Expression Of Hn Gene Of Goose-Host Paramyxo Virus Strain E01and Study Of Immune Effect

The study was to clone HN gene from E01strain of goose paramyxovirus and analyze their sequences. At First, the RNA of virus was exstracted. According to the full nucleotide sequence of ZJ1strain of goose paramyxovirus, a pair of primers named HNF1/HNR1was designed to amplify the HN gene by RT-PCR, which was from E01strain of goose paramyxovirus isolated from diseased goose in JiangSu, the amplified product was ligated into pMD18-T vector and sequenced. Positive plasmid named T-HN.The tested result showed the homologues of HN gene sequence and amino acid sequence were about65.7%～99.0%and84.6%-99.0%relatively. Phylogenetic tree show that isolated strain E01matches to virulent APMV-1strain, belonging to genotype VII of APMV-1strain.We designed the second pair of primer HNF2/HNR2by inserting two restricted enzyme sites that EcoR I/Sal I at the5’ends. HN gene ORF used for prokaryotic expression was amplified by PCR, which inserted into pET32a vector and transformed into competent E. coli BL21(DE3). The recombinant bacteria was induced by IPTG and analyzed with SDS-PAGE. The result showed that gene encoding HN was expressed in E.coli at high level. Western blot were used to detect the expression of target proteins.Then we designed the third pair of primers HNF3/HNR3by inserting EcoR I/Not I with similar method, and HN gene ORF used for eukaryotic expression was amplified by PCR, which was cloned into multiple clone site of the eukaryotic expression vector pVAX I.The positive recombinant plasmids were transformed into E. coli DH5a competent cells, which were named pVAX-HN.Then the recombinant plasmids were amplified and purified and transfected into vero cells with lipofectamine.The indirect immunofluorescence analysis and RT-PCR were used to detect the expression of target protein. Expression of HN gene in prokaryotic and eukaryotic expression systems could be used to advance subunit gene vaccine and improve the development of GPMV.In order to observe immuno-efficiency of HN protein and DNA vaccine, we injected7-day SPF chickens for three times with the largely extracted expression product of HN gene and pVAX-HN plasmid, at the same time, empty vector pVAXland sterile saline were injected as negative group. We collected blood sample before immunization, and tested them by indirect ELISA. The results of indirect ELISA showed that the specific humoral immune response of the chicken in the test group was increased significantly compared to chicken in the control groups. It means that prokaryotic expression product HN protein and DNA vaccine can simulate immune react,which could be foundation for proventing of GPMV...