Expression Of SeV-F In E.coli And Development And Application Of An Indirect ELISA For Detection Of Anti-SeV Antibodies

Sendai virus (SeV), a parainfluenza virus of the paramyxovirus family, has a linear single-stranded RNA genome with negative polarity and commonly infects laboratory mice, rats, and hamsters. Because of its subclinical infection as well as strong contagion and easy diffusion, SeV is one of the most difficult viruses in laboratory rodent to control. Infected with SeV, the cell-mediated immune response in vivo will be changed and then the results of experiments tend to be interfered. Thus, it is necessary to establish a quick and accurate diagnostic method.Serological methods, such as the enzyme-linked immunosorbent assay (ELISA), are commonly used to determine whether laboratory animals are infected with SeV. SeV has been propagated in many cell lines such as LLCMK2 and BHK21, but riral yields in vitro cell culture systems are poor. Maximum virus production is achieved by infection of embryonated chicken eggs. Unfortunately, this process is labor intensive, and purified virus preparation may remain contaminated with egg proteins, resulting in false-positive reactions when these viral preparations are used as antigens in serological tests. In our study , SeV- FP(S) is expressed by prokaryotic expression system pQE31 and we establishes new ELISA method using the purified recombinant antigen .The serum antibodies of SeV can be detected easily and quickly.1 Cloning and expression of F gene of Sendai virus and preparation antigen PCR primers were designed according to fusion protein of Sendai virus (gi:9627219) . FP(S) cDNA specific for the primary antigen form of fusion gene of Sendai virus was produced by reverse transcription polymerase chain reaction (RT-PCR) and was cloned into pMD18-T vector for sequence analysis .The pMD18-T-FP(S) was digested by restriction enzymes and then subcloned into the prokaryotic expression vector pQE31.The FP(S) was expressed in E.coli M15 by IPTG induction and purified, which was confirmed by SDS-PAGE and western-blot. The results suggested that the recombinant protein FP(S) was about 26kD and could react with mice-anti Sendai virus serum.2 Development and Application of An Indirect ELISA for Detection of Anti-SeV AntibodiesThe anti-serum was prepared by injecting 6-week BALB/c mice for five times with the purified FP(S) protein. An indirect ELISA was established using the purified recombinant FP(S) as the antigen and the anti-serum to FP(S) protein as antibody for detection of SeV-specific antibodies .As the results showed, the ELISA with high sensitivity and speciality could detect the antibody of SeV efficiently.