论文部分内容阅读
目的构建人类8型疱疹病毒(HHV-8)基因开放读码框(ORF)_(73C)重组原核表达质粒并诱导表达,获得HHV-8潜伏态相关核抗原重组蛋白,并对其免疫活性进行初步鉴定。方法软件设计引物,分别在引物两端添加特异的酶切位点,以pGEM-Teasy/ORF_(73)质粒为模板,聚合酶链反应(PCR)扩增ORF_(73C)基因序列,克隆入原核表达载体pET-28a(+),构建原核表达质粒pET28a- ORF_(73C)；转化E．coli DH_(5α),经酶切、测序鉴定其插入序列的正确性,转入E．coli BL21(DE3),并诱导表达,以多聚组氨酸亲合层析柱纯化,凝胶蛋白电泳、Western印迹方法行表达蛋白的分析和抗原特异性免疫鉴定。结果测序结果表明构建的pET28a-ORF_(73C)原核表达质粒连接正确,插入的ORF_(73C)基因片段为453 bp。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)表明ORF_(73C)基因成功表达,在相对分子质量20000处有表达条带；Western印迹分析显示重组蛋白能被卡波济肉瘤患者阳性血清识别,具有良好的抗原性。结论成功构建了pET28a-ORF_(73C)原核表达质粒,获得的纯化HHV-8潜伏态相关核抗原蛋白与卡波济肉瘤患者血清具有特异性识别,显示其作为检测抗原的可能,但其敏感性和特异性则有待进一步的验证。
Objective To construct a recombinant prokaryotic expression plasmid of human herpesvirus type 8 (HHV-8) gene and to induce the expression of HHV-8 latent-related nuclear antigen recombinant protein, and to study its immunocompetence Preliminary identification. Methods Primers were designed by software to insert specific restriction sites at both ends of the primer. The pGEM-Teasy / ORF_ (73) plasmid was used as a template to amplify the ORF_ (73C) gene sequence by polymerase chain reaction (PCR) The expression vector pET-28a (+) was constructed and the prokaryotic expression plasmid pET28a-ORF_ (73C) was constructed. coli DH_ (5α), the correctness of the inserted sequence was identified by enzyme digestion, sequencing, transferred to E. coli BL21 (DE3) was induced and induced to express. The recombinant protein was purified by polyhistidine affinity chromatography, gel electrophoresis and Western blotting. Results The sequencing results showed that the prokaryotic expression plasmid pET28a-ORF_ (73C) was correctly ligated and the inserted ORF_ (73C) gene fragment was 453 bp. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that ORF_ (73C) gene was expressed successfully and expressed at a molecular weight of 20000. Western blot analysis showed that recombinant protein could be detected by Kaposi’s sarcoma Patients positive serum identification, has good antigenicity. Conclusion The prokaryotic expression plasmid pET28a-ORF_ (73C) was successfully constructed and the purified HHV-8 latent nuclear protein correlated specifically with the serum of Kaposi’s sarcoma, which indicated that it could be used as an antigen. However, its sensitivity And specificity is subject to further validation.