Expression And Identification Of Of Nucleoprotein Of Rabies Virus In Prokaryotic Cell

Objective Rabies is an important public health problem in the whole globe. The death rate ofhuman rabies deaths of China ranked the second place in the world. In this test to clone N genefragment of Rabies virus (RV) and construct the prokaryotic expression vector expressingNucleoprotein (NP) of RV CVS strain with a 6×His-tag, expressing Nucleoprotein in Escherichia coliin order to provide the necessary antigens for the development of sero-diagnostic kids for Rabies.Methods a pair of primers was designed based on the sequences of the sequences of N gene fragmentof Rabies virus, and the primers were ended with NdeI and BamHI restriction endonuclease sites at the5 end. By using the PCR technique, the target genes of NP were amplified from Teasy—RVNPrecombinant plasmid, then the PCR product was digested with NdeI and BamHI and inserted intoprokaryotic expression vector. As demonstrated by the sequencing, the recombinant plasmidpET-15b-NP was transferred into E. coli BL21 cells and the recombinant vector was expressed in E. colistrain BL21. Optimizing the the range of induction temperature, induction time and revulsivumconcentration, expression of the recombinant protein were analyzed with fermentation bottle Theexpressed protein was detected by SDS-PAGE and western blotting with polycloned antibodies., Results It was found that after transformation the host strain of bacteria could express successfullythe target protein with a molecular size of 51kDa. and mainly expressed as inclusion body inEscherichia coli. Western blotting analyses demonstrated that this recombinant protein presented thespecific antigenicity Conclusion It suggests that the construct could be expressed on E.coli BL21cells, and the immune sera from mouse immunized with Rabies Virus could specifically recognize theexpressed protein. It prOvide a solid basic for product high purity and cheap RV NP, and could beused in diagnostic for RVNP.