mGM-CSF-mMIP-3α重组质粒的构建、表达及鉴定  

Recombinant Plasmid with mGM-CSF and mMIP-3α:Construction,Expression and Identification

作　　者：刘畅[1] 孙林英[1] 刘秀丽[1] 邵晓婷[1] 谭晓华[1] 刘端祺[2] 

机构地区：[1]北京军区总医院血液科 [2]北京军区总医院肿瘤诊疗中心,北京100700

出　　处：《华北国防医药》2006年第1期1-4,F0003,共5页Medical Journal of Beijing Military Region

基　　金：国家自然科学基金资助项目(No.30271185;30371627);北京市自然科学基金资助项目(No.7042062)

摘　　要：目的:构建pCDNA3.1mGM-CSF-mM IP-3α重组质粒,为mGM-CSF-mM IP-3α基因治疗肿瘤的研究奠定基础。方法:利用本实验室保存的pCDNA3.1mGM-CSF和pCDNA3.1mM IP-3α质粒,采用分子生物学技术构建了pCDNA3.1mGM-CSF-mM IP-3α重组质粒。用PCR、酶切进行鉴定,然后用脂质体转染B16细胞,用G418筛选后通过ELISA和W estern b lot检测该融合蛋白。结果:重组质粒中含有mGM-CSF-mM IP-3α基因,转染B16细胞后,其表达产物能分泌到肿瘤细胞外,分泌到细胞外的产物用ELISA和W esternb lot检测证明能与特异性mGM-CSF和mM IP-3α抗体结合。结论:成功构建含mGM-CSF-mM IP-3α真核表达重组质粒,有助于进一步研究其抗肿瘤作用。Objective：To construct recombinant plasmid pCDNA3, 1mGM-CSF-mMIP-3α and lay a primary foundation for further study of mGM-CSF-mMIP-3α gene therapy of tumors. Methods：pCDNA3.1 mGM-CSF plasmid and pCDNA mMIP-3α plasmid in our laboratory were constructed through molecular biology technology into recombinant pCDNA3, lmGM CSF mMIP-3α plasmid, The recombinant plasmid was then transfected into B16 cell line via Iipsome after being identified through PCR, restriction enzyme digesting and DNA sequencing. The transfeeted cells,B16,were screened by C418. And then ELISA and Western blot were performed to identify the fusion protein. Results：The recombinant plasmid contained mGM-CSF-mMIP-3α gene,which was expressed in B16/G418 cells in vitro. The expressed protein was secreted out of the ceils, The protein was proved to bind with specific antibodies against mGM-CSF and mMIP-3α. Conclusions：The successful eukaryotic expression of recombinant plasmid pCDNA3.1 mGM-CSF-mMIP3α would be helpful to the further study on its anti-tumor effect.

关 键 词：粒细胞-巨噬细胞集落刺激因子 重组 巨噬细胞爽性蛋白3α 重组质粒 基因疗法 

分 类 号：R392.1[医药卫生—免疫学]