Cloning And Prokaryotic Expression Of H5N1 Subtype Avian Influenza Virus HA1 Gene

Highly pathogenic avian influenza (HPAI) is an avian infection and/or disease syndrome endangering farming and human healthy. In recent years, HPAI had broken out many times in Anhui province, which not only has caused huge economic losses in poultry industry, also made human infect or die. So the prevention from the disease of HPAI and human AIV has been unprecedently put forward in the public hygiene field and it is very urgent to find a perfect protective antigen, which is used to diagnose and prevent this disease now.Hemagglutinin(HA) is a major surface glycoprotein of AIV, which plays a key role in adsorpting and fusing the cytoplasmic membrane of host target cells. HA1 has the characteristic of binding to specific receptors on target cells. HA1 is also the main protective antigen of HA, there are four epitopes situating on the surface of HA1 among five HA major antigenic sites. It is of great importance to study HA1 protein to reveal the mechanism of AIV and manufacture HA1 vaccine with antiadsorption, which is one of the focus in the research of AI immunoprevention now. In order to reveal the HA1 gene homology of AIV Anhui strain A/Chicken/Anhui/M(H5N1) with other referent strains isolated from home and abroad at the molecular level, inquire into the structure of HA1 protein and obtain abundant recombinant HA1 protein, which would supply the basis to further prepare the McAb of HA1, research its function and prepare HA1 vaccine, in this study HA1 gene of A/Chicken/Anhui/M(H5N1)was cloned, sequenced and biological information analysis, recombinant plasmids pGEX-4T-HA1 and pET-32a-HA1 were constructed and prokaryotic expressed.Firstly, HA1 gene was amplified, cloned and sequenced and biological information analysis. According to mRNA of AIV-HA1 published by Genbank, a pair of primers was designed. HA1 gene of A/Chicken/AnHui/ M (H5N1) AIV was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA extracted from brain tissue of sicken chicken. The PCR products were cloned into pMD-18T vectors. The recombinant plasmid was identified by restriction enzyme analysis and PCR and then sequenced. The results showed that HA1 gene of A/Chicken/AnHui/ M (H5N1) contains an ORF about 1 038 bp, encoding 346 amino acids. There are a signal peptide consisting of 16 amino acids at the 5'end and six basic amino acids(RRRKKR) inserting into the cleavage site, revealing A/Chicken/AnHui/ M (H5N1) AIV is a highly pathogenic one. The sequence of HA1 gene had been submitted to Genbank (No.DQ356886). The nucleotide and deduced amino acid sequences homology of the HA1 gene are 97.3%-97.9% and 96.3%-97.1% respectively between A/Chicken/AnHui/ M (H5N1) AIV and other referent strains isolated from home and abroad. Anhui isolated strain have the closest relationship with A/tree sparrow/Henan/2/2004(H5N1)(AY741217), with the homology of 97.5%.Secondly, different biological analysis softwares were chosen to analysis the characteristics of HA1 protein sequence. The results showed that HA1 protein is composed of 346 amino acids, its molecular weight is 39.291kD, its isoelectric point (PI) is 8.369 and the number of hydrophilic amino acids and hydrophobic amino acids are 148 and 143 respectively. There is a 5aa~15aa transmembrane region and an obvious hydrophobic region at N-terminal. There are 6 N-glycosylation- site, 5 CK2- phospho-site, 4 myrlstyl-site and 3 PKC-phospho-site in the structure of HA1 protein.The second structure of HA1 protein is mainlyα-helix andβ-sheet and partlyβ-turn. There may be some potential antigenic determatints at 199 aa -203aa, 205 aa -210aa, 237 aa -242aa, 291 aa -296aa and 336-344aa.Finally, recombinant plasmids pGEX-4T-HA1 and pET-32a-HA1 were constructed and prokaryotic expressed. A new pair of primers without signal was designed according to the ORF sequences of HA1 gene, HA1 protein structure and ORF sequences of plasmid vectors. The HA1 gene was amplied by RT-PCR and then inserted into the bacterial plasmid pET-32a and pGEX-4T-1. The constructed recombinant plasmids pET-32a-HA1 and pGEX-4T-HA1 were transformed into E.coli BL21 (DE3) respectively and expressing products were identified and analysed. The results showed that the recombinant fusion proteins Trx-HA1 were highly expressed in E.coli BL21 after induced with 1.0mmol/L IPTG at 37℃4h. Most of Trx-HA1 were expressed in the form of inclusion bodies, and a few were solubility. GST- HA1 were highly expressed in E.coli BL21 after induced with 1.0mmol/L IPTG at 37℃5h. GST- HA1were expressed in the form of inclusion bodies.Western-blot analysis with HI antibodies against H5 subtype showed that the two recombinant proteins have good antigencity.Above all, in this research AIV-HA1 gene was successfully cloned, sequenced and biological information analysis. There is high homology between Anhui isolation strain and other referent strains isolated from home and abroad at the molecular level. Recombinant plasmids pET-32a-HA1 and pGEX-4T-HA1 were constructed successfully and then HA1 protein was effectively expressed by Trx and GST fusion expression system. All these can provide a solid foundation for preparing AIV-HA1 McAb and studying the fuction of HA1 protein, also for further developing the HA1 vaccine.