人巨细胞病毒pp65基因片段的原核表达及鉴定

目的构建人巨细胞病毒(HCMV)pp65基因片段(363～505位氨基酸)的原核表达质粒,并鉴定所表达蛋白的反应原性。方法以含有HCMVpp65全长基因的pGEM-T-pp65质粒为模板,PCR扩增pp65基因第1087～1515位核苷酸片段,酶切后插入pET-21a(+)载体,转化大肠杆菌BL21(DE3)。经IPTG诱导表达,纯化后进行Western blot鉴定。结果酶切分析和测序证明原核表达质粒pET-21a(+)-pp65构建正确。表达的融合蛋白主要以包涵体形式存在,以1.0mmol/L IPTG诱导5h,目的蛋白的表达量最高。纯化后的蛋白具有良好的反应原性。结论已成功构建了HCMV pp65基因片段原核表达质粒,并在大肠杆菌中表达了融合蛋白。

Prokaryotic Expression of Human Cytomegalovirus pp65 Gene Fragment and Identification of Expressed Product

Objective To construct the prokaryotic expression vector for human cytomegalovirus(HCMV) pp65 gene fragment and test for the reactogenicity of express product. Methods Amplify the nucleotides 1087-1515 of HCMV pp65 gene by PCR using plasmid pGEM-T-pp65 containing the full-length of HCMVpp65 gene as template, identify by restriction analysis and insert into vector pET-21a(+). The constructed recombinant plasmid pET-21a(+)-pp65 was transformed to E. coli DH5α, and positive clones were screened and identified by PCR, from which recombinant plasmid was extracted and identified by sequencing, then transformed to E. coli BL21(DE3) for expression under induction of IPTG. Purify the expressed product by affinity chromatography and identify by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pET-21a(+)-pp65 was constructed correctly and the expression level of target protein reached a peak value after induction with 1. 0 mmol / L IPTG for 5 h. The expressed fusion protein mainly existed in a form of inclusion body, and showed good reactogenicity after purification. Conclusion The prokaryotic expression vector for HCMV pp65 was successfully constructed and expressed in E. coli.