检验医学与临床
檢驗醫學與臨床
검험의학여림상
JOURNAL OF LABORATORY MEDICINE AND CLINICAL SCIENCES
2014年
z1期
51-53
,共3页
分枝杆菌属%基因%聚合酶链反应%连接酶
分枝桿菌屬%基因%聚閤酶鏈反應%連接酶
분지간균속%기인%취합매련반응%련접매
mycobacterium%genes%polymerase chain reaction%ligase
目的:构建结核分枝杆菌 H37Rv 的 ligC 原核表达载体,并进行表达纯化及酶学测定。方法以结核分枝杆菌 H37Rv 基因组 DNA 为模板,PCR 法扩增 ligC 基因,构建 pQE-30-ligC 重组质粒,在表达宿主菌 E.coli M15中诱导表达蛋白,经 Ni2+-NTA 亲和层析柱纯化,最后 ATP 酶活性检测。结果成功构建了 ligC 原核表达载体 pQE-30-ligC,并能在宿主菌 E.coli M15中表达,纯化后具有 ATP 酶活性。结论LigC 基因克隆到原核宿主菌中能进行表达纯化,纯化后的蛋白具有 ATP 酶生物学活性。
目的:構建結覈分枝桿菌 H37Rv 的 ligC 原覈錶達載體,併進行錶達純化及酶學測定。方法以結覈分枝桿菌 H37Rv 基因組 DNA 為模闆,PCR 法擴增 ligC 基因,構建 pQE-30-ligC 重組質粒,在錶達宿主菌 E.coli M15中誘導錶達蛋白,經 Ni2+-NTA 親和層析柱純化,最後 ATP 酶活性檢測。結果成功構建瞭 ligC 原覈錶達載體 pQE-30-ligC,併能在宿主菌 E.coli M15中錶達,純化後具有 ATP 酶活性。結論LigC 基因剋隆到原覈宿主菌中能進行錶達純化,純化後的蛋白具有 ATP 酶生物學活性。
목적:구건결핵분지간균 H37Rv 적 ligC 원핵표체재체,병진행표체순화급매학측정。방법이결핵분지간균 H37Rv 기인조 DNA 위모판,PCR 법확증 ligC 기인,구건 pQE-30-ligC 중조질립,재표체숙주균 E.coli M15중유도표체단백,경 Ni2+-NTA 친화층석주순화,최후 ATP 매활성검측。결과성공구건료 ligC 원핵표체재체 pQE-30-ligC,병능재숙주균 E.coli M15중표체,순화후구유 ATP 매활성。결론LigC 기인극륭도원핵숙주균중능진행표체순화,순화후적단백구유 ATP 매생물학활성。
Objective To construct LigC prokaryotic expression vector from mycobacterium tuberculosis H37Rv,detect its activity.Methods LigC gene was amplified from H37Rv genomic DNA by polymerase chain reac-tion(PCR)and then cloned into expression vector pQE-30.Finally the recombinant plasmid was expressed in E.coli M15.After induction by IPTG,the fusion protein of LigC was produced,the protein was purified by Ni2+-NTA afini-ty chromatography,detected its ATPase activity.Results LigC was successfully constructed and expressed in E.coli M15.It could be purified,the purified ligase showed ATPase activity.Conclusion LigC gene could be cloned into the host bacterium and successfully expressed and purified,the purified protein was ATPase biologically active.