RESEARCH ARTICLE

Molecular Cloning and Characterization of the Gene EncodingHeat Shock Protein 70 from the Chicken (Gallus gallus)

Elza Neelima Mathew • Gurvinder Singh Brah •

B. V. Sunil Kumar • Chandra Sekhar Mukhopadhyay •

Prem Prakash Dubey

Received: 27 June 2013 / Revised: 9 August 2013 / Accepted: 26 September 2013

� The National Academy of Sciences, India 2013

Abstract Heat shock protein 70 (HSP70) is a predomi-

nant member of the HSP family of proteins which play a

variety of functions in the cells and are responsible for

cytoprotection under stress conditions. In this study, in-

tronless gene of chicken HSP70 was amplified, cloned in

E. coli and characterized. The HSP70 gene contains

1,905 bp ORF. Sequence analysis of the HSP70 gene using

Mega 4 and DNA STAR softwares showed a high sequence

homology among different species indicating that the gene

is evolutionarily conserved. Synonymous substitution (dS)

in the HSP70 gene was higher than non-synonymous sub-

stitution (dN), suggesting that the gene is not under positive

selection and that no advantageous mutations had any

significant role in its evolutionary adaptation. The pre-

dicted Swiss-model of HSP70 protein consisted of 3 a-

helices, 19 b-turns, 7 G-turns and 4 hairpins. The G-factor

score for the HSP70 protein model was -0.82 for dihedral

bonds, -0.05 for covalent bonds and -0.45 overall, which

suggest that the model obtained for HSP70 is a reliable

one.

Keywords Heat shock proteins � HSP70 �Thermal stress � Chaperones � Poultry

Introduction

The mechanism responsible for the cell survival in stressful

conditions (as in case of hyperthermia) leads to quick and

transient responses at transcriptional and translational lev-

els [1]. In response to a variety of stresses, a highly con-

served set of polypeptides are synthesized by the cells from

virtually all organisms, known as heat shock proteins

(HSPs), accumulation of which was initially associated

with thermotolerance.

HSPs are molecular chaperones associated with the

folding of proteins. HSPs are expressed at normal body

temperature as well as under stress (e.g., heat shock) and

therefore are critical for both normal cell function and

survival under stress. The first heat shock proteins to be

discovered were with molecular masses of 70 and 26 kDa

[2]. HSPs have distinct locations and functional properties.

They are present in the cytosol, mitochondria, endoplasmic

reticulum, and nucleus. Although, these locations vary

depending on the particular protein. The principal HSPs

range in molecular mass from 8 to 150 kDa and are divided

into groups based on both size and function [3]. The most

well-studied and understood HSPs in mammals are those

with molecular masses of 10, 27, 40, 60, 70, 90 and

110 kDa. A number of stimuli other than hyperthermia like

energy depletion, hypoxia, acidosis, ischemia-reperfusion,

reactive oxygen species (ROS), reactive nitrogen species

such as nitric oxide, and viral infection are known to

induce HSP70 transcription [4].

Since birds are being constantly challenged by heat

stress in hot countries, many studies involving HSP70

expression in broilers have been made. The expression of

HSP70 in chickens is observed to be affected by heat stress.

It is tissue and allele dependent [5]. HSP70 expression was

shown to be tissue dependent when the levels of this

E. N. Mathew � G. S. Brah (&) � B. V. Sunil Kumar �C. S. Mukhopadhyay

School of Animal Biotechnology, Guru Angad Dev Veterinary

and Animal Sciences University, Ludhiana, Punjab, India

e-mail: gsbrah@yahoo.co.in

P. P. Dubey

Department of Animal Genetics and Breeding, Guru Angad Dev

Veterinary and Animal Sciences University, Ludhiana,

Punjab, India

123

Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.

DOI 10.1007/s40011-013-0252-0

protein were studied in different organs of chick embryos

subjected to heat stress [6]. Early age conditioning by

exposure of chicks induces a long term mechanism that

acts to reduce hyperthermia during heat challenge and

therefore reduces the induction of HSP [7].

HSP70 can be used as a potential indicator for adapta-

tion to stress. The cytoprotective effects of HSP70 can be

used for applications like organ preservation during trans-

plantation and anticancer therapeutics. Besides its role in

cytoprotection, this protein is also considered a cellular

thermometer [8]. Keeping in view all these points the

present study was planned to clone and characterize

chicken cytosolic HSP70 gene (DNAK type molecular

chaperone).

Material and Methods

Collection of Blood and DNA Isolation

Blood samples in 2.7 % EDTA were collected from the

wing vein of apparently healthy 6 weeks old chicken using

sterile 24 gauge needle and syringe.

Genomic DNA was isolated from chicken blood as per

the standard protocol with slight modifications [9]. The

absorbance of the extracted DNA was measured at 260 and

280 nm using a Nanodrop� spectrophotometer (Thermo

Scientific, USA). The ratio of OD260 and OD280 was cal-

culated to check purity of the extracted DNA.

Cloning and Sequencing of HSP70

Using GENETOOL software [10], oligonucleotide primers

were synthesized for amplifying the gene encoding HSP70

mature peptide corresponding to 1,905 bp. Restriction sites for

the enzymes NcoI and XhoI were incorporated at the 50 ends of

the forward (50-CGCCCATGGCCATGTCTGGCAAAGGG

CCGG-30) and reverse (50-CGCCTCGAGTTAATCTACTTC

TTCAATGGTTGG-30) primers, respectively, for directional

cloning. The reverse primer also contained a stop codon.

Addition of RE sites to the 50 ends of the primers gave rise to

1,927 bp amplicon.

The HSP70 protein encoding region was amplified by

PCR using Taq polymerase (Invitrogen, USA). PCR was

carried out in a 25 lL reaction containing 100 ng of DNA,

1 mM of each primer, 0.4 mM dNTPs, and 2.5 Units of

Taq polymerase (Invitrogen, USA). The reaction started

with an initial denaturation at 94 �C of 5 min, followed by

30 cycles of denaturation (94 �C for 1 min), annealing

(55 �C for 1 min) and extension (72 �C for 2 min), along

with one final extension step at 72 �C for 10 min. The PCR

products were analyzed in 1.0 % agarose gel along with

DNA molecular weight marker (Fermentas, USA). The

PCR product was gel purified successfully using gel

extraction kit (Qiagen, Germany) which was ligated at 4 �C

overnight with pGEM�-T Easy cloning vector (Promega,

USA) using T4 DNA ligase (Promega, USA). The ligated

product was transformed into competent E. coli DH5acells. The transformed cells were then plated on to LB agar

plates supplemented with ampicillin (100 mg/mL) X-Gal

(25 mg/ml) and IPTG (100 mM). The plates were incu-

bated at 37 �C for 12–14 h for the development of colo-

nies. White colonies harboring the recombinant plasmid

were grown overnight in LB broth containing ampicillin

(100 mg/mL) at 37 �C with vigorous shaking. Plasmids

were isolated from cultured clones using alkaline lysis

method [9]. Isolated plasmids were checked for the pre-

sence of insert by PCR amplification using specific prim-

ers. Clones were sent for sequencing and the obtained

sequence of HSP70 gene was submitted to NCBI and an

accession number JX827254 was obtained.

Sequence Characterization and Phylogenetic Analysis

The nucleotide sequence of the 1905 of Gallus gallus HSP70

(JX827254) was subjected to BLAST search [11]. Using the

blastn program and from the top 50 hits, representative

nucleotide sequences, Coturnix coturnix (EU622852.1),

Numida meleagris (AB096696.1), Coturnix japonica

(AB262971.1), Anas platyrhynchos (EU678246.2), Capra

hircus (JN656106.1), Canis lupus familiaris (XM_5374

79.3), Equus caballus (NM_001256923.1), Mus musculus

(NM_008301), Ovis aries (JN604434), Bos taurus

(BTU09861), Rattus norvegicus (L16764) and Oryctolagus

cuniculus (XM_002719559.1) were chosen for the study.

The UPGMA, Neighbor-Joining and Maximum Parsimony

programs in the Mega 4.0 package were used for the analysis

[12]. The Boot-strapping option with 1,000 replicates was

used with P distance model and with pair-wise deletion of the

gaps/missing data. The in silico translated protein sequence

from the HSP70 internal sequence was subjected to protein

blast and from the top 50 hits representative sequences C.

coturnix (ACC85671.1), N. meleagris (BAC24791.1), C.

japonica (BAF38390.1), A. platyrhynchos (ACD47154.2),

C. hircus (AFP43992.1), C. l. familiaris (XP_537479.1), E.

caballus (A2Q0Z1), M. musculus (AAC84168.1), O. aries

(AEX55801.1), B. taurus (AAA73914.1), R. norvegicus

(AAA17441.1) and O. cuniculus (XP_002719605.1) as

above were chosen for phylogenetic analysis. The MegAlign

(DNA star, Madison, WI, USA) program was used for the

generation of the multiple sequence alignments. The nucle-

otide sequences of HSP70 were used for estimation of the

number of synonymous substitutions per synonymous site

(dS) and the number of nonsynonymous substitutions per

non-synonymous site (dN). Estimates were computed using

a maximum likelihood (ML) method [13] and several

E. N. Mathew et al.

123

counting methods [14, 15] implemented in the CODEML

program of the PAML package Version 4.1 [16] ML analysis

was performed with runmode-2. Positive (dS \ dN) or

purifying (dS [ dN) selection was tested with a codon-based

z-test, using the Nei–Gojobori method (P distance) in

MEGA 4.0 [12].

Homology modelling program Swiss-model was

employed to generate a three-dimensional (3D) structure

model of heat shock protein 70 [17, 18]. Swiss-model is a

server for automated comparative modelling of 3D protein

structures. No other refinements were applied. Swiss PDB

viewer software was employed as a tool to envisage the

generated structural model. The generated 3D-model was

evaluated at various structure verification servers viz.

PROCHECK [19] that relies on Ramachandran plot [20]

which is a way to visualise backbone dihedral angles wagainst the amino acid residues in protein structure [21].

Results and Discussion

PCR amplification of the gene using specific primers

resulted in an amplicon of 1,927 bp size upon analytical

agarose gel electrophoresis (Fig. 1). The amplified HSP70

gene fragment was cloned into pGEM�-T Easy cloning

vector and transformed into DH5a cells. PCR amplification

of the isolated plasmids using specific primers resulted in a

single specific band on 1.0 % gel (Fig. 2). Cloned HSP70

gene was custom sequenced, and the sequence was sub-

mitted to NCBI (accession number JX827254).

Earlier pGEM�-T Easy cloning vector has been suc-

cessfully used for the cloning of 1,926 bp bovine HSP70

gene which upon sequencing showed a point mutation

(941, T ? C) in the orf [22]. It was also used for the

cloning of 1,926 bp buffalo HSP70 [23] and the cDNA

PCR amplicons of heat shock protein 70-1 gene of goat (C.

hircus) [24].

On nucleotide sequence BLAST search of the 1,905 bp

of chicken HSP70 (JX827254) using the blastn programme,

the sequence shared 96.4 % similarity with C. coturnix

(EU622852.1), 96.2 % with N. meleagris (AB096696.1),

96.4 % with C. japonica (AB262971.1), 91.8 % with A.

platyrhynchos (EU678246.2), 81.1 % C. hircus

(JN656106.1), 81.6 % with C. l. familiaris (XM_537479.3),

76.7 % with E. caballus (NM_001256923.1), 81.7 % with

M. musculus (NM_008301), 75.5 % with O. aries

(JN604434), 75.2 % with B. taurus (BTU09861), 75.4 %

with R. norvegicus (L16764) and 81.7 % O. cuniculus

(XM_002719559.1). Further, the sequence showed vari-

ability at various nucleotide positions. For phylogenetic

analysis, Neighbor-Joining, Maximum parsimony and UP-

GMA (unpaired group mean average) methods were used.

Both the neighbor joining and the maximum parsimony

trees were consistent in generating trees with similar

topologies. The neighbor joining tree was with relatively

higher bootstrap values providing a clear resolution of all

the nodes (Fig. 3).

The deduced protein showed 99.2 % identity with C.

coturnix (ACC85671.1), 99.0 % with N. meleagris

(BAC24791.1), 99.2 % with C. japonica (BAF38390.1),

99.1 % with A. platyrhynchos (ACD47154.2), 92.9 % with

C. hircus (AFP43992.1), 93.1 % with C. l. familiaris

(XP_537479.1), 85.6 % with E. caballus (A2Q0Z1),Fig. 1 PCR amplification of HSP70 gene from chicken

Fig. 2 Specific amplification of the PCR product from isolated

plasmid

Molecular Cloning and Characterization of the Gene Encoding

123

93.0 % with M. musculus (AAC84168.1), 85.3 % with O.

aries (AEX55801.1), 85.3 % with B. taurus (AAA739

14.1), 84.9 % with R. norvegicus (AAA17441.1) and

92.9 % with O. cuniculus (XP_002719605.1) HSP70 pro-

tein sequences. The chicken HSP70 sequence from this

study was found close to C. coturnix and C. japonica. The

estimates of the number of synonymous (dS) and

non-synonymous (dN) differences in a pair of protein-

coding sequences are given in Table 1. The dS was found

to be significantly higher than the dN in the analyses of

nucleotide sequences of all the other HSP70 sequences

(P B 0.05).

The extent to which selection affects genes and genomes

is a key question in genetics and molecular evolution.

Selection may modulate gene sequence evolution in dif-

ferent ways, for example, by constraining potential changes

of amino acid sequences (purifying or negative selection)

or by favoring new and adaptive genetic variants (positive

selection). To quantify selection in the simplest case and to

test whether positive selection is operating on the HSP70

protein, the relative abundance of synonymous (dS) and

non-synonymous (dN) substitutions that have occurred are

compared. The dS was significantly higher than the dN in

the analyses of nucleotide sequences of all other HSP70

sequences (P \ 0.05), suggesting that these nucleotide

sequences were under purifying selection. The authors

conclude that the gene encoding HSP70 is not under

positive selection and no advantageous mutations have

played an important role in evolutionary adaptation.

The 3D-structure model of heat shock protein 70 was

generated by homology-modelling programme Swiss-

model. The predicted model of heat shock protein 70

(Fig. 4) depicted in the form of ribbons is composed of 10

strands, 3 a-helices, 19 b-turns, 7 G-turns and 4 hairpins.

The assessment of the predicted model using the Ra-

machandran plot showed that the modeled HSP70 protein

has 64.3 % residues in the most favourable regions, 30 %

residues occurring in the allowed regions and 1.4 % resi-

dues in the disallowed regions. Such figures assigned by

Ramachandran plot represent a good quality of the pre-

dicted model (Fig. 5). All Ramachandrans showed 23

labeled residues out of 157, whereas chi1-chi2 plots

showed 5 labelled residues out of 95. The main chain and

side-chain parameters for all of them were found to be

concentrated/convoluted in the ‘‘better’’ region. One bad

Fig. 3 Neighbour joining tree obtained from HSP70 amino acid

sequence data of different species

Table 1 Estimates of synonymous and non-synonymous substitution

rates under realistic evolutionary models

Species–Species dS ± SE dN ± SE

Coturnix coturnix 0.148* ± 0.018 0.003 ± 0.001

Numida meleagris 0.157* ± 0.018 0.003 ± 0.002

Coturnix japonica 0.148* ± 0.018 0.003 ± 0.001

Anas platyrhynchos 0.334* ± 0.022 0.006 ± 0.003

Capra hircus 0.663* ± 0.028 0.040 ± 0.006

Canis lupus familiaris 0.661* ± 0.027 0.038 ± 0.005

Equus caballus 0.631* ± 0.025 0.097 ± 0.010

Oryctolagus cuniculus 0.638* ± 0.026 0.040 ± 0.006

Mus musculus 0.638* ± 0.026 0.041 ± 0.006

Ovis aries 0.653* ± 0.025 0.098 ± 0.010

Bos taurus 0.672* ± 0.025 0.099 ± 0.010

Rattus norvegicus 0.660* ± 0.025 0.101 ± 0.010

Values are mean ± standard error

dS number of synonymous substitutions per synonymous site, dN

number of non-synonymous substitutions per non-synonymous site* Values differ significantly (P B 0.05)

Fig. 4 Homology model of the chicken HSP70 protein

E. N. Mathew et al.

123

contact was detected in the modeled structure. To define a

model reliable, the score for G-factor (a log odds score

based on the observed distribution of stereochemical

parameters such as main-chain bond angles, bond length

and phi-psi torsion angles) should be above -0.50. The

observed G-factor score for the present model was -0.82

for dihedrals bonds, -0.05 for covalent bonds and -0.45

overall. The distribution of the main-chain bond lengths

and bond angles were 99.9 % and 92.9 % within the limits,

respectively. These parameters suggest that the predicted

model obtained for the chicken HSP70 was a reliable one.

Acknowledgments The authors are thankful to the Director, School

of Animal Biotechnology, GADVASU, and DBT-HRD for providing

necessary facilities to carry out the work.

Conflict of interest None of the authors of this paper has a financial

or personal relationship with other people or organisations that could

inappropriately influence or bias the content of the paper.

References

1. Burdon RH (1986) Heat shock and the heat shock proteins.

J Biochem 240:313–324

2. Tissieres A, Mitchell HK, Tracy U (1974) Protein synthesis in

salivary glands of Drosophila melanogaster: relation to chro-

mosome puffs. J Mol Biol 84:389–398

3. Benjamin IJ, McMillan DR (1998) Stress (heat shock) proteins

molecular chaperones in cardiovascular biology and disease. Circ

Res 83:117–132

4. Kregel CK (2002) Heat shock proteins: modifying factors in

physiological stress responses and acquired thermotolerance.

J Appl Physiol 92:2177–2186

Fig. 5 Ramachandran plot of the predicted model of chicken HSP70 protein

Molecular Cloning and Characterization of the Gene Encoding

123

5. Zhen FS, Du HL, Xu HP, Luo QB, Zhang XQ (2006) Tissue

and allelic-specific expression of hsp70 gene in chickens: basal

and heat-stress-induced mRNA level quantified with real-time

reverse transcriptase polymerase chain reaction. Br Poult Sci

47:449–455

6. Givisiez PEN, Silva MM, Mazzi CM, Ferro MIT, Ferro JA,

Gonzalez E, Macari M (2001) Heat or cold chronic stress affects

organ weights and Hsp70 levels in chicken embryos. Can J Anim

Sci 82:83–87

7. Yahav S, Shamay A, Horev G, Bar-ilan D, Genina O, Friedman-

einat M (1997) Effect of acquisition of improved thermotolerance

on the induction of heat shock proteins in broiler chickens. Poult

Sci 76:1428–1434

8. Segal BH, Wang XY, Dennis CG, Youn R, Repasky EA, Manjili

MH, Subjeck JR (2006) Heat shock proteins as vaccine adjuvants

in infections and cancer. Drug Discov Today 11(11–12):534–540

9. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory

manual, 3rd edn. Coldspring Harbour, Coldspring Laboratory

Press, NY

10. Wishart DS, Stothard P, Van Domselaar GH (1999) Prep-

TToolTM and GeneToolTM. Bioinformatics methods and pro-

tocols, vol 132, 1st edn. Humana Press, New Jersey, pp 93–113

11. Altschul SF, Thomas LM, Alejandro AS, Jinghui Z, Zheng Z,

Webb M, David JL (1997) Gapped BLAST and PSIBLAST: a

new generation of protein database search programs. Nucleic

Acids Res 25:3389–3402

12. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular

evolutionary genetics analysis (MEGA) software version 4.0.

Mol Biol Evol 24:1596–1599

13. Goldman N, Yang ZH (1994) A codon-based model of nucleotide

substitution for protein-coding DNA sequences. Mol Biol Evol

11:725–736

14. Nei M, Gojobori T (1986) Simple methods for estimating the

numbers of synonymous and nonsynonymous nucleotide substi-

tutions. Mol Biol Evol 3:418–426

15. Yang Z, Nielsen R (2000) Estimating synonymous and nonsyn-

onymous substitution rates under realistic evolutionary models.

Mol Biol Evol 17:32–43

16. Yang ZH (2007) PAML 4: phylogenetic analysis by maximum

likelihood. Mol Biol Evol 24:1586–1590

17. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISSMOD-

EL: an automated protein homology-modeling server. Nucleic

Acids Res 31:3381–3385

18. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-

MODEL workspace: a web based environment for protein

structure homology modelling. Bioinformatics 22:195–201

19. Laskowski RA, MacArthur MW, Moss D, Thornton JM (1993)

PROCHECK: a program to check the stereochemical quality of

protein structures. J Appl Crystallogr 26:283–291

20. Ramachandran GN, Sasisekharan V (1968) Conformation of

polypeptides and proteins. Adv Protein Chem 23:283–289

21. Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963)

Stereochemistry of polypeptide chain configurations. J Mol Biol

7:95–99

22. Cai YF, Wang GL (2005) Point mutation, molecular evolution

and prokaryotic expression in E.coli DE3 of bovine HSP70 gene.

Curr Zoolog 51(6):1080–1090

23. Pawar HN, Brah GS, Agrawal RK, Verma R (2013) Molecular

and immunological characterization of heat shock protein 70

(HSP70) gene from buffalo. Proc Natl Acad Sci India Sect B

83(2):163–169

24. Gade N, Mahapatra RK, Sonawane A, Singh VK, Doreswamy D,

Saini M (2010) Molecular characterization of heat shock protein

70-1 gene of goat (Capra hircus). Mol Biol Int 2010:1–7

E. N. Mathew et al.

123