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,温玉明,华成舸,李珉,王刚,王昌美,李龙江,陈绍维
Author：PANJian*,WEN Yuming,HUAChengge,et al.
收稿日期：2003-08-25 年卷（期）页码：2003,21(04):321-
期刊名称：华西口腔医学杂志
Journal Name：West China Journal of Stomatology
关键字：血管内皮生长因子-C,克隆,原核表达,
Key words：vascular endothelial growth factor (VEGF)-C,clone,prokaryotic expression,
基金项目：本课题为国家自然科学基金资助项目(编号39970796)
中文摘要
目的 探讨从人舌癌组织中克隆到的血管内皮生长因子-C(vascular endothelial growth factor,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1·1 kb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 56 000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。
英文摘要
Objective To evaluate whether the vascular endothelial growth factor (VEGF)-C cDNAwhich cloned from a patient with squamous cell carcinoma (SCC) of tongue can encode a functional protein or not.Methods RT-PCRwas employed to clone the functional VEGF-C fragment from the surgical specimen of a lingual SCC patient. Then it was subcloned into expres- sive plasmid vector pBKCMV, whichwas transfected intoE.colito examine its expression.Results Atruncated human VEGF- C cDNA fragment was amplified from the lingual SCC. The sequencing results of the fragment demonstrated that it had 99.6% similaritywith the reported human VEGF-C cDNA (representing the 559~1611 bp according the sequence of Genbank Entry X94216). Induced with IPTG, theE.coliXL1-Blue MRF′containing the recombinant pBK-VEGF-C expressed a 56 000 fusion protein, which can be recognized by polyclonic anti-humanVEGF-C antibody.Conclusion Afunctional fragmentVEGF-C cDNA was cloned from a lingual SCC. Itwill promote more intensive research on the function of VEGF-C and its relationshipwithmetas- tasis of oral SCC.
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