Prokaryotic Expression Of Glycoprotein Of Spring Viremia Of Carp Virus(svcv) And Preparation Of The Monoclonal Antibodies

Spring Viraemia of Carp（SVC）is an immediately and infectious disease which can affectsorts of Cyprinidae and result in much losses. The Glycoprotein that locates on the cystmembrane of SVCV is not only the main antigen epitopes, but associates with the process ofviropexis. Monoclonal Antibody（mAb） is produced by B lymphocytes, only specific for certainantibody epitope. Mabs have features of specificity, high sensitivity and can be prepared in alarge number, which have broad application prospects in the detection of the virus, the detectionmethods of OIE are built on the basis of mAbs. In this paper, the recombinant G proteinfragment will express to prepare mAbs against it. Compared to the total viral antigen,monoclonal antibody based on the Glycoprotein antigen showed more advantages. It cansimplify the preparation process, improve the positive rate and reduce the preparation cycle.There have not been reports of preparation of the monoclonal antibodies with recombinantexpressive Glycoprotein. Here are the main results.1、the expression and purification of G protein fragment. By RT-PCR method, the fragmentof G gene was amplified from SVCV Chinese isolate (0504). Constructed expression plasmid ofpET-28a-G and achieved highly expression of G protein in E. coli, while the expression productexsited in form of inclusion body that is consistent with the results of Zhang Lin. By denaturing,washing and purifying processes, obtained denaturation protein and renatured after dialysis.Finally we obtained the immunization protein antigens. Yang zhenhui immunized New Zealandrabbits with tapping recycling inclusion and successfully prepared a polyclonal antibodyanti-SVCV, indicating the presence of G protein linear epitopes, which is the theory basis of thispaper.2、Preparation of anti-G mAbs. We immunized Balb/c rats with Purified G protein. After5immunization, fused spleen cells were with SP2/0cells. Then harvested300strains fused cells,the fusion rate reached about50%. Adopted indirect ELISA method combined with indirect IFAto filter out the specific anti-G protein mAbs, the former suited for mass screening, while theIIFA was established to verify whether the specific monoclonal antibody reacts with the naturalvirus. In the results, we selected two hybridoma cell lines secreting anti-G protein mAbs, namedas1C5,2D5.3、The characteristics of mAbs. Two hybridoma cell lines belonged to IgG，and the mediumsupernatant titers reached1:2560and1:5120, respectively. Western-blot showed that the mAbscould react specifically with recombinant Glycoprotein. Stability analysis showed that twohybridoma cell lines could secret antibodies after several passages.On the conclusion, hybridoma cell lines that secreted anti-G McAb had been preparedsuccessfully, which will lay foundation for the establishment of the new SVCV detectionmethods.