Cloning And Sequence Analysis And Construction Of Prokaryotic Expression Vector Of HN Gene And F Gene From Goose Paramyxovirus

Goose paramyxovirus disease is an acute contagious disease caused by GPMV, a member of Rubulavirus family. GPMV has been found in many areas since it was reported by Wang Yongkun of Yangzhou university in 1997, which was characterized by the lesion of digestive tract with high mortality and occurrence, and has caused a large loss in economy. The primary study show that there is a difference in pathogenicity between GPMV and traditional NDV, and the common NDV vaccine doesn't provide an effective protection against disease. So it is of great interest to study the pathogen from the molecular biology.HN(Hemagglutinin-Neuraminidase) protein and F(Fusion) protein are two important membrane glycosylated proteins. HN protein has hemagglutinin activity and neursminidase activity. The cleavage site of F protein is the mainly decisive factor in virulence. In this study, according to the complete genome sequence of GMPV SFO2 isolate and other NDV strains published in GenBank, two pairs of primers were designed to amplify HN gene by RT-nested PCR, and one pair of primers were designed to amplify F gene by PCR. The specific DNA products were cloned into PMD18-T vector, and transformed into competent cell TG1, and the positive recombinant plamids were screened by AMP/IPTG/X-gal ,and endonuclease digesting and PCR identification. The sequence analysis showed that HN gene is 1776bp in length, encoding 571 amino acid residues; the homology of nucleotide between JS/1/97/Go strain and other strains varies from 82.1%-98.4%, and the homology of the deduced amino acid varies from88.3%-99.5%; the F gene is 1662bp in length, encoding 553 amino acid residues, the homology of nucleotide varies from 84.2%-99.8%, and the homology of the deduced amino acid varies from 87.7%-99.8%. The amino acid duduced from HN gene has four glycosylated sites, three among them are very conservative. The amino acid duduced from F gene has six glycosylated sites, five among them are very conservative. The amino acid sequence of cleavage site is 112R-R-Q-K-R-F117 according with the virulent NDV strain. The epitopes were predited by DNAstar software according to the sequencing results of HN and F. A single primer was designed to amplify the 5'terminus of HN gene(named as HNp)which includes three epitopes. The sequencing result of HNp gene show there is only two variations in nucleotide and deduced amino acid residues respectively. The HN gene and HNp gene were subcloned into prokaryotic expression vector pProEXHTb respectively ,then were transformed into competent cell DH5α, the positive colony containing recombinant plamsid were screened by extracting plasmid and endonuclease digesting and PCR identification. The F gene were subcloned into prokaryotic expression vector pET-30a(+) and pGEX-6P-1 respectively, then was transformed into competent cell TG1 and screen positive colony.In this research, HN gene and HNp gene and F gene were cloned and analyzed sequence, and their homologous comparision with NDV were done. According to the sequencing results ,the epitopes of HN gene and F gene were predicted ,providing a theoretical foundation for studying the relationship between GPMV and NDV. Three kinds of prokaryotic expression vector containing HN gene, HNp gene and F gene were constructed respectively, laying a base for expessing and further differentiating diagnosis and developing epitope vaccine .Candidate:Gao HongliMajor:Preventive Veterinary ScienceSupervisor:Prof.Wang Junwei...