大蒜A病毒CP基因的原核表达及抗血清制备

Prokaryotic Expression of Garlic virus A CP Gene and Preparation of Its Antiserum

根据GenBank报道的大蒜A病毒(GarV-A)序列设计引物、扩增其外壳蛋白基因并进行序列分析。结果表明,GarV-A的CP基因与目前已报道的两种GarV-A不同分离物CP基因的核苷酸序列同源性为98%-99%;氨基酸序列同源性均为98%。将GarV-A CP基因插入表达载体pSBET,在大肠杆菌BL21(DE3)Plys E菌株中诱导表达。CP经12%SDS-PAGE和5%-20%SDS-PAGE两次纯化,免疫小鼠获得抗CP血清,Western blotting分析表明确定制备的抗体对CP具有高度特异性,ELISA检测表明制备的抗体能够与天然病毒离子结合,因此可以作为该病毒的检测。

Specific primer was designed to amplify Garlic virus A Liuan isolate.The multiple aligment shows that GarV-A shared 98%-99% nucleotide acids identities and 98% amino acids identities with the two sequences of CP genes reported on GenBank.Then the CP gene was inserted into pSBET and expressed in Escherichia coli BL21（DE3） plys E strain.The object protein was purified by 12% SDS-PAGE firstly and subsequently 5%-20% gradient SDS-PAGE.The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blotting analysis.ELISA result indicated that the antiserum is suitable for GarV-A detection.