
Expression of the NA gene Initial activity of partially purified recombinant virus was confirmed by an NA
enzyme assay. Purified recombinant virus stock was obtained for the kinetic analysis of recombinant NA expression. Maximal NA activity was found in the supernatant and continued to increase until the end of the experiment (Fig. 1). The activities of recombinant NA (supernatant) and the NA from influenza virions were inhibited by polyclonal antisera to the same extent (Fig. 2). One out of 4 monoclonal antibodies, specific for NA sub-type 2, also inhibited activity (mono- clonal 15, data not shown).
The polyclonal and three out of four monoclonal antibodies recognised recom- binant NA (non-denatured) when applied to nitrocellulose (Fig. 3).
Immunofluorescence analysis of fixed and unfixed cells using polyclonal antisera specific for NA, subtype 2, indicated that NA was expressed throughout the cell and on the cell surface (Fig. 4).
The characterisation of the recombinant protein by immunoprecipitation and SDS-PAGE is shown in Fig. 5. There appear to be two different-sized forms of NA. The larger, cell-associated NA appears to be approximately the same size as
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NA ACTIVITY ASSAY Bacuiovirus expressed NA
1400
n m
1200.
0
I 1000-
";: 800 -
r e 600-
I e a 400-
*
z 200-
cell pellet
O+-- / i
0 20 40 100 120 140
Fig. 1. Time course of NA activity in recombinant virus-infected cells (m.o.i. - 5). Lysed cell pellets and supernatants were assayed for activity at various times ~st-infection. The NA activity is given in
arbitrary fluorescence units. Activity is calculated for the total volume of cell pellet or supernatant.
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Baculo X.4 Supernatant
0 2 4 6 a 10
Log? RecLiprotal Dilution of Antisera Fig. 2. Inhibition of NA activity by the presence of polyclonal anti-NA sera. Native influenza virus (A/NWS/Tokyo, NA sub-type 2) and recombinant NA (supernatant) were incubated with different
dilutions of antisera and then tested for activity (See Methods).
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A B C D E F G H
10 15 17 PC Fig. 3. Non-denatured recombinant and native influenza virus (N2 subtype) were blotted onto nitrocel- lulose. The blot was then reacted with either specific anti-neuraminidase (subtype21 polyclonal antisera (PC) or monoclonal antibodies (4, 10, 15 or 17). The nitrocellulose was then reacted with the appropriate conjugate and the TMB substrate (See Methods). Rows labelled A and B are decreasing amounts of native virus (subtype N2), C and D are decreasing amounts of recombinant supernatant (4 days p.i.), E is wild-type supernatant, F is partially purified recombinant NA from supernatant, G is
partially purified recombinant NA from the cell pellet, H does not contain any protein.
the expected size of native NA, - 67 kDa, whilst the supernatant appears to contain a smaller form of NA, - 50 kDa.
Western blot analysis of unlabelled partially purified recombinant NA (Fig. 6) also showed that there was a 67-kDa form in the cell pellet and a - 50-kDa form in the supernatant, which co-migrated with pronased purified NA heads.
Culture medium was separated by centrifugation into a viral pellet and super- natant. Activity was found in both the viral pellet and the clarified supernatant. Western analysis of purified extracellular recombinant virus indicated that it contained the 67-kDa form of NA (Fig. 6). NA activity co-purified with the sucrose-gradient purified extracellular virus.
Electron microscope analysis of purified extracellular virus labelled with im- munogold (Fig. 7) indicated that recombinant NA was present on the membrane capsule (Fig. 7c and d) and not expressed on the nucleocapsid (Fig. 7b).
The limit of detection of the ELISA assay was 40 pg/ml of purified influenza NA. Comparison of the absorbances obtained with known concentrations of influenza NA enabled estimation of the recombinant product yield. Wild-type baculovirus-infected cell supernatant did not react in this ELISA. Recombinant-in- fected supernatant contained - 75 ng of NA per litre (Table 1). Recombinant NA
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Fig. 4. Immunofluorescence of NA expressed by recombinant virus-infected cells. Unfixed cells were infected with recombinant virus or wild-type virus at a low multiplici~ of infection and incubated with polyclonal anti-neuraminidase sera and anti-rabbit FITC. (See Methods). Cells were examined using a BioRad MRCSOO Confocal Microscope. (a) Recombinant infected cells, the left panel showing the fluoresence image, the right panel shows the phase contrast image of the same cells. (b) Control. Wild-type infected cells, the left panel showing the fluorescence image and the right panel shows the
phase contrast image of the same cells. Arrow points to a cell with polyhedra.
baculovirus pelleted from the supernatant contained approximateiy 1% of the NA. The yield of recombinant NA in the supernatant was approximately l/25 of levels detected in allantoic fluid from A/NWS/Tokyo infected eggs (Table 1).
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1 2 3 4 Fig. 5. Radioimmunoprecipitation of recombinant NA by polyclonal anti-NA sera (A/NWS/Tokyo, NA subtype 2). Lanes labelled P and S represent cell pellet and supernatant respectively. Lane 1 is uninfected Sf celis (pellet and supernatant respectively), lane 2 is wild-type infected cells and lanes 3 and 4 are different loadings of recombinant baculovirus-infected cehs. The arrows point to the 67-kDa
NA species (lanes 3P and 4PI and to the * 50-kDa smaller species (lanes 3s and 4s).
110 84
47
33
24
MW\T’ NA P EV IMW
110 84
33
24
Fig. 6. Analysis of partly purified supernatant, cell pellet and ECV forms of recombinant NA. Western blots of an SDS-12% actylamide gel of different loadings of purified supematant (9 and cell pellet (PI NA forms and purified extracellufar virus (EVI. The blots were reacted with a polyclonal sera/mono- clonal pool against NA, sub-type 2. Fig. 6. (a) Lane NA is purified pronase treated native NA (subtype 2) and lane MW refers to molecular weight marker proteins t&e-stained, Bio-Rad) with apparent sizes indicated in kilodaltons (to the left of lane MWI. Arrows point to the two different-sized forms of NA. Fig. 6. (b) Lane I refers to wild-type influenza virus (NWS/Tokyo, NA subtype 2, allantoic fluid).
Arrow points to NA.
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A recombinant AcNPV virus was constructed which expressed the NA gene, sub-type 2. The recombinant protein retained a number of properties of native influenza NA including enzyme activity.
Recombinant NA was i~unologically related to native influenza neu- raminidase as shown by the recognition of non-denatured recombinant NA and by inhibition of activity by monoclonal antibodies and polyclonal antisera. One out of
a b
d Fig. 7. ~mmunoelectron microscope analysis of purified recombinant extracellufar vnus. Virus was visualised after incubation with rabbit polyclonal anti-NA/Goat anti-rabbit 10 nm gold particle complex followed by staining with uranyl acetate. Panel (a) virus incubated with goat anti-rabbit-gold only, (b), (c) and (d) virus plus rabbit anti-NA/goat anti-rabbit-gold. Arrows in (a) point to rod-shaped virion and its membrane capsule. The arrow in (b) points to a virus nucleocapsid and the arrows in Cc)
and (d) point to a virion (with its membrane capsule).
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TABLE 1
NA capture ELBA
NA pg/ml
A/NWS/ Tokyo (allantoic fluid) 2000
Recombinant NA b/virus cell s’nt 7.5
Purified recombinant NA b/virus 1
ELBA estimation of the amount of lrecombinant NA in cell culture supernatant and purified recombinant purified virus versus egg grown influenza virus (see Methods).
four monoclonal antibodies did not recognise the recombinant NA which may reflect differences in the glycosylation of NA.
The yield of supernatant recombinant NA estimated from an NA ELISA was 75 ng per litre. Recombinant baculovirus pelleted out of the supernatant contained only a fraction of the NA. In an NA activity assay the recombinant NA appeared to be more active than expected from the NA ELISA results, suggesting that the recombinant product has a higher specific activity compared to influenza virus-de- rived NA.
Recombinant NA was found on the insect cell surface similar to native NA which is transported to the cell surface of influenza-infected cells. Recombinant NA was also located on the extracellular baculovirus membrane capsule. It was also similar in size to the cell-associated form (- 67 kDa) and may therefore be a product of virus budding through the Sf cell membrane.
In contrast to native influenza NA, there are two different-sized forms of recombinant NA, the smaller form (- 50 kDa) in the supernatant and the expected size (N 67 kDa) in the cell pellet. Since cell viability was similar to uninfected cells during the early phases of infection, it suggests that the NA present in the supernatant was not due to lysis of the cells. The discrete size of the supernatant form suggests that it is an immature or cleavage product. In this respect the baculovirus-expressed NA gene product is similar to influenza NA which can be cleaved by pronase treatment without loss of enzyme activity (Laver, 1978). Further investigation is required to determine whether the NA is being cleaved and/or secreted from the cell.
Cleavage of type 1 signal sequences and subsequent secretion of foreign proteins in the baculovirus system has been demonstrated (e.g. tissue plasminogen activator, Jarvis and Summers, 1989, alpha-interferon, Maeda et al., 1985). Recom- binant haemaglutinin(HA)-NA of parainfluenza type 3, another type II membrane glycoprotein, was also found to be enzymatically active, cell-surface expressed and immunogenic (Van Wyke Coelingh et al., 1987). However, Van Wyke Coelingh et al. did not monitor expression of HA-NA in the extracellular medium. Nagy et al. (1990) expressed the HANA gene of Newcastle Disease Virus, another type II glycoprotein, which is produced predominantly intracellularly and also in the
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extracellular medium. Nagy et al. (1990) also found two different-sized forms of HANA corresponding to an immature and mature form, presumably due to differences in glycosylation. The HANA also co-purified with the recombinant extracellular AcNPV from the extracellular medium, suggesting that the virus membrane contains the HANA and the protein is not secreted per se.
A recent report (Weyer and Possee, 1991) has described the co-expression of the influenza HA and NA genes in the baculovirus system. The authors however only observed a single-sized NA cell membrane product and did not investigate the cell supernatant or the baculovirus virions for the presence of NA.
Our results suggest that the baculovirus expressed NA gene product is both cell-associated and present in the supernatant. The expected size form is found in the cell pellet and a smaller form in the supernatant. In addition extracellular recombinant baculovirus has NA activity and contains the larger NA form. Elec- tron microscopy indicates that NA is also present on the membrane capsule of extracellular recombinant baculovirus.
Experiments are in progress to purify and characterise the supernatant form. Since the type II signal/membrane anchor functions in baculovirus-infected cells, other foreign proteins could be engineered with the NA signal sequence for the display of recombinant proteins on the surface of Sf cells or on the virion membrane capsule.
Acknowledgements
The authors would like to thank Deidre Marshall for generously providing the monoclonal antibodies, 4, 7, 10 and 15. We are also grateful to Bob Irving, Elizabeth Cartwright and Bruce Caldwell for valuable discussions during the course of this project.
References
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sation of an avian influenza virus nucleoprotein expressed in E. coli and in insect cells. Arch. Virol.
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Jarvis, D.J. and Summers, M.D. (1989) Glycosylation and secretion of human tissue plasminogen
