Study On Expression Of G(n+c) Gene Of RVFV Using Insect Baculovirus And Its Immunogenicity

RVF(Rift Valley Fever, RVF)is a sever viral zoonotic disease.The bite of mosquitoes is the main transmission mode of the Rift Valley fever, RVFV was characterized by high mortality rates among foetuses, neonates, and juvenile livestock and causes a severe febrile illness also in man.RVF was one of list legal notice disease of OIE and was classified as category A infectious disease in China, because RVF outbreaks are characterized by economically disastrous “abortion storms”and public health crisis. It has no reports show to this disease was discovered in China still now.With expanding the scale of China Africa Cooperation ? global commerce and humans travel frequently, it is hard to prevent effectively the disease from coming into our country. So it is important to go in fo research that develop efficient RVF and more safer vaccine and improve some diagnostic methods to RVF for the potential pandemic in China.The Rift Valley fever virus belonging to genus p Hlebovirus from Bunyaviridae family, has only one serotype. G(n+c) is the main structural protein and protective immunity of RVFV. The protein of G(n+c) is encoded by M segment. It was resolved into Gn and Gc subunit after translation. In this paper, we express the partial protein of G(n+c) in E.coli and the whole G(n+c) in Bac-to-Bac expression system of Bcaulovirus to lay the foundation for develop an virus-like particulate vaccine of RVFV.1 Expression and immunogenicity analysis of truncated glycoprotein Gn and Gc. According to the references and the biological information software, the four major antigenic regions of G(n+c)were determined. The Major Antigenic Region fragments were designed and amplified with PCR according to the M whole genome sequence of Rift Valley fever Virus in Gen Bank(Gen Bank ACCESSION: DQ380206). The prokaryotic expression vector p Cold I-Gc2, p Cold I-Gn2,p Cold I-Gc1 and p Cold I-Gn1 were constructed with Gn2, Gc1, Gn1 and Gc2 fragments using In-Fusion? HD Cloning Kit. The recombinant protein was induced by 1Mm IPTG after the recombinant plasmid was transformed into E.coli BL21 competent bacteria. The results showed that the recombinant protein prokaryotic expression of truncated mainly existed in the form of inclusion body by SDS-PAGE.The purified protein was injected new Zealand white rabbits to prepare the corresponding high serum free of serum.The expressed protein was purified with His-Tag Ni+. The results of ELISA test showed that the serum titer of the high immunity serum was more than1:51200.The results of Western-blot demonstrate that the Purification of protein was successfully expressed with the well antigenicity. The researches make a huge contribution to develop an ELISA kit for detecting Rift Valley fever Virus.2 Expression and immunogenicity analysis of G(n+c) gene in Bac-to-Bac expression system.The G(n+c) gene of RVFV was inserted into the transponson vector p Fast Bac TM HT A to construct the recombinant transposon vectors p Fast Bac TM HT A-G(n+c). Positive recombianants were screened in LB/AMP+ culture after transofrmation into E.coli DH5?and further identified with restriction endonucleases digestion, PCR and sequenced. To generate recombiant bacmids, the positive p Fast Bac TM HT A-G(n+c) was transformed into E.coli DH10 Bac. White bacterial colonies were selected as positive recombiant expression vectors after screening in GM, Ka, Tet, X-gal and IPTG selection LB ager plates. Then recombiant bacmids r-Bacmid-G(n+c) were transfected into SF9 cell to generate recombinant baculovirus that express G(n+c) protein of RVFV. The results of SDS-PAGE and Western-blot showed that the expressed G(n+c) protein cleavages into Gn and Gc were specific with molecular weight 56 KD and 58 KD and have good bilological activity and immunogencity.