Expression Of HA Gene Of Avian Influenza Virus With Six His-tag In Baculovious Expression Vector System

Avian influenza (AI) is a fowl disease,which is caused by type A influenzavirus. The fatal disease, named "highly pathogenic avian influenza",is mainly caused by subtypes H5 and H7 of influenza A virus. For a long time,scientists believe that avian influenza virus couldn't infect human,but the 97'events happened in Hong Kong demonstrated that HPAI virus of H5N1 subtype could infected human directly,and caused them dead. Together with those events in recently years,which draw much attention in the public hygiene field. The hemagglutinin (HA) gene is the biggest variability in AIV genes,which determined the antigenicity and pathogenicity of AIV in a certain degree. Hemaglutinin plays an important role in the process of cohesion and transmembrane of AIV that makes the host produces the neutralizing antibody,neutralizing the infection of virus.Humoral immunity against HA is the main determinant for protecting the host against disease. Based on the safty of subunit vaccine and the good research results we have made previously,we have improved the method to express the HA gene of AIV with 6×His tag in BESV in this paper that contains two parts.ExpermentⅠ:Study on expression of HA gene of avian influenza virus with 6×His tag in Bac–N-Blue expression system of baculovirus. The complementary DNA of 1.7 kb HA gene drived from the A/Goose/GuangDong/1/96 (H5N1) AIV was amplified by RT-PCR,then cloned the amplified fragment into the plasmid vector pMD18-T and sequenced. The HA gene absent the sequence of signal peptide was subcloned into the baculovirus transfer vector pBlueBacHis4.5. The recombinant plasmid was screened at random by picking colonies and designated rpBachisH5HA. The rpBacHisH5HA was sequenced and co-transfected the sf9 cell with the lined Bac-N-BlueTM DNA by the technique of cationic liposome mediated transfection. The recombinant baculovirus was purified by three cycles of plaque assay with the chromogenic substracte X-gal in the low temperature melting agarose overlay and analyzed by PCR. The expression of HA gene in High Five cells was identified by HA assay,SDS-PAGE and western-blot,which proved that HA gene have expressed successfully and with good biological activity and immunogenicity. We had attempt to purify HA protein,but failed.ExpermentⅡ:Study on expression of HA gene of avian influenza virus with 6×His tag in Bac-to-Bac expression system of baculovirus. The HA gene of A/Goose/GuangDong/1/96 (H5N1) AIV was inserted into the transposon vector pFastBacTMHTA to construct the recombinant transposon vectors pFastBacHT-H5HA. Positive recombinants were screened in LB/AMP±culture after transofrmation into E.coli DH5αand further identified with restriction endonucleases digestion,PCR and sequenced. To generate recombinant bacmids , the positive pFastBacHT-H5HA was transformed into E. coli DH10Bac.White bacterial colonies were selected as positive recombinant expression vectors after screening in GM,Ka,Tet,X-gal and IPTG selection LB agar plates. Then recombinant bacmids rBacmid-H5HA were transfected into sf-9 cell to generate recombinant baculovirus that express the HA protein of AIV. The results of SDS-PAGE,HA assay and Western-blot showed that the expressed HA protein were specific with a molecular weight 65KD and have good biological activity and immunogenicity. But The purified HA protein only obtained under denatured condition. The application of recombinant HA protein was restricted within narrow limits.