Prokaryotic Expression And Generation Of Monoclonal Antibodies Of Glycoprotein Extracellular Domain Of Rabies Virus Strain AG

Rabies, also known as hydrophobia, is an acute, potent and natural of zoonotic viral disease causedby the causative agents of rabies virus (Rabies virus, RABV). Rabies is still epidemic in a number ofcountries and regions of the world, it occurring mainly in developing countries in Asia and Africa. Ourcountry is also the high incidence of rabies, it is reported the number of died people from rabies foreight consecutive year has topped among the37kinds of statutory reporting of infectious diseases.Monoclonal antibody diagnostic methods of high sensitivity, specificity, simple operation, low prices,so are widely used in clinical detection and treatment of many diseases. In this study, monoclonalantibody technology was adapted and aG strain of the rabies virus as research materials was used. Thisresearch laid the foundation for detection of rabies virus antigen and antibody ELISA kit through genecloning, sequence analysis, codon optimization, prokaryotic expression, protein purification andmonoclonal antibodies.In this paper, the Glycoprotein Extracellular Domain fragment was successfully cloned from aGstrain of rabies virus, the sequencing results showed the fragment of1317bp encoding440amino acids.Sequence comparison with several related virus strain in GenBank was completed by using the NCBIblast software analysis, the nucleotide sequence similarity with4aG strain, PG strain, Chinese vaccinestrain,334strain and Nishigahara strain was more than97%, the nucleotide sequence similarity with theinternational standard strain (CVS) was93%.As the exogenous gene may contain transcription termination signal of E. coli, when it is expressedin E.coli, the expression level may not high. To improve expression levels, the target fragment wasanalyzed with rare codon Calculator (RACC) and39group of E. coli rare codon in the sequenceoptimized into E. coli normal codon. The optimized sequence was synthesized and constructed intoprokaryotic expression vector pET-28a (+) and transformed into the expression system of Rosseta (DE3)inducted by IPTG. Studies have shown that recombinant protein was significantly at higher levels ofexpression when recombinant bacteria induced with IPTG to a final concentration of0.6mM incubatingfor6h at30℃. The recombinant protein was proved to have good antigenicity through western boltting.To establish an indirect ELISA method for detection of animal RV antibody, recombinant protein waspurified by Merck’s His·Tag affinity chromatography. The best antigen-coated amount is determined at1.725μg/mL, serum dilution of1:200, and HRP-labeled secondary antibody dilution of1:50,000.The mixture of purified recombinant protein and Freund adjuvant immunized Balb/c mice(subcutaneously and intraperitoneally) for three times, and then given a booster with the recombinantprotein. Taken spleen cells of mice fusion with sp2/0cells and after three subclones and indirect ELISAassay screening, two stably secreting hybridoma cell lines positive monoclonal antibody (4B7and1F10)were obtained. ELISA experiments shown that the two hybridoma secreted antibody subtypes were thelgG2b and IgM; the chromosomes numbers were95and103, respectively; the titers of purified asciteswere1:25600; the McAbs has high specificity and high stability. The study paves the way for thedevelopment of RV antigen detection kits.