Cloning And Expression Of Hn Gene Of Newcastle Disease Virus7793

Background and ObjectiveNewcastle disease virus (NDV) belongs to Avulavirus genera of the Paramyxoviridae family, which is enveloped virus containing single-stranded negative-sense RNA. Hemagglutinin-neuraminidase as a fiber protein on NDV envelope has the absorption function to host cells. It can activate immune cells, which is closely related to antitumor effects. In recent years Newcastle disease virus has been interested increasingly for its strong antitumor effects and high security. Some scholars have already used NDV to infect tumor cells for obtaining modified autologous tumor vaccine, which was used to immunize patients with tumors and obtained some therapeutic effects.The virus used in this study is a NDV strain isolated in wild duck in Jiangxi which is named after NDV WDK/JX/7793/2004(hereinafter referred to as NDV7793). Our previous studies showed that NDV7793is a weak virulence strain that has strong selective destruction of many tumor cells. It has a potential to anti-tumor treatment. Moreover, our lab has analyzed the whole genome sequence of NDV7793(GenBank accession number is HM125898). The purpose of this study is to clone and express HN gene of NDV7793strain, which will further elucidate the anti-tumor mechanism of virus and lay the foundation for the development of genetic engineering vaccine.Materials and methodsThe NDV was inoculated in chick embryo for amplification of virus. Viruses from the allantoids fluid were purified through high speed centrifugation. Total RNA of NDV7793was prepared by RNA extraction kit. According to the HN gene sequence of NDV7793in Genbank, a pair of primers was designed to amplify1851bp fragment containing the coding sequence (CDS) of HN gene by using the method of RT-PCR. And then1851bp fragment was cloned into the pGEM-T easy vector to built pGEM-T-HN recombinant plasmid.Based on the relevant literatures and software analysis, we predicted that the major antigenic determinants of HN gene of NDV7793were located between142bp and1849bp (the size is1707bp). By using pGEM-T-HN recombinant plasmid as a template, a pair of primers with restriction sites was used to amplify1707bp fragment by using the method of PCR. After then1707bp fragment was cloned into pET-30a prokaryotic expression vector to obtain pET-HNa combinant plasmid. Finally pET-HNa was transformed into E.coli BL21(DE3). Induced by IPTG, the expression product of HN protein was checked by SDS-PAGE.ResultsA1707bp fragment without cytoplasmic and transmembrane region of the HN gene was successfully cloned. Compared with the HN gene logged on the Genbank, the homology reaches99%. Digestion of recombinant plasmid with restriction enzymes showed that we succeeded in building a recombinant expression plasmid pET-HNa containing the antigenic determinant fragment of the HN gene. A68kD HN fusion protein was obtained by prokaryotic expression system under the induction of IPTG.ConclusionExpression of the major antigenic determinant of NDV7793HN protein lays the foundation for the further elucidation of immunogenicity of NDV HN protein in molecular level and its antitumor mechanism.