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.
出处
《中国癌症防治杂志》
CAS
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)
关键词
肝肿瘤
CNN2
基因工程
表达
纯化
Liver neoplasms
CNN2
Genetic engineering
Expression
Purification
