非洲猪瘟病毒外膜蛋白CD2v的原核表达及其免疫特性的研究

Prokaryotic expression and immunological characterization of African swine fever virus out membrane CD2v protein

本研究参考非洲猪瘟病毒(ASFV)国内分离株SY18株的EP402R基因(编码CD2v蛋白),优化其序列,设计特异性引物扩增。将去除跨膜保留胞外区或胞内区的两种截短体克隆到原核表达载体p ET-28a中,再转化至大肠杆菌BL21(DE3)感受态细胞。经IPTG诱导,铁蛋白融合CD2v胞外区(CD2vN-Fe)和铁蛋白融合CD2v胞外-胞内区(CD2v-NC-Fe)均获得高效表达。以该融合蛋白为抗原免疫BALB/c小鼠制备多克隆抗体,通过酶联免疫吸附实验(ELISA)和间接免疫荧光(IFA)方法检测其灵敏度和特异性。结果表明:CD2vN-Fe多抗特异性较强。本研究为建立ASFV的血清学鉴别检测方法奠定了基础。

In this study,the sequence of EP402 R gene encoding CD2 v from African swine fever virus(ASFV)Chinese strain SY18 was optimized and synthesized.The extracellular domain of CD2 v fused with ferritin(CD2 v N-Fe)and the extracellular-intracellular domain of CD2 v fused with ferritin(CD2 v-NC-Fe)were amplified by fusion PCR.Different fragments were subcloned into prokaryotic expression vector p ET-28 a and transformed into E.coli BL21(DE3)competent cells.Induced by Isopropylβ-D-thiogalactoside(IPTG),CD2 v-NC-Fe and CD2 v N-Fe fragments were highly expressed.The fusion proteins were used as antigen to immunize Balb/c mice to prepare antibodies.The sensitivity and specificity of antisera were detected by ELISA and IFA.The results showed that the specificity of antibodies against CD2 v N-Fe protein was strong,which laid a foundation for the establishment of a serological method for the detection of ASFV.