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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 the human cytomegalovirus UL32 and UL44 truncated genes by polymerase chain reaction (PCR) and construct the expression vector pET-32a-UL32-UL44. The recombinant plasmid was transformed into E. coli BL21 (DE3) Purified, labeled with HRP for detection of IgM antibodies. Methods Based on the pp150 and pp52 protein sequences of HCMV in Gene-bank, Protean computer software was used to analyze the hydrophobicity and antigenicity of HPMV. The suitable segments were selected and designed according to the nucleotide sequence of UL32 and UL44. After sequencing, the recombinant plasmid pET-32a-UL32-UL44 was cloned, and the recombinant plasmid was introduced into E. coli BL21 (DE3) strain to induce expression. The target protein was purified and labeled with ELISA The target protein-HRP markers such as sensitivity and specificity were evaluated. Results The recombinant plasmid pET-32a-UL32-UL44 was successfully constructed and introduced into BL21 (DE3) strain. After induction, the target protein was expressed in the supernatant. HRP was purified and labeled. The ELISA method was accurate and specific Detected HCMV serum samples, 80 serum test results showed that the sensitivity of up to 92.5%, specificity of 97.5%. Conclusion HCMV genetically engineered recombinant protein has good immunogenicity, which lays the foundation for the development of HCMV detection kit which replaces the traditional whole virus antigen with the genetically engineered recombinant antigen.