Expression Of Nipah Virus Structural Proteins F1 And G And Preparation Of Hyperimmune Antisera Against Two Proteins

Nipah virus is an newly emerged paramyxoviruses and was identified in Malaysia 1999. The broad species tropisms and the ability to cause fatal disease in both animals and humans have distinguished NiV and HeV from all other known paramyxoviruses, these two viruses have been classified in a separate genus Henipavirus. It has been identified that Flying foxes are the natural host of NiV. In recent years, NiV has continued to reemerge, and the epidemic investigation suggests that the virus could be widely present in the Flying foxes, especially Pteropus lylei in Tailand and Cambodia, which are close to China, moreover, the similar enviorment and Flying foxes species, distribution in the south-east area poese a great threat to pig industry and public health of China, based on the above issues, there is a pressing need for diagnostic technologies on the nipah virus.In order to prevent their potential outbreak in China, in this study, NiV fusion protein gene (F) and NiV glycoprotein gene (G) fragments without the sequences coding signal peptide and transmembrane domain were amplified by PCR from the plasmid imported from Australia, and cloned into the pGEX-6p-1 expressing vector, transformed to host strain BL21(DE3), SDS-PAGE shows NiV F1 protein and G protein were expressed effectively in E.coli. After the purification of NiV F1 and G proteins, the I-ELISA and Western blot results showed the recombinant NiV F1 protein and G protein the great reactivity of both with the rabbit antiserum against intact NiV viron. So, this implied that all of them can be the detective antigenic candidates for NiV antibody.The NiV F1 and NiV G genes were also cloned into a modified transfer vector pFastBa-Mels-6His, resulting in the expression in baculovirus system of F1 and G proteins fused with His-tag at its C terminal. In order to get the recombinant Bacmid Bac-NF1 and Bac-NG , then the modified transfer vectors were transformed to DH10Bac host cell. And the Bacmid Bac-NF1 and Bac-NG were transfected with sf9 cells and got the recombinant baculovirus rAcNPV-NF1 and rAcNPV-NG. Then using rAcNPV-NF1 and rAcNPV-NG. transfected sf9 cells, CPE was observed after 72h transfected. The indirect immunofluorescent assay (IFA) using rabbit anti-NiV serum could identify the cells expressing the F1 and G proteins on sf9 cell monolayers, showing that NiV F1 and G proteins were exactly expressed. So, it can develop a new mothed to detect the serum antibody of NiV.We successful expressed NiV F1 and G proteins in E.coli and recombinant baculovirus. In order to get sera of anti NiV F1 and G, the application of purified E.coli-expressed F1 and G proteins to respectively immunize rabbits lead to the preparation of specificity polyclonal antiserum directed to each protein. The results of ELISA showed that the titer of sera are 1∶6 400(F1 protein) and 1∶12800(G protein), respectively.After concentration of supernatant that were expressed through recombinant baculovirus, the Western blot showed that antisera had good reactivity. At the same time, the results of IFA also suggested that antisera had good reactivity. So, the antisera could be used in virus neutralization test and it can develop a new mothed to detect the serum antibody of NiV.