【摘要】 目的人免疫缺陷病毒(human immunodeficiency virus,HIV)-1逆转录酶是由p66和p51亚单位组成的异二聚体,抑制其活性可阻断HIV-1的复制,是治疗获得性免疫缺陷综合征(acquired immunodeficiency syndrome,AIDS)的重要靶点。获得大量纯化的HIV-1 p66亚单位蛋白有助于研究其活性,为抗HIV-1药物研究提供药物筛选平台。文中构建HIV-1逆转录酶基因的原核表达系统,获取高纯度高活性的HIV-1 p66亚单位重组蛋白。方法用PCR从含HIV-1 HXB2标准株全基因的质粒中扩增出编码HIV逆转录酶p66亚单位的基因,通过酶切、连接等方法构建重组质粒pET-28a(+)-p66,并转化到宿主菌大肠埃希菌BL21(DE3)中,用异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达。表达产物经镍柱亲和层析纯化后获得HIV逆转录酶p66亚单位蛋白。结果所构建了pET-28a(+)-p66质粒酶切片段为1680 bp,测序符合HIV逆转录酶p66亚单位编码基因序列。在IPTG诱导下表达相对分子质量为66 000的HIV逆转录酶p66亚单位蛋白。结论成功构建了pET-28a(+)-p66质粒,具有较高的HIV逆转录酶p66亚单位蛋白表达效率及活性。更多还原

【Abstract】 Objective Reverse transcriptase,a heterodimer composed of subunits p66 and p51,is considered as an important target for AIDS therapy.HIV-1 replication can be blocked by inhibition of the reverse transcriptase activity.The obtainment of purified HIV-1 p66 protein will help to study its activity and develop a screening assay for anti-HIV-1 drugs.In the present study,we constructed a p66 prokaryotic expression plasmid,and obtained highly pure and active HIV-1 p66 recombinant protein.Methods The sequence encoding HIV-1 p66 protein was amplified by PCR from the plasmid containing the HIV-1 HXB2 genome,and then cloned into the pET-28a(+) vector by restriction enzyme digestion and ligation to construct the prokaryotic expression plasmid pET-28a(+)-p66.Then it was transformed into E.coli BL21(DE3) strain.IPTG was used to induce the expression of p66.Finally,nickel affinity gel was used to purify the p66 protein.Results A 1680 bp fragment was identified by restriction enzyme digestion.The sequencing result indicated that the HIV-1 p66 gene was cloned into pET-28a(+) correctly.Conclusion The prokaryotic expression plasmid pET-28a(+)-p66 was constructed successfully.Highly expressed and active HIV-1 p66 recombinant protein was obtained.更多还原