Structure Analysis Of Nucleoprotein And Glycoprotein Genes Of Rabies Virus And Expression Of Nucleoprotein In E.coli

The genome of Rabies virus is a single-chain and minus-strand, which encodes five kinds of protein: nucleoprotein, matrix protein, large protein, glycoprotein and phosphoptotein. Nucleoprotein is the most conserved and stable protein among the five protein, which have best antigenicity and can induce specific reaction mediated by B and Th cell. Glycoprotein is the only antigen which can induce protective neutralization antibody and cellular immunity. Research indicated that there is a close relation between the protein and pathengicity, nucleotides and amino acids change frequencely, so Glycoprotein gene is often studied for engineering vaccine.In view of the importance of NP and GP among all the proteins, we obtained the NP and GP genes of rabies virus Flury LEP strain through RT-PCR, sequenced and identified, and then NP was expressed in prokaryotic systems.Firstly, two pairs of primers was designed according to the published sequences of the LEP strain of rabies virus, by using the RT-PCR assay, obtained the whole N and G genes fragment, and then they were cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET-N and pET-GSecondly, by comparing N and G genes with the published sequences of different fixed strains, the system phylogenetic tree was drawn, calculating the hydrophilicity and antigenic index and finding the difference of amino acids of chief antigen site. As it is known that N gene is conserved much more than G in different virus strains, the chief antigen site III of G is most typical, the 333th site of Arginine of GP is substituded by Glycin, which is the main difference between the strain of LEP and street virus.Lastly, the recombinant plasmid PET-N was transformed to BL21(DE3) and expressed after induced by IPTG Confirmed the best optimal expression condition within different time of inducing and different IPTG concentration.The protein expressed is hoped to use as the diagnosis antigen to establish a way to detect the RV antibody by Indirect ELISA. Sequence analysis of NP and GP genes could help to analyze the ability of immunoprotection of vaccine strains for animals in china, and provided the basis for the epidemiology research.These data can provide foundation for evulating the adeptability and validity of the present vaccine for animals, as well as for choosing new kind of inactived vaccine.