非洲猪瘟病毒p54抗原C端的原核表达及多克隆抗体的制备与鉴定

Prokaryotic Expression of C-terminus of p54 Antigen of African Swine Fever Virus and Characterization of Its Polyclonal Antibodies

利用大肠埃希菌表达非洲猪瘟病毒核心抗原p54蛋白C末端55个氨基酸的肽段(命名为p54-2),制备了多克隆抗体并进行了纯化,以进一步用于非洲猪瘟ELISA试剂盒的研发。将PCR扩增获得的p54-2目的片段与表达载体pGEX6p-1质粒均经过限制性内切BamHⅠ和XhoⅠ双酶切后,构建pGEX6p-1-ASFV-P54-2原核表达质粒。将该质粒转化到大肠埃希菌BL21中,诱导蛋白大量表达,并纯化获得高纯度的蛋白。将纯化的p54-2蛋白免疫小鼠,获得p54-2多克隆抗体并进一步纯化多抗。用ELISA、Westernblot对纯化的多克隆抗体进行效价滴定及特异性鉴定。结果显示,成功构建了pGEX6p-1-ASFV-P54-2重组质粒,在大肠埃希菌中IPTG诱导下,能够高效表达重组p54-2蛋白。用p54-2蛋白免疫Balb/c小鼠获得的多克隆抗体经ELISA检测其抗体效价为1∶128000,说明该蛋白有很好的免疫原性。Westernblot结果显示,制备的多克隆抗体能特异性识别非洲猪瘟病毒p54胞内区,具有较高反应性和特异性,可以用于ELISA检测方法研究和p54抗原表位的鉴定。

In this study,the C-terminus of p54 antigen of African swine fever virus was expressed using prokaryotic(Escherichia coli)expression system.The C-terminal 55 amino acid peptide of p54 protein(named as p54-2)was expressed,purified and used as antigen to generate polyclonal antibodies,which would be further applied in the development of ELISA kit.The plasmid named as pGEX6p-1-ASFV-P54-2 was constructed.This plasmid was further transformed into Escherichia coli BL21 to induce the expression of the protein.The purified p54-2 protein was used to immunize mice to obtain p54-2 polyclonal antibodies.The purified polyclonal antibodies were titrated and characterized by ELISA and Western blot.The results showed that the pGEX6p-1-ASFV-P54-2 recombinant plasmid was successfully constructed.Under the induction of IPTG in Escherichia coli,the plasmid can express recombinant p54-2 protein efficiently.The result of ELISA showed that the titer of the polyclonal antibodies was 1∶128000,which indicated that the protein had good immunogenicity.The results of Western blot showed that the prepared polyclonal antibodies can specifically recognize the intracellular region of p54 in African swine fever virus,which indicated that the protein and polyclonal antibodies can be used for development of ELISA kit.