Prokaryotic Expression Of VP2Gene Of Aleutian Mink Disease Virus And Establishment Of Indirect ELISA For Detection Of Antibody

Aleutian mink disease also known as plasma cell histiocytosis is a chronic infectious disease caused by Aleutian mink disease virus persistently infected mink, which disordered its immune system. Since Aleutian disease has been discovered, it has not yet developed a genetically engineered vaccine to prevent the disease effectively, and it has caused huge economic losses to the mink industry at home and abroad for a long period of time. This disease has become one of three viral infectious diseases which harm mink industry. This study aimed at the insufficient of detection methods of AD for this stage, accomplished prokaryotic expression of the VP2gene of ADV and thus established indirect enzyme-linked immunosorbent assay for detecting ADV antibody. The main contents included the following aspects:(1) Referencing the ADV sequence published in the GenBank, truncated gene fragment with antigenic epitope after analysis of VP2gene by use of biological software. Designed a pair of specific primers, amplified target fragment with the size of620bp, cloned into pMD18-T vector, identified the construction of recombinant plasmids by PCR, enzyme identification and sequencing analysis, then cloned the correct target fragment into the prokaryotic expression vector pGEX-4T-1, the recombinant plasmid was named pGEX-4T-VP2, same by PCR, enzyme identification and sequencing analysis, the results showed that the target fragment was imported into the expression vector successfully.(2) Transformed the recombinant plasmid pGEX-4T-VP2and empty vector pGEX-4T-1into E.coli BL21, induced expression with37℃,lmol/L IPTG, detected by SDS-PAGE electrophoresis, the molecular weight of the target protein was57kD, the protein expression reached a maximum at5h and the protein had reactogenicity after detected by Western Blot. Adopted the purification method of the gravity flow column, purified the recombinant protein by balancing, integrating and eluting, then detected by SDS-PAGE electrophoresis and obtained a single target band, the size was as same as expected result, the result showed that the target protein was purified successfully.(3) Used purified fusion protein as antigen to coat ELISA plates, through optimized reaction conditions which included concentration of antigen, blocking liquid, serum, IGg-HRP and substrate, established detection method of indirect ELISA. The optimum reaction system of indirect ELISA initially:the concentration of antigen envelope liquid of recombinant protein was1:200,37℃for1h, overnight at4℃,5%BSA37℃closed2h, the dilution of serum was1:100,37℃for1h, the dilution of HRP-labeled rabbit-anti-mink IgG was1:2000,37℃for1h, chromogenic TMB substrate buffer which is kept in dark place act20min. After terminating the reaction, determined the OD450value. Specificity, sensitivity and repeatability test also had been implemented, which acquired good effects.