非洲猪瘟病毒EP153R蛋白的原核表达及其多克隆抗体的制备

试验旨在构建非洲猪瘟病毒（African swine fever virus，ASFV）EP153R基因原核表达系统，表达EP153R重组蛋白（rEP153R），并制备抗rEP153R的多克隆抗体。根据ASFV Georgia 2007/1的EP153R基因序列（GenBank登录号：FR682468.1）合成EP153R基因序列，并将其插入pET-28a载体，构建pET-28a-EP153R重组质粒，经双酶切和测序鉴定后，将重组质粒转化大肠杆菌BL21（DE3）感受态细胞，分别在16和37 ℃条件下经IPTG诱导12 h进行最适诱导温度筛选。在最适温度下进行重组蛋白的表达，并利用SDS-PAGE、Western blotting进行初步鉴定。切取SDS-PAGE蛋白胶25 ku处条带，利用液相色谱－质谱联用技术鉴定重组蛋白是否正确表达。用纯化的rEP153R免疫小鼠，制备抗rEP153R的多克隆抗体，通过间接ELISA检测其抗体效价，Western blotting鉴定抗体特异性。结果显示，重组菌在16 ℃表达量较高，并以包涵体形式表达，产物在25 ku处出现明显的条带，与预期蛋白大小相符，表明rEP153R成功表达。液相色谱－质谱联用技术鉴定结果显示，匹配到YIGLINK、NESVLLR和KYIGLINK 3个rEP153R的特异性肽段，证实该蛋白为rEP153R。间接ELISA检测多克隆抗体效价为1∶128 000；Western blotting结果显示，制备的抗体具有较好的特异性。以上结果表明本研究成功表达了rEP153R，并制备了其特异性抗体，为进一步研究该蛋白生物学功能、非洲猪瘟活载体疫苗鉴定等提供了技术支持和材料。

Prokaryotic Expression of EP153R Protein of African Swine Fever Virus and Preparation of Its Polyclonal Antibodies

The aim of this study was to construct the prokaryotic expression system of EP153R gene of African swine fever virus (ASFV),express the recombinant protein EP153R (rEP153R),and prepare the polyclonal antibody against rEP153R.EP153R gene sequence was synthesized according to EP153R gene sequence of ASFV Georgia 2007/1 (GenBank accession No.:FR682468.1),and inserted into pET-28a vector to construct pET-28a-EP153R recombinant plasmid.After identification by double enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3) competent cells,which were induced by IPTG at 16 and 37 ℃ for 12 h respectively.The recombinant protein was expressed at the optimum temperature and identified by SDS-PAGE and Western blotting.The 25 ku bands of SDS-PAGE protein gel were cut,and the expression of recombinant protein was identified by liquid chromatography-mass spectrometry LC-MS.The polyclonal antibody against rEP153R was prepared by immunizing mice with purified rEP153R.The antibody titer was detected by indirect ELISA,and the specificity was identified by Western blotting.The results showed that the expression level of the recombinant bacteria was high at 16 ℃,and it was expressed in the form of inclusion body,and the product appeared obvious bands at 25 ku,which was consistent with the expected protein size,indicating that rEP153R was successfully expressed.The results of LC-MS showed that the specific peptides of YIGLINK、NESVLLR and KYIGLINK were matched,and the protein was confirmed to be rEP153R.The titer of polyclonal antibody was 1∶128 000 by indirect ELISA.Western blotting results showed that the antibody had good specificity.The above results showed that rEP153R was successfully expressed and its specific antibody was prepared,which provided technical support and material for further study of biological function of the protein and identification of live carrier vaccine of African swine fever.