羊口疮病毒B2L和VIR基因原核表达及抗原性鉴定

Prokaryotic Expression of Orf Virus B2L,VIR Genes and Antigenicity Evaluation

为检测羊口疮病毒(Orf virus)B2L和VIR蛋白的抗原性,通过PCR方法扩增出羊口疮病毒B2L和VIR基因,与原核表达载体pET-32a连接,将重组质粒转化到BL21(DE3)感受态中,对其进行BamHⅠ和HindⅢ双酶切鉴定和测序验证。对鉴定为阳性的菌液进行诱导表达,并进行SDS-PAGE分析。用灭活的羊口疮病毒免疫家兔,制备多克隆抗体,Western blot、ELISA鉴定B2L和VIR蛋白的抗原性。结果显示B2L、VIR蛋白均能与兔抗羊口疮病毒抗血清结合,证明B2L、VIR蛋白具有与羊口疮病毒共同的B细胞表位。

To detect the antigenicity of B2L and VIR proteins of Off virus,B2L and VIR genes were ampli- fied by PCR, then PCR products were connected to the pET-32a vector. The recombinant plasmids were transformed into the competent cell BL21（DE3）,then digested with BamH I and Hind Ⅲ and sequenced. The positive clone was induced tO express the target protein,meanwhile analyzed by SDS-PAGE. The rab-bits were immuned with inactivated Orf virus to prepare polyclonal antibodies,then the antigenicity of B2L and VIR proteins were evaluted by Western blot and indirect ELISA. The result suggested the recombinant B2L and VIR proteins can specifically combine with the polyclonal antibodies of rabbit anti-orf virus and B2L,and VIR proteins have the same B cell epitope as Off virus.