Rabies is an old known disease to mankind in recorded history. Approximately 55,000 individuals die from rabies, caused by rabies virus (RABV), worldwide per annum (1). The ~12 kb genome of RABV is composed of five genes that encode the structural proteins of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), matrix protein (M), and large protein (L) (2). The cDNA of L protein encodes a long polypeptide of 2128 amino acids with a relative molecular weight of 243.09 kDa (silver-haired bat-associated strain (SHBRV)). L protein (named after its large molecular weight) is considered to work with the phosphorylated non-catalytic subunit of viral RNA polymerase, P protein, and as a catalytic subunit of RNA polymerase (3). The polymerase complex of L and P displays all the enzymatic activity of transcription, including co-transcriptional modifications of RNAs, such as capping and polyadenylation, and the initiation and elongation of transcripts (4). L protein functions were mostly predicted from studies on vesicular stomatitis virus. In recent years, reports about RABV were mainly focused on the viral G (5-7), P (8-14), M (15-18), and N (9,19-24) proteins. However, the work on L protein of RABV has been relatively limited. Its potential role in the virus cycle and host factors interacting with L protein remains to be determined.In our previous study (9), we have established 11 mAbs, including three neutralizing antibodies, one anti-nucleoprotein antibody and seven mAbs against phosphoprotein, through a strategy of suckling mouse brain antigen immunization. The large molecular size of L protein and the lack of commercially available antibodies against RABV L protein restricted the progression of RABV L protein research work. Development of an effective antibody is especially Background: Rabies virus (RABV) is a deadly neurotropic virus that causes the disease of rabies in humans and animals. L protein is one of the large structural protein of rabies virus, which displays multiple enzymatic activities, and is required for viral transcription and replication. Objectives: A truncated L protein of Rabies virus is being cloned, expressed and purified to produce relevant polyclonal antibody. Materials and Methods:The gene fragment of L protein of RABV was subcloned into prokaryotic expression vector pET28a and transformed into E. coli Rosetta DE3 host strain. The recombinant L protein of RABV was expressed and characterized by SDS-PAGE and western blot analysis using anti-his tag antibody. Mice were immunized with the purified recombinant L protein, the reaction of the anti-serum was checked by immunofluorescence and dot-blot, respectively. Results:The results of PCR and sequencing confirmed that the fragment of L gene of RABV was successfully cloned into the expression vector. The expression of recombinant L protein fragment induced by IPTG was confirmed by the band of 43 kDa in SDS-PAGE and western blot. The antiserum of purified L protein immunized mice was reacted with RABV infected N2a cells and suckling mouse brai...

BackgroundHerpes B virus (BV) is a zoonotic disease caused by double-stranded enveloped DNA virus with cercopithecidae as its natural host. The mortality rate of infected people could be up to 70% with fatal encephalitis and encephalomyelitis. Up to now, there are no effective treatments for BV infection. Among the various proteins encoded by monkey B virus, gD, a conserved structural protein, harbors important application value for serological diagnosis of frequent variations of the monkey B virus.ObjectivesThis study aimed to expressed the gD protein of BV in Escherichia coli by a recombinant vector, and prepare specific monoclonal antibodies against gD of BV to pave the way for effective and quick diagnosis reagent research.Materials and MethodsThe gD gene of BV was optimized by OptimWiz to improve codon usage bias and synthesis, and the recombinant plasmid, pET32a/gD, was constructed and expressed in E. coli Rosetta (DE3). The expressed fusion protein, His-gD, was purified and the BALB/c mice were immunized by this protein. Spleen cells from the immunized mice and SP2/0 myeloma cells were fused together, and the monoclonal cell strains were obtained by indirect enzyme-linked immunosorbent assay (ELISA) screening, followed by preparation of monoclonal antibody ascetic fluid.ResultsThe optimized gD protein was highly expressed in E. coli and successfully purified. Five monoclonal antibodies (mAbs) against BV were obtained and named as 4E3, 3F8, 3E7, 1H3 and 4B6, and with ascetic fluid titers of 2 × 106, 2 × 105, 2 × 105, 2 × 103 and 2 × 102, respectively. The 1H3 and 4E3 belonged to the IgG2b subclass, while 3E7, 3F8 and 4B6 belonged to the IgG1 subclass.ConclusionsThe cell lines obtained in this work secreted potent, stable and specific anti-BV mAbs, which were suitable for the development of herpes B virus diagnosis reagents.