Characterization and Expression of Outer Membrane Protein A I Gene of Aeromonas veronii

Wang Hai-juan, and Wang Li

1Institute of Qinghai-tibet Plateau, Southwest University for Nationalitities, Chengdu 610041, China

2College of Life Science and Technology, Southwest University for Nationalitities, Chengdu 610041, China

Characterization and Expression of Outer Membrane Protein A I Gene of Aeromonas veronii

Wang Hai-juan1, and Wang Li2*

1Institute of Qinghai-tibet Plateau, Southwest University for Nationalitities, Chengdu 610041, China

2College of Life Science and Technology, Southwest University for Nationalitities, Chengdu 610041, China

The outer membrane protein, ompA, of Aeromonas veronii has a role in the virulence of the organism and is a potential candidate for vaccine development. In this study, ompAⅠof Aeromonas veronii strain WA106 was cloned and sequenced, then, it was expressed in Escherichia coli BL21. The nucleotide sequence of ompAⅠgene was 1 023 base pairs (GenBank Accession NO.KC748024), which showed 100% homology with that of A. veronii (NO.AB290200.1). This predicted protein was composed of 340 amino acid residues. Its molecular weight was 35.78 ku and isoelectric point was 5.18. The protein was a hydrophilic protein containing alpha helix and random coil with percentage of 35.0% and 49.7%, respectively. The tertiary structure, quaternary structure prediction showed that ompAⅠprotein contained two peptide chains. SDS-PAGE showed that the actual value of the fusion protein was consistent with the expected result. It will facilitate further study of the role of ompAⅠprotein.

Aeromonas veronii, ompAⅠ, gene cloning, sequencing, prokaryotic expression

Introduction

Aeromonas veronii is a kind of conditional pathogenic bacteria, generally infected the immunocompromised and lower immunocompetent patients (Ko et al., 2000). It is also a kind of fish pathogen and has been isolated from Zebra fish, Ictalurus punctatus (Huang et al., 2010), Tincaeus (Gou et al., 2012), Acipenser baerii (Ma et al., 2009), Sclizothorax prenanti (Liu et al., 2012), Anguilla japonica (Yang et al., 2012), Cyprinus carpio L. (Qin et al., 2008), Oreochromis niloticus (Li et al., 2011), Andrias davidianu (Wang et al., 2010), salmon, turbot, angler, perch, etc (Bin Kingombe et al., 2004). Therefore, developing a vaccine to prevent and cure the fish bacterial diseases can be very important. A few of virulence factors may contribute to the pathogenicity of this kind of bacterium. The virulence factors of Aeromonas veronii included aerolysin, hemolysin, enterotoxins, adhesis, extracellaluar proteases, invasins, phospholipase and lipase. They have been detected in bacteria from fish, human, food and potable water (Parker and Shaw, 2011; Nawaza et al., 2010; Senderovich et al., 2012; Pablos et al., 2009; Albert et al., 2000 ).

Outer membrane proteins (OMPs) being highly conserved and the most immunogenic can be used as candidates for vaccine development in aquaculture. Outer membrane protein A (ompA) is an important kind of OMPs which are highly conserved and has been presumed to be as a pathogen-associated vector in anti-infectious and anti-tumor vaccines inEnterobacteriaceae family (Jeannin et al., 2002). In Aeromonas, Costello et al. (1996) first accomplished the cloning of ompA successfully and found two tandem paralogues in Aer. salmonicida, the pathogen of furunculosis. The two allelic forms, ompAⅠand ompAⅡ(Krishnan et al., 2012), have specific differences in the amino acids (Smith et al., 2007). In this paper, ompAⅠof Aeromonas veronii was cloned and expressed in Escherichia coli host cell BL21 (PLYs) with pET32a expression vector, which would benefit the study of the function of ompA.

Materials and Methods

Strains, plasmids and enzymes

Aeromonas veronii WA106 was obtained in Chengdu (Sichuan Province, China) and was used for cloning and protein expression of ompAⅠ. Escherichia coli DH5α was used as the host for recombinant plasmids during cloning and sequencing analyses. Escherichia coli BL21 (PLYs) was used as the host for expression of cloned genes. Plasmid PMD-19 T-Vector was used for PCR cloning vector. E. coli DH5α and PMD-19 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) and were stored at –80℃. Taq polymerase, T4DNA ligase and restriction enzymes were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China) and were used according to the manufacturers' instructions.

PCRamplificationofompAIgene

To amplify ompAⅠgene of Aeromonas veronii WA106, primers were designed as follows: the forward prime sequence, 5'-CCCAGATCTATGATG ATGAAAATGGCTCCTTC-3', containing a BglⅠrestriction enzyme site (underlined). The reverse prime sequence, 5'-CTCGGATCCTAAATGTTCTTCAA GTCTTCATT-3', containing a BamHⅠrestriction enzyme site (underlined). DNA extraction was accomplished according to the method of AXYGEN Bacteria DNA Kit (Beijing). PCR reaction system was performed in a final volume of 25 μL containing 12.5 μL of 2×Taq PCR MasterMix (0.1 U Taq polymerase, 500 μmol ? L-1dNTP each, 20 mol ? L-1Tris-HCl, pH 8.3, 100 mol ? L-1KCl, 3 mol ? L-1MgCl2), 1.0 μL of the primers, respectively, 1.2 μL of template DNA and 9.3 μL of ddH2O. PCR run in a programmable thermocycler having an initial delay at 94℃ for 5 min followed by 34 cycles of denaturation at 94℃ for 1 min, annealing at 59℃ for 1 min, extension at 72℃for 1.5 min, then a final delay at 72℃ for 10 min and finally stored at 4℃. PCR amplified products were resolved by electrophoresis on 1% agarose gel and analyzed using a gel documentation system.

CloningandsequencingofompAIgene

The open reading frame (ORF) of ompAⅠgene was amplified by using the forward and reverse primers. PCR product was digested and directly ligated to PMD19-T Vector (TaKaRa). Reactions were conducted at 16℃ for 8 h with 4.5 μL of PCR product, 5.0 μL of SolutionⅠand 0.5 μL of PMD19-T vector. The ligation was mixed with E. coli DH5α competent cells and placed on the ice for 30 min followed by heated at 42℃ for 45 s, immediately iced for 1 min. The cells were added to the 890 μL of LB liquid medium and shaken at 37℃ for 1.5 h. The recombinant transformants were selected on LB agar plates containing X-Gal (2 ug ? mL-1), IPTG (5 ug ? mL-1), and Amp (10 ug ? mL-1). The white colonies bearing the recombinant plasmid were cultured in LB broth medium and identified by PCR method. The positive clone was sequenced by Sangon Biotech (Shanghai) Co., Ltd. The identity of the nucleotide sequences and amino acid sequences of ompAⅠwere analyzed by using Blast program of NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi).

AnalysisofompAIprotein

Multiple sequence alignment of ompAⅠprotein was generated by using ClustalX2.0 software. Phylogenetic tree was constructed from MEGA5.0 program using neighbour-joining method and P-short correction model of bootstrap 1 000 times (Boostrap). DNAMANsoftware was used for the primary structure analysis and ProtScale program with Hphob./Kyte & Doolittle scale (http://www.ExPASy.org/cgi-bin/protscale. pl) for hydrophobicity analysis. Signal peptide predictions were accomplished by SignalP-4.1 server. The positions of the linear B-cell epitopes were predicted to be located the residues with BepiPred 1.0b Server (www.cbs.dtu.dk/services/BepiPred). The secondary structure prediction of ompAⅠprotein was accomplished by online server HNN(http://npsapbil.ibcp.fr/cgi-bin/npsa_automat.pl? page=/NPSA/ npsa_hnn.html). The tertiary and quaternary structure of ompAⅠprotein was predicted by using SWISSMODEL (http://swissmodel.expasy.org/).

Constructing recombinant plasmids

The positive plasmid PMD-19-ompAⅠwas digested with BglⅡ-BamHⅠand ligated into pET32a vector. The ligation reactions were performed in a 20 μL mixture consisting of 8 μL of PMD-19-ompAⅠ, 1 μL of pET32a vector, 3 μL of buffer, 7 μL of ddH2O and 1 μL of T4ligase and were incubated overnight at 16℃. The ligated product was transformed into E. coli DH5α competent cells by heat shock at 37℃for 1 h. The recombinant transformants were screened on plates with ampicillin (50 μg ? mL-1) for selection. The white colonies were selected and incubated in LB liquid medium with ampicillin (100 μg ? mL-1) at 37℃overnight. The recombinant plasmid was examined by PCR and digested by restriction endonuclease.

Expressionofrecombinantprotein

Overnight grown cultures of recombinant ompAⅠwere inoculated into LB medium with ampicillin (50 μg ? mL-1) at 37℃ until OD600reached 0.5-0.7. The cells were induced with 1 mmol ? L-1isopropyl thiogalactoside (IPTG) after the addition of it for 1 h, 2 h, 4 h and 6 h, 1mL of each culture was obtained by centrifugation. The protein sample was washed by distilled water and suspended in 5×SDS sample buffer [250 mmol ? L-1Tris-HCL (pH 6.8), 5% β-mercaptoethanol, 10% sodium dodecyl sulfate, 0.5% bromophenol blue, 50% glycerol] and boiled at 100℃for 5 min before analyzing by means of SDS-PAGE on 12% (w/v) gel. At last, the protein was stained overnight with coomassie brilliant blue G250.

Results

ompAIgene cloning and sequencing

A 1 023 bp fragment was successfully amplified (GenBank Accession NO.KC748024) by PCR method using two specific primers and was cloned into PMD-19T plasmid (Fig. 1). Then, PMD-19T plasmid was used in the construction of the final pET32a-ompA I plasmid. Phylogenetic analysis indicated that ompA I gene of WA106 strains showed 100% homology with ompA genes of A. veronii (NO. AB290200.1) ( Fig. 2).

Fig. 1　Agarose gel electrophoresis of ompAIproduct

Bioinformaticanalysisstructureandfunction prediction

The amplified fragment of ompA I gene was predicted to encode 340 amino acid residues. The relative molecular weight was 35.78 ku and isoelectric point was 5.18. Amino acid composition analysis of ompAⅠ

indicated that Ala, Gly, Val and Leu were plentiful, and the content of Met, His, Cys and Trp were relatively poor. The most frequency used rare codon was CCG with the used percentage of 3.24%, while the using frequencies of CTC, CGA, AGA and ATA were zero.

Fig. 2　Phylogenetic tree generated from an alignment of ompAI

The maximum value of the protein hydrophobicity was 2.856 in the 12th amino acids, while the minimum value in the 169th amino acid was 2.311. There were larger hydrophobicity from the 11th to the 14th amino acids, other parts had no obvious hydrophilicity and hydrophobicity. The result indicated that ompA I protein was a hydrophilic protein. Analysis of signal peptide of ompA I proetin indicated that it had a signal peptide with 24 amino acid residues in N-terminal and the cleavage site was between 24 and 25. The possible linear B-cell epitopes of ompA I proetin were located at 8-11, 31-45, 59-61, 63-83, 91-93, 104-107, 111-130, 150-163, 178-180, 182-197, 214-222, 234-241, 248-262, 283-301, and 315-318. The secondary structure of ompA I protein contained alpha helix and random coil with percentage of 35.0% and 49.7%, respectively.

The tertiary structure, quaternary structure prediction showed that ompAⅠprotein consisted of two peptide chains, A and B, joining together with two disulfide linkages. A was from the first amino acid to the 191th amino acid, and the protein model number was 2 kola, Z value was –7.53. B was from the 208th to the 335th amino acids, amino acids protein model number was 4 erha, and Z value was 1.71 (Fig. 3).

ExpressionofompAIgene in E. coli

The recombinant plasmids ompAⅠ-PMD-19T was digested by BamHⅠand BglⅡ. Therefore, two different sizes of fragment about 2 692 bp and 1 023 bp were obtained, respectively. The produce of the recombinant plasmid pET32a-ompAⅠwas digested by enzymes (BamHⅠand BglⅡ) in 1% agarose gel electrophoresis, it is shown in Fig. 4 (Lane 1). The size of DNA fragment approximately 5 863 bp obtained from pET32a-ompAⅠwas consistent with the theory value, which indicated pET32a+ompA recombinant plasmid was successfully built.

Fig. 3　Predictive tertiary structure of ompAI

Fig. 4　PCR detection of pET32a+ ompAI

ompAⅠgene was expressed in E. coli BL21 containing the construction of pET32a-ompAⅠfor analyzing the function of recombinant protein. Recombinant plasmid pET32a-ompAⅠwas theoretically translated to 340 amino acids of ompAⅠgene and 119 amino acids of pET32a harboring its expression on Trx ? Tag and His ? Tag and S ? Tag. The theoretical molecular weight of the protein expression was about 48.87 ku. Expression of the recombinant plasmid in E. coli BL21 with different induced time is shown in Fig. 5. SDSPAGE showed that protein molecular weight was about 49 ku (Fig. 5). In the experiments, the recombinant cells were shown to start the expression of protein at 1 h and reached a maximum level at 6 h. Thus, the optimal time for IPTG-induced protein expression was 6 h after the chemical induction.

Fig. 5　SDS-PAGE of expression products

Discussion

Outer membrane proteins of gram-negative bacteria included ompA, ompX, general porins (PhoE, OmpF), phospholipase A, substrate-specific porins (LamB, ScrY) and TonB-dependent receptors (FhuA, FepA) (Koebnik et al., 2000). Omp plays a key role in bacterial pathogenesis by enhancing the adaptation of pathogens to a variety of the environment (Lin et al., 2002). They are characterized as adhesion factors that can be contributing to the pathogenesis of Aeromonas veronii (Vàzquez-Juárez et al., 2004) and they also interact with the immune cells, such as antigen presenting cells (APCs) (Jeannin et al., 2002). Thus, ompA is selected as potential candidate for vaccine development. Recently, ompA protein is well characterized in Escherichia coli (Nicholson et al., 2009), Cronobacter sakazakii (Kim et al., 2010), Haemophilus parasuis (Zhang et al., 2009), Klebsiella pneumonia (Llobet et al.,2009), Edwardsiella tarda (Maiti et al., 2011, Kumar et al., 2007) and Acinetobacter baumannii (Choi et al., 2008).

In this study, the gene encoding ompAⅠfrom Aeromonas veronii WA106 strain was cloned and sequenced. The fragment length of ompAⅠgene for WA106 strain in our study and CY0806 strain from Cyprinus carpio (Shan et al., 2011) had been higher homology, which showed that ompAⅠgene was highly conservative. ompAⅠproteins of the two strains were both the hydrophilic proteins. However, there were still some subtle differences between two isolated strains in isoelectric point and structure, which might be associated with genetic diversity between the strains. It was reported that the production of ompAⅠin Aeromonas veronii was related to low cultivationtemperature (Namba et al., 2008). We discovered that the cultivation temperature of ompAⅠ in WA106 was 37℃. It suggested that two growth modes of ompAⅠwere in Aeromonas veronii. The gene encoding ompA of Klebsiella pneumonia was introduced into two expression vectors pABPm and pSPPABPm of Staphylococcus carnosus, it demonstrated that ompA was efficiently expressed in both systems (Hansson et al., 2002). In this experiment, ompAⅠwas efficiently expressed in E. coli expression system. It indicated that ompA gene could be expressed well in different expression systems.

Signal peptides consisting of short peptides had been located at N-terminus part of proteins. The stability of the signal peptide were capable of influencing the viability of cells and protein expression (Nagano and Masuda, 2014). ompAⅠprotein ofAeromonas veronii had a signal peptide with 24 amino acid residues in N-termina. Signal peptides analysis in this experiment would provide useful data for further researches on the heterogeneous expression of ompAⅠprotein. The detection and features of conformational and linear B-cell epitopes played a significant role in immunodiagnosis, antibody production and vaccine development (EI-Manzalawy et al., 2008). It was reported that 11 possible linear B-cell epitopes, which could trigger B-cell's response, were located at ompAⅠprotein from CY0806 strain (Shan et al., 2011). Fifteen potential linear B-cell epitopes were also detected in WA106 strain. These data demonstrated the existence of antigenic variability between Aeromonas veronii ompAⅠproteins.

ompA of Edwardsiella tarda had two protein bands of 36 ku and 40 ku (Maiti et al., 2011) and the 36 ku which was also previously reported by Hansson et al. (2002) with obtained from Klebsiella pneumonia, and 36 ku might correspond with 35.78 ku protein in our study. During the induction phase, the efficient expression of foreign proteins could be affected by the induced temperature of host cell, the concentration of IPTG, ion concentration of culture medium, pH and induction time. Optimizing the expressive condition of recombinant protein, we found that the expression level of protein reached the highest when the induction temperature was 30℃ and the optimal IPTG concentration was 1 mmol ? L-1. Furthermore, the best induction time was 6 h which was more than the incubation time of ompA protein of Edwardsiella tarda (Maiti et al., 2011).

ompA protein of Edwardsiella tarda had a high antigenicity in both fish and rabbit (Maiti et al., 2011). The recombinant ompA protein of Haemophilus parasuis was strong immunogenic cross-reactivity between two serovars, 4 and 5. In the study, it demonstrated that 15 possible linear B-cell epitopes, which had an important role in immunodiagnosis and antibody production, were located at ompAⅠprotein of WA106 strain. The data indicated ompA protein was an high immunogenic protein which might be useful for the development of fish vaccine.

In conclusion, ompAⅠgene of Aeromonas veronii in WA106 strain was cloned and sequenced. The recombinant ompAⅠprotein was successfully constructed and expressed in E. coli BL21. It would be helpful for further investigation on the function of ompAⅠ.

References

Albert M J, Ansaruzzaman M, Talukder K A, et al. 2000. Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls and the environment. Journal of Clinical Microbiology, 38(10): 3785-3790.

Bin Kingombe C I, Huys G, Howalda D, et al. 2004. The usefulness of molecular techniques to assess the presence of Aeromonas spp. harboring virulence markers in foods. International Journal of Food Microbiology, 94(2): 113-121.

Choi C H, Lee J S, Lee Y C, et al. 2008. Acinetobacter baumannii invades epithelial cells and outer membrane protein A mediates interactions with epithelial cells. BMC Microbiology, 8: 216.

Costello G M, Vipond R, MacIntyre S. 1996. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, ompA. Journal of Bacteriology, 178(6): 1623-1630.

EI-Manzalawy Y, Dobbs D, Honavar V. 2008. Predicting linear B-cell epitopes using string kernels. Journal of Molecular Recognition, 21(4): 243-255.

Gou X L, Hou X Y, W Y, et al. 2012. Study on the identification and biological characteristics of Aeromonas veronii from Tincaeus. Sichuan Animal & Veterinary Sciences, 6: 26-28.

Hansson M, Samuelson P, Nguyen T N, et al. 2002. General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae. FEMS Microbiology Letters, 210(2): 263-270.

Huang X L, Wu C Y, Deng Y Q, et al. 2010. Pathohistological observation of Ictalurus punctatus infected with Aeromonas veronii. Chinese Veterinary Science, 40(7): 738-742.

Jeannin P, Magistrelli G, Goetsch L, et al. 2002. Outer membrane protein A (OmpA): a new pathogen-associated molecular pattern that interacts with antigen presenting cells-impact on vaccine strategies. Vaccine, 20(Suppl 4): A23-27.

Kim K, Kim K P, Choi J, et al. 2010. Outer membrane proteins A (OmpA) and X (OmpX) are essential for basolateral invasion ofCronobacter sakazakii. Applied and Environmental Microbiology, 76(15): 5188-5198.

Ko W C, Lee H C, Chuang Y C, et al. 2000. Clinical features and therapeutic implications of 104 episodes of monomicrobial Aeromonas bacteremia. The Journal of Infection, 40(3): 267-273.

Koebnik R, Locher K P, Van Gelder P. 2000. Structure and function of bacterial outer membrane proteins: barrels in a nutshell. Molecular Microbiology, 37(2): 239-253.

Krishnan S, Prasadarao N V. 2012. Outer membrane protein A and OprF: versatile roles in gramnegative bacterial infections. The FEBS Journal, 279(6): 919-931.

Kumar G, Rathore G, Sengupta U, et al. 2007. Isolation and characterization of outer membrane proteins of Edwardsiella tarda and its application in immunoassays. Aquaculture, 272(1/2/3/4): 98-104.

Li J, Ye X, Lu M X, et al. 2011. Isolation and identification of Aeromonas veronii from infected Tilapia fry and analysis of its drug sensitivity. Journal of Hydroecology, 32(3): 132-136.

Lin J, Huang S X, Zhang Q J. 2002. Outer membrane proteins: key players for bacterial adaptation in host niches. Microbes and Infection, 4: 325-331.

Liu G B, Wang L, WangJ K, et al. 2012. Sequencing analysis of 16S rDNA genes of Aeromonas veronii isolated from Schizothorax prenanti. Journal of Hydroecology, 33(1): 92-96.

Llobet E, March C, Giménez P, et al. 2009. Klebsiella pneumoniae ompA confers resistance to antimicrobial peptides. Antimicrobial Agents and Chemotherapy, 53(1): 298-302.

Ma Z H, Y H, Li T L, et al. 2009. Isolation and identification of pathogenic Aeromonas veronii isolated from infected Siberian sturgeon (Acipenser baerii). Acta Microbiologica Sinica, 49(10): 1289-1294.

Maiti B, Shetty M, Shekar M, et al. 2011. Recombinant outer membrane protein A (ompA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp. Microbiological Research, 167(1): 1-7.

Nagano R, Masuda K. 2014. Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells. Biochemical Biophysical Research Communications, 447(4): 655-659.

Namba A, Mano N, Takano H, et al. 2008. ompA is an adhesion factor of Aeromonas veronii, an optimistic pathogen that habituates in carp intestinal tract. Journal of Applied Microbiology, 105(5): 1441-1451.

Nawaza M, Khan S A, Khan A A, et al. 2010. Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiology, 27(3): 327-331.

Nicholson T F, Watts K M, Hunstad D A. 2009. ompA of uropathogenic Escherichia coli promotes postinvasion pathogenesis of cystitis. Infection and Immunity, 77(12): 5245-5251.

Pablos M, Rodríguez-Calleja J M, Santos J A, et al. 2009. Occurrence of motile Aeromonas in municipal drinking water and distribution of genes encoding virulence factors. International Journal of Food Microbiology, 135(2): 158-164.

Parker J L, Shaw J G. 2011. Aeromonas spp. clinical microbiology and disease. Journal of Infection, 62(2): 109-118.

Qin G M, Zhang X J, Chen C Z, et al. 2008. Infection of Aeromonas veronii from diseased Cyprinus carpio L. and the biological characteristics of its pathogen. Journal of Anhui Agriculture Science, 36(19): 8115- 8117.

Senderovich Y, Ken-Dror S, Vainblat I, et al. 2012. A molecular study on the prevalence and virulence potential of Aeromonas spp. recovered from patients suffering from diarrhea in Israel. PLoSOne, 7(2): e30070.

Shan X F, Wu T L, Meng Q F, et al. 2011. Cloning and bioinformatic analysis of outer membrane protein gene A 1 of Aeromonas veronii CY0806 from Cyprinus carpio. Chinese Journal of Veterinary Drug, 45(10): 3-6.

Smith S G, Mahon V, Lambert M A, et al. 2007. A molecular Swiss army knife: ompA structure, function and expression. FEMS Microbiology Letters, 273(1): 1-11.

Vàzquez-Juárez R C, Romero M J, Ascencio F, et al. 2004. Adhesive roperties of a LamB-like outer-membrane protein and its contribution to Aeromonas veronii adhesion. Journal of Applied Microbiology, 96(4): 700-708.

Wang X, Yan Q G, Lei Y, et al. 2010. Isolation and identification on pathogenic bacteria of "rotten skin" disease in Chinese giant salamander (Andrias davidianus). Chinese Journal of Zoonoses, 26(10): 944-948.

Yang Q H, Guo S L, Zou W Z, et al. 2012. Isolation and identification of pathogenic Aeromonas veronii from Anguilla japonica. Biotechnology Bulletin, 7: 134-140.

Zhang B, Tang C, Yang F, et al. 2009. Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis. Veterinary Microbiology, 136(3/4): 408-410.

R37 Document code: A Article ID: 1006-8104(2015)-02-0015-07

10 February 2015

Supported by the Science & Technology Department of Sichuan Province (2013FZ0014); the Construction Project of Postgraduate Academic Degree in Southwest University for Nationalities (2015XWD-S071007)

Wang Hai-juan (1983-), female, engaged in the research of gene engineering and molecular biology. E-mail: wanghaijuan08@163.com

. Wang Li, Ph. D, associate professor, engaged in the research of zoopathology and molecular biology. E-mail: qinxin916@ aliyun.com