人巨细胞病毒UL32和UL44融合基因的表达及其抗原性分析

Cloning and expression of UL32 gene and UL44 gene of HCMV and its antigenicity

目的用聚合酶链式反应(PCR)扩增人巨细胞病毒UL32、UL44截短基因,构建pET-32a-UL32-UL44表达载体,转化宿菌大肠杆菌BL21(DE3)进行表达,对目的蛋白进行纯化、标记HRP,用于检测IgM抗体。方法根据Gene-bank中HCMV的pp150、pp52蛋白序列,利用Protean计算机软件分析其亲疏水性和抗原性,选取适合的区段,根据其对应的UL32、UL44基因核苷酸序列设计合成引物,PCR扩增目的基因序列,经测序证明无误之后,克隆pET-32a-UL32-UL44重组质粒,将重组质粒导入大肠杆菌BL21(DE3)菌株,进行诱导表达,纯化目的蛋白,标记HRP,用ELISA捕获法对目的蛋白-HRP标记物的灵敏性和特异性等指标进行评价。结果成功构建pET-32a-UL32-UL44重组质粒,导入在BL21(DE3)菌株中,经诱导后目的蛋白表达于上清中,经纯化、标记HRP,标记物用于ELISA捕获法能够准确、特异地检测HCMV血清标本,80份血清检测结果显示其灵敏度可达92.5%,特异性达97.5%。结论 HCMV基因工程重组蛋白具有较好的免疫活性,为研制以基因工程重组抗原代替传统全病毒抗原的HCMV检测试剂盒打下了基础。

Objective To amplify and the construct truncated fragments of UL32, UL44 gene of human ctomegalovirus （HCMV）, construct the pET-32a-UL32-UL44 prokaryotic expression vector and prepare recombinant antigen for detection of IgM antibody in human serum. Methods According to the gene sequence of HCMV pp15-0, pp52 in the Genebank, the hydrophobicity and amigenicity of HCMV were analyzed by using protean computer software. The appropriate section corresponding to the nueleotide sequence of UL32 and UIA4 gene was selected, PCR primers was designed and synthesized. A PCR amplification was executed. After the confirmation of gene sequences, the target protein was expressed, labeled by HRP and used for determine the sensitivity and specificity by the antibody-capture enzyme-linked immunosorbent assay （AC-ELISA）.Results Recombinant plasmid pET-32a-UL32-UL44 was successfully constructed, transferred into B-L21 （DE3） strain, the induced target protein was in the supernatant, after purification, labeled by HRP, and tested by capture ELISA .80 human serum were tested. The sensitivity and specificity recombinant antigen were 92.5% and 97.5% respectively. Conclusion The cloned HCMV truncated chimeric gene fragments and the protein possess a relatively good immunological reactivity to have set foundation for substitution of traditional whole virus antigen with recombinant genetic antigen..