血管内皮生长因子-C表达载体的构建和原核表达  

Prokaryotic Expression of Vascular Endothelial Growth Factor-C

作　　者：潘剑[1] 温玉明[1] 华成舸[1] 李珉[2] 王刚[2] 王昌美[1] 李龙江[1] 陈绍维[1] 

机构地区：[1]四川大学华西口腔医院口腔颌面外科,610041 [2]四川大学生命科学院

出　　处：《华西口腔医学杂志》2003年第4期321-323,共3页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目 (编号 39970 796)

摘　　要：目的　探讨从人舌癌组织中克隆到的血管内皮生长因子_C(vascularendothelialgrowthfactor,VEGF_C)片段cDNA表达载体在原核细胞中的功能表达 ,为进一步研究VEGF_C在口腔鳞癌中表达的意义及与淋巴转移的关系打下基础。方法　以RT_PCR法从舌鳞癌组织中克隆的VEGF_C基因功能片段cDNA与表达载体pBK_CMV构建表达质粒 ,转化并诱导其在大肠杆菌中的表达 ,并通过SDS_PAGE及Western_blotting进行检测。结果　从人舌癌组织总RNA中克隆到约 1 1kb大小的VEGF_C基因cDNA ,与Genbank公布的人VEGF_C基因cDNA序列有 99 6 %同源性 ,以之构建出的重组质粒pBK_VEGF_C转染大肠杆菌经IPTG诱导后 ,检测到稳定表达的相对分子质量约5 6 0 0 0的融合蛋白 ,该融合蛋白与VEGF_C多抗具有强阳性免疫印迹反应。结论　证实克隆得到VEGF_C基因功能片段cDNA可在原核细胞中成功表达 ,为进一步研究VEGF_C在口腔癌颈淋巴结转移中的功能效应提供了物质基础。Objective To evaluate whether the vascular endothelial growth factor (VEGF)-C cDNA which cloned from a patient with squamous cell carcinoma (SCC) of tongue can encode a functional protein or not.Methods RT-PCR was employed to clone the functional VEGF-C fragment from the surgical specimen of a lingual SCC patient. Then it was subcloned into expressive plasmid vector pBKCMV, which was transfected into E.coli to examine its expression. Results A truncated human VEGF-C cDNA fragment was amplified from the lingual SCC. The sequencing results of the fragment demonstrated that it had 99.6% similarity with the reported human VEGF-C cDNA (representing the 559～1611 bp according the sequence of Genbank Entry X94216). Induced with IPTG, the E.coli XL1-Blue MRF′ containing the recombinant pBK-VEGF-C expressed a 56 000 fusion protein, which can be recognized by polyclonic anti-human VEGF-C antibody.Conclusion A functional fragment VEGF-C cDNA was cloned from a lingual SCC. It will promote more intensive research on the function of VEGF-C and its relationship with metastasis of oral SCC.

关 键 词：血管内皮生长因子-C 表达载体 构建 原核表达 舌鳞癌组织 

分 类 号：R346[医药卫生—基础医学]