非洲猪瘟病毒p72蛋白可溶性表达及鉴定

Soluble Expression and Identification of African Swine Fever Virus p72 Protein

为获得可溶性的非洲猪瘟病毒(ASFV)p72蛋白,将重组质粒pColdⅠ-p72分别与5种分子伴侣质粒进行融合表达,SDS-PAGE鉴定表达产物;以重组蛋白His-p72免疫小鼠制备多克隆抗体,通过Western blot和间接免疫荧光方法鉴定多克隆抗体与真核表达蛋白的免疫反应性。结果显示,重组蛋白His-p72与伴侣蛋白pG-Tf2或pTf16共表达后,实现了可溶性表达。以可溶性His-p72蛋白免疫小鼠后,获得His-p72多克隆抗体,抗体效价达到1∶512000;Western blot和间接免疫荧光的试验结果显示多抗血清可特异性识别真核表达的p72蛋白。说明利用分子伴侣融合表达的ASFV p72可溶性蛋白具有较好的免疫反应性,为抗ASFV药物的研发提供了候选蛋白。

In order to obtain soluble p72 protein of African swine fever virus(ASFV),the recombinant plasmid pColdⅠ-p72 was fused and expressed with five kinds of molecular chaperone plasmids,and the expression products of different expression strains were identified by SDS-PAGE.The mice were immunized with the recombinant protein His-p72 to prepare polyclonal antibody,and the immunoreactivity of the polyclonal antibodies with eukaryotic expression protein was detected by Western blot and indirect immunofluorescence assays.As results,the soluble expression of recombinant protein His-p72 was obtained by co-expression with partner plasmids pG-Tf2 or pTf16.After immunizing mice with soluble His-p72 protein,the His-p72 polyclonal antibody was obtained,and the antibody titer reached 1:512000.The results of Western blot and indirect immunofluorescence showed that the polyclonal antibody serum can specifically recognize the eukaryotic expressed p72 protein.In conclusion,the soluble ASFV p72 protein expressed by fusing with molecular chaperone has good immunoreactivity,and can provide the candidate protein for the development of anti-ASFV drugs.