Preparation Of Phosphoprotein Monoclonal Antibody 1C9 Against Rabies Virus And Analysis Of Its Antigenic Epitope

Rabies is a kind of fatal zoonosis caused by rabies virus(RABV) with a mortality virtually up to 100%. China presents a high incidence of rabies, and the prevalent area has been expanding since 1990 s. Fluorescent antibody staining was the gold standard method for rabies diagnosis. At present, the FITC conjugated-polyclonal antibody was comprehensively used as diagnostic reagent around the world. A diagnostic kit based on FITC-conjugated monoclonal antibody(Mc Ab) against RABV nucleoprotein(N) was developed in our laboratory, and was applied for RABV detection countrywide. A series of Mc Abs with good reactionogenicity to multiple strains were obtained during screening of Mc Abs, and showed good properties for application.In this study, RABV challenge standard strain CVS-11 was inactivated and used as immunogen to prepare the Mc Ab against rabies virus. The spleen cells of immunized mice were collected and fused with SP2/0(myeloma cell). A hybridoma cell strain that could stably produce Mc Ab against RABV was obtained after multiple screening, namely 1C9. The ascites was prepared by intraperitoneal injection of BALB/c mice, and the titer was determined as 1: 2.56 × 104. Western-blot assay verified it as an anti-RABV phosphoprotein(P) Mc Ab. Ig G was purified by affinity chromatography and identified to be Ig G2 a. 1C9 was then labeled using FITC. The optimal working dilution of the FITC-conjugated 1C9 was determined as 1: 800. Parallel detection of 916 suspected samples and varies RABV cultures using FITC-conjugated 1C9 and anti-RABV N demonstrated that 1C9 was not only sensitive to most RABVs, but also sensitive to RABV-related lyssavirus, such as Irkut virus.Given its excellent perspective for application, we further analyzed the linear epitope of RABV P recognized by 1C9. Full length and serial C-terminal truncated P gene fragments were amplified by PCR and were inserted into pc DNA3.1 using the restriction sites Nhe I and Kpn I. The recombinant plasmids were transfected to BHK-21 cells respectively after identification with accurate sequences. Forty-eight hours later, the expressed polypeptides were analyzed using IC9 by direct fluorescent assay and western blotting respectively. Results showed that the site on RABV P related for 1C9 recognition mapped to aa131-133. Sequence alignment showed that the Asn132 and Phe133 were highly conserved among different RABV strains and Irkut virus. Conclusionly, Asp132 and Phe133 were the key amino acids related to 1C9 recognition.In summary, a Mc Ab against RABV P was obtained successfully in the present study and showed high specificity to RABV and Irkut virus, providing a sensitive and reliable detection tool for these viruses. The key amino acids of RABV responsible for Mc Ab 1C9 recognition were also analyzed, which laid foundation for further functional study of RABV P and the application of 1C9.