Expression Of S1 Gene Of Reovirus Type 3 And Establishment Of Indirect ELISA For Detection Of Antibodies Against Reo-3

Reovirus type 3(Reo-3), a orthoreovirus of the Reoviridae, has a linear double -stranded RNA genome. Reo-3 is the must detection project for the SPF level laboratory animal, such as rat, mouse, Cavia procellus and hamster. Its nature infection host is quite widespread, such as human, rat, mouse, monkey, cattle and so on, and can silent infect animal and produce the antibody in vivo. It brings difficulty for animal test and the external pathogenic factor examination such as blood product, monoclonal antibody, cell culture etc. 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 Reo-3. The antigen gain method mainly is the cell culture, but is very difficult to obtain the high titer the virus; Although may multiply in the chicken embryo, but does not have the regularity. In our study ,δ1 is expressed by prokaryotic expression system pQE31 and we establishes new ELISA method using the purified recombinant antigen .The serum antibodies of Reo-3 can be detected easily and quickly.Based on hydrophilicity and antigenity analysis of the amino acids ofδ1 using biosoftware DNAstar, PCR primers were designed according to gene order of Reo-3 S1(gi:61780). SR gene was amplified by one step reverse transcription polymerase chain reaction (RT-PCR), and was cloned into pMD18-T vector for sequence analysis .The masculine recombinant plasmid was digested by restriction enzymes Sph I+Sal I and then was sub-cloned into prokaryotic expression vector pQE-31, and was induced expression in E.coli M15(pREP-4).The expressed SR-δ1 protein was identified by SDS-PAGE and Western-blot analysis. The results showed that the molecular weight of the expressed protein was 32 kD and the protein could specifically react with antiserum against Reo-3.The indirect ELISA for detection of antibodies of Reo-3 was established using SR-δ1 as coated antigen with the optimal working parameters, including 0.6μg/mL of the SR-δ1 protein antigen for coating, testing sera dilution at 1:100, second antibody (Sigma) dilution at 1:8000, the standard of determining as positive sample S/P≥0.292 and others. It revealed a negative reaction with the positive sera of EcT, SeV, MHV and PVMT. demonstrating specificity of the diagnosis method is excellent. The variation coefficient of inter- and inner- batch antigen production testing quality control serum which can guarantee the quality of antigen and the accuracy of testing results is below 10 percents, demonstrating that repeatability of the antigen is good. Compared with the Reo-3 ELISA kit by national monitoring center of the quality laboratory animals, the two methods had 98.7% agreement by detecting 75 samples. the speciality of the test is 98.5%, the sensitivity, 100%. The result shows that the ELISA assay has excellent specificity , high sensitivity and excellent reproducibility, and could be used to detect the antibody against Reo-3.In conclusion, Reo-3 S1 gene was successfully cloned and expressed in E.coli and purified. They lay the foundation for further studies of the protein structure and function ofδ1. The simple and fast diagnosis method using SR-δ1 expressed as diagnosis antigen can be a technique support for further developing the commercial kit of diagnosis and quality control for Laboratory Animals.