肝癌相关抗原H2-calponin基因重组质粒的构建与表达  

Construction and expression of hepatocellular carcinoma-associated antigen H2-calponin

作　　者：范升龙[1] 黄朋[1,2] 涂镇波[1] 李玲[1] 张胜昌[1] 陆巧巧[1] 赵赫[1] 周素芳[3] 

机构地区：[1]广西医科大学研究生学院,南宁530021 [2]广西壮族自治区妇幼保健院遗传代谢研究室 [3]广西医科大学生物化学与分子生物学教研室

出　　处：《中国癌症防治杂志》2012年第4期311-315,共5页CHINESE JOURNAL OF ONCOLOGY PREVENTION AND TREATMENT

基　　金：国家自然科学基金资助项目(81160362);广西自然科学基金资助项目(2011GXNSFA018261);广西科学研究与技术开发计划项目(桂科攻10124001A-2)

摘　　要：目的应用基因工程的方法构建H2-calponin(CNN2)基因工程表达质粒,体外表达、分离、纯化CNN2蛋白。方法以PCR扩增CNN2cDNA序列,用基因工程技术将CNN2基因重组到表达质粒pColdⅢ中,转化BL21(DE3)pLysS宿主菌,以SDS-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamideg elelectrophoresis,SDS-PAGE)检测目的蛋白的表达情况,优化表达条件,以较大体积培养基因工程菌,菌体经超声波破菌后,过Ni-NTA亲和柱纯化,Western-blot检测表达纯化的融合蛋白。结果转化有pColdⅢ-CNN2重组质粒的基因工程菌能诱导表达CNN2蛋白,表达的目的蛋白占总菌体蛋白的35%;SDS-PAGE检测发现菌体上清液和沉淀物中均有目的蛋白片段,证实部分CNN2蛋白以可溶性形式表达;Western-blot检测结果提示表达纯化的蛋白能与抗CNN2抗体发生反应。结论成功构建出表达可溶性CNN2蛋白的重组质粒,诱导表达的蛋白具有CNN2免疫原性。Objective To construct a pCold III-hepatocellular carcinoma-associated antigen H2-calponin （CNN2） expression plasmid, and to express and purify CNN2 from bacteria. Methods CNN2 cDNA was amplified and constructed into the pCold III expression vector.The resulting pCold III-CNN2 expression plasmid was transformed into E. coli BL21 （DE3）pLysS; optimal conditions for culturing E. coli BL21 （DE3） pLysS/pCold III-CNN2 and for inducing CNN2 expression were explored.SDS-PAGE was used to track the HIS- CNN2 fusion, which was purified on Ni-NTA resin. The identity of the purified protein was checked by Western blotting with anti- CNN2 antibody. Results CNN2 protein was overexpressed in E.coli BL21 （DE3） pLysS/pCold III-CNN2.Under optimal culture and induction conditlons,the expressed protein HIS-CNN2 accounted for 35% of total bacterial protein. The fusion protein was found in both the sol-uble and insoluble fractions of cell lysate,indicating that CNN2 protein was expressed in soluble form.Western blotting with anti-CNN2 antibody detected the purified 14is-CNN2 fusion,suggestlng thai the overexpressed protein retains its immunogenicity. Conclusion The pCold III-CNN2 expression plasmid was successfully constructed and antigenic CNN2 protein was obtained.

关 键 词：肝肿瘤 CNN2 基因工程 表达 纯化 

分 类 号：R735.7[医药卫生—肿瘤]