人巨细胞病毒gp52蛋白基因片段的克隆及表达

Cloning and Expression of Human Cytomegavirus gp52 Gene in E. coli

目的为获得高表达人巨细胞病毒 ｇｐ５２蛋白基因的工程菌。方法通过计算机分析，筛选出巨细 胞病毒蛋白ｇｐ５２中优势抗原决定簇，用ＰＣＲ技术扩增克隆了含强抗原决定簇的基因片段。将克隆的基因片段插 入表达载体ｐＥＴ２８（ａ）内，转化大肠杆菌ＢＬ２１，经Ｓｅｐｈａｒｅｒｙ１ Ｓ－２００分子筛及Ｎｉ－ＮＴＡ Ｓｅｐｈａｒｏｓｅ螯合层析进行纯化。结 果获得的工程菌所表达的 ｇｐ５２蛋白片段相对分子质量约为 ２１ ０００，约占菌体可溶性蛋白的 ２８％。获得的表达蛋 白纯度达９５％，电泳显示单一条蛋白带，ＥＬＩＳＡ分析证实有较强的抗原性和较好的特异性。结论本研究对人巨细 胞病毒感染，尤其是对孕妇及献血员等感染的检测有重要意义。

Objective To obtain engineering E. coli for the high expression of human cytomegavirus gp52 gene. Methods The human cytomegalovirus (HCMV) gp52 gene fragment containing 2 dominant antigen dormains confirmed by computer analysis was cloned by PCR and inserted into plasmid vector pET28(a),then the recombinant plasmid was transformed to E. coli BL21 and purified by Sephacry1 S-200 and Ni-NTA Sepharose chromatographies. Results About 28% of ed soluble somatic proteins were expressed, the molecular weight of the expressed gp52 protein was abaut 21 000. After being purified, the purity of it reached 95%. SDS-PAGE profile showed a single protein band,and ELISA showed good antigenicity and specificity of the expressed protein. Conclusion The study is of great signficance in examination of HCMV, especially in pregnant women and blood donors.