人巨细胞病毒UL135蛋白的原核表达及其多克隆抗体的制备和鉴定

Prokaryotic expression of UL135 protein of human cytomegalovirus and its preparation and identification of polyclonal antibody

目的:制备人巨细胞病毒(HCMV)UL135原核表达蛋白及其多克隆抗体。方法:生物信息学分析HCMV UL135蛋白的跨膜区结构,经原核密码子优化后对胞外序列进行全基因合成,将序列克隆至原核表达载体构建pET21a(+)/HCMV UL135重组质粒,IPTG诱导蛋白表达后,Ni-NTA亲和层析法纯化UL135重组蛋白,免疫新西兰兔制备多克隆抗体,ELISA、Western blot法检测抗体灵敏度和特异性。结果:HCMV的UL135基因在原核表达系统中成功表达,经Ni-NTA亲和纯化后获得高纯度的目的蛋白;免疫新西兰兔诱导产生特异性的多克隆Ig G抗体,且效价为1:12 800;经ELISA以及Western blot证实制备的兔抗多克隆抗体可特异性识别UL135蛋白。结论:成功实现了HCMV UL135的重组表达并制备了UL135兔多克隆抗体,为进一步研究UL135蛋白的生物学功能奠定基础。

Objective： To construct a prokaryotic expression vector expressing UL135 protein of human cytomegalovirus （HCMV） and prepare a UL135 specifc polyclonal antibody. Methods： Bioinformatics methods were used to analyze the transmembrane domain of UL135 protein, and then the cytoplasmic domain synthesized by fully-synthetic gene was cloned into vector pET21a （＋） after the prokaryotic codon optimization. The pET21a （＋）/UL135 recombinant plasmid was transformed into E.coli BL21 （DE3） and then induced by isopropyl β-D-1-thiogalactopyranoside （IPTG）. To prepare specifc polyclonal antibody, New Zealand rabbits were immunized by the UL135 recombinant protein which was purifed by Ni-NTA affnity chromatography primarily. The sensitivity and the specifcity of the antibody were determined by ELISA and Western blot assays. Results： HCMV UL135 protein was successfully expressed in prokaryotic system and highly purifed target protein was obtained through Ni-NTA affnity chromatography. New Zealand rabbits could generate specifc polyclonal IgG antibody after im-munized. ELASE and Western blot analysis determined that the preparative polyclonal IgG antibody could spe-cifcally identify the UL135 protein. Conclusion： HCMV UL135 protein is successfully expressed in prokaryotic expression system, and anti-UL135 rabbit polyclonal antibody is successfully prepared for the further research.