【摘要】 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/ORF₇₃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。更多还原

【Abstract】 Objective To construct the prokaryotic expression plasmid for pET28a open read- ing frame（ORF）₇₃,and to purify and identify its fusion protein.Methods Using recombinant plas- mid pGEM-Teasy/ORF₇₃as the template,EcoRⅠand XhoⅠrestriction digestion sites were ampli- fied by PCR and subsequently inserted into prokaryotic vector,pET-28a（+）.The recombinant plas- mid was transformed into E.coli BL21（DE3）and then identified by double enzyme digestion and se- quencing.The fusion protein was expressed by induction with isopropy-β-D-thiogalactoside（IPTG）. The expressed recombinant protein was purified using polyadenylic histiding affinity chromatography. Purification of the target protein was confirmed by Western blot.Results The insert target DNA was 453 bp.The fusion protein was relative molecular mass 20 000 by SDS-PAGE comfirmation and then recog- nized by serum samples from patients with Kaposi’s sarcoma.Showed good imrnunication distinguish in Western blot analysis.Conclusions pET28a-ORF₇₃was successfully constructed and ORF₇₃gene encode fusion protein was qualified for the further lab comfirmation.更多还原