Prepared The Rabies Virus N And G Polyclonal Antibodies

Rabies is a zoonotic and acute infectious disease caused by rabies virus. A person infected with RV, once appearing neurological symptoms, death rate is almost hundred percent. Every year more than1,000people died of rabies, so rabies was a serious threat to human. With the extensive application of molecular biology, genomics, and proteomics, function of each RV protein, pathogenesis, budding process has made great progress, but many RV-host interaction mechanisms are still not completely clear. Thorough study of these mechanisms will help to improve the quality of rabies vaccine drugs. Currently, due to lack of suitable RV monoclonal or polyclonal antibodies, the pace of our research was seriously hindered, so the preparation of antibodies has become the applicable prerequisity. Nucleoprotein (N) is essential for the transcription and replication of viral protein, the preparation of N antibody is use for N protein detection, and then evaluating replication-competent viral transcription. Glycoprotein (G) is the major protective antigen of RV, while it is closely related to virus neurotropic and pathogenicity, so the preparation of G protein antibody is necessary. The main structure of N protein antigen area is located in360-383amino acid residues, including6linear distribution antigen epitope. This paper takes the RC-HL plant of N gene plasmid as a template, using two pairs of primers RV-NF1/R1, RV-NF2/R2for amplification N1-2(350-350amino acids), N3-4(360-451amino acids). Reuse the RV-NF1and RV-NR2two complement at the end of the primer and N1-2, N3-4fragment of recycling products for overlapping PCR, amplified the N1-4fragment. The N1-4fragment was cloned into pMD18-T vector, after plasmid extraction and double enzyme and then cloning to pET-32a prokaryotic expression vector. Transformed into E. coli Rosetta and using IPTG inducible expression, SDS-PAGE analysis of recombinant N protein found that mainly expressed as inclusion bodies. Recombinant N protein was purified by Ni+metal chelate affinity chromatography with inclusion body wash solution containing different concentrations of urea. Purified protein with gradient dialysis, and made its refolding. Protein renaturation with freund’s adjuvant according to reasonable immunity procedure was carried on the mice, so as to get preparation of polyclonal antibody. Used Western blot to detect the N protein expression of infection RC-HL strain in BHK cells, appeared50.5kd purpose bands; Indirect immunofluorescence test found that the N protein antibody can get good response, appeared specific fluorescence. The diluted times N protein polyclonal antibody reacted with the48h infection BHK cells with RC-HL, found1:2000dilution of antibody could still occur target band and N protein responded well, showing a clear and specific target band.G protein251tryptophan (Trp251) is a linear epitope. Using Fmoc solid-phase peptide synthesis method of the G protein epitope G15-13peptide fragment (aa245-264), in accordance with the procedures established by immunization of mice for five times, collected serum to get G protein polyclonal antibody preparation. Western blot detected G protein of48h infectious RC-HL strain in BHK cells, appeared56kd purpose bands; indirect immunofluorescence experiments showed that the prepared antibodies could be favorable response, specific fluorescence was detected well.