Expression Of MHV-N In E.coli And Development Of An Indirect ELISA For Detection Of Anti-MHV Antibodies

Mouse Hepatitis Virus(MHV), a coronavirus of coronavirus genus, has a linear single-stranded RNA genome with positive polarity and causes a variety of diseases, such as enteritis, hepatitis and demyelinating encephalomyelitis in susceptible rodents depending on the virus strain, animal species and immune state of animals, etc. The infectious animals usually have no symptom and spread all over the world which determines MHV to be one of the most difficult diseases in rodents to control. Infected with MHV, the parameters of 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, for example the enzyme-linked immunosorbent assay (ELISA), are commonly used to determine whether laboratory animals are infected with MHV. MHV has been propagated in cell culture system and purified to be used as coated antigen. Because of its low virus titer and complicated purification procedure, a new ELISA is developed using recombinant protein as coated antigen. It has been demonstrated that the new method is more specific and sensitive than the traditional one.According to N gene sequence of MHV published on GenBank, a pair of primer was designed to amplify a part of N gene of MHV using RT-PCR technique. The PCR product was purified and cloned into pGEM- T- easy vector. After testified the N gene was further subcloned into prokaryotic expressing vector pGEX-6p-1 and transfected into E.coli strain BL21 . The transformant was induced by IPTG to express recombinant protein. The recombinant protein was purified and then identified by SDS-PAGE and Western-blotting analysis. The result revealed that the protein had a molecular weight of 56×103, which could be specifically recognized by anti-MHV antibody. Because of its good immunogenicity, the recombinant protein can be used as an antigen to detect anti-MHV antibody in sera of mice in ELISA.The indirect ELISA for detection of anti-MHV antibodies was established using recombinant protein as coated antigen. The optimal concentration of the coated antigen, the dilution of serum and second antibody, the standard of determining as positive sample were determined. The diagnosis method was demonstrated specific and sensitive by specificity test, sensitivity test, stability test and contrast test. The result above confirmed the assay to be specific and sensitive, suggesting an application prospect.