Preparation Of The Recombinant HA1 Protein Of Different Subtype Swine Influenza Virus And Development Of An ELISA Assay For H5-subtype Specific Antibody

In this study,swine influenza virus(SIV) subtypes H1N1,H3N2,H5N1 and H9N2 were propagated in 9-11 day SPF chicken embryos,respectively.The virus culture of allantioc fluid was collected 24-96h post-inocubation and its. hemagglutination titers was determined.The SIV immunogens were obtained after deactivated by 2%formaldehyde at 37℃for 48 h and concentrated.By immunizing the SIV-negative pigs with the SIV antigens,250-300ml of each SIV-positive serum specific for H1,H3,H5 of H9 subtype was produced respectively,whose HI titer was determined at about 1:640.The HA1 gene of SIV Zhejiang isolate subtypes H1N1, H3N2,H5N1,H9N2 were amplified by reverse transcriptase-polymerase chain reaction(RT-PCR),and subcloned into a prokaryotic expression vector of pET-28a(+) or pET-32a(+) to construct recombinant plasmids,designated as pET28a-HA1 /H1, pET28a-HA1 /H3,pET32a-HA1/H3,pET28a-HA1/H5,pET32a-HA1/H9.After transformed with the recombinant plasmids,Escherichia coli(E.coli) BL21(DE3) was induced by isopropy-β-D-thiogalactoside(IPTG) to express the interest protein of HA1.SDS-PAGE and Western blot analyses showed that the recombinant plasmids pET28a-HA1/H1,pET28a-HA1/H3,pET32a-HA1/H3,pET28a-HA1/H5 and pET32a-HA1/H9,successfully expressed a fusion proteins of His-HA1,Trx-His-HA1 with molecular weight of 45kDa(His-HA1/H1),45kDa(His-HA1/H3),58kDa (Trx-His-HA1/H3),45kDa(His-HA1/H5) and 56kDa(Trx-His-HA1/H9) as inclusion bodies in E.coli,which was specifically recognized by anti-His monoclonal antibody as well as SIV positive serum indicating its well immunologic reactivity.After dissolved in 8M urea solution,the expressed proteins were purified by a Ni-chelated affinity chromatograph and the yield was calculated to be about 18mg/L (pET28a-HA1/H1),15mg/L(pET28a-HA1/H3),10mg/L(pET32a-HA1/H3),15mg/L (pET28a-HA1/H5),12mg/L(pET32a-HA1/Hg) of bacterial culture.Using the purified recombinant protein(HA1)of H5N1,an indirect ELISA for detection of anti-SIV antibodies was developed and its optimal reaction conditions were determined as follow:coating antigen for 37℃for 2 hours at a concentration of 0.31μg/well(1:200);the optimal serum sample dilution werel:100,the 0.05M Tris-HCl buffer at pH8.5 was the best coating buffer;the 5%skimmed milk was the optimal blocking buffer;the phosphate buffer containing 0.05%tween-20 and 0.1% BSA was the best diluent;the serum sample conjugate being incubated at 37℃for 45 rain,and the conjugate being incubated at 37℃for 30min.the substrate for ELISA being incubated at 37℃for 10 min.The ELISA assay was confirmed to have a good reiteratively,specificity and sensitivity by reiteratively test,cross test.The HA1-ELISA assay possesses well specificity and sensitivity for SIV H5 subtype antibody,whose accuracy was determined as 81.4%compared with the classical hemagglutination inhibition assay(HI).In addition,360 serum samples collected from swine farms were detected by the developed assay,The material base was established for assembling test kit for diagnosis of SIV further.Offered a kind of good technological means for diagnosis and monitoring of the disease of SIV.