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中国癌症防治杂志

中国癌症防治杂志 ›› 2012, Vol. 4 ›› Issue (4): 311-315.doi: 10.3969/j.issn.1674-5671.2012.04.03

• 基础研究 • 上一篇    下一篇

肝癌相关抗原H2-calponin基因重组质粒的构建与表达

  

  1. 广西医科大学研究生学院；广西医科大学生物化学与分子生物学教研室
  • 出版日期:2012-12-25 发布日期:2013-01-11
  • 通讯作者: 周素芳 E-mail:zfs20000@yahoo.com.cn
  • 基金资助:

    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）

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

  • Online:2012-12-25 Published:2013-01-11

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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CNN2 cDNA序列，用基因工程技术将CNN2基因重组到表达质粒pCold Ⅲ中，转化BL21（DE3）pLysS宿主菌，以SDS-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamidegel electrophoresis，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pCold Ⅲ-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。

关键词: 肝肿瘤, CNN2, 基因工程, 表达, 纯化

Abstract: 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 conditions，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 His-CNN2 fusion，suggesting that the overexpressed protein retains its immunogenicity. Conclusion The pCold III-CNN2 expression plasmid was successfully constructed and antigenic CNN2 protein was obtained.

Key words: Liver neoplasms, CNN2, Genetic engineering, Expression, Purification