摘要
目的构建人黏蛋白1串联重复区(MUC1 VNTR)17重复序列肿瘤抗原真核表达载体,并在COS-7、HeLa和Lewis细胞中表达。方法设计1对分别含SalⅠ和XhoⅠ酶切位点的引物,PCR法合成1重复MUC1 VNTR片段,克隆入pGEM-Teasy载体,依据SalⅠ和XhoⅠ酶切产生相同黏性末端的特性,依次连接,构建17重复MUC1 VNTR(17m),再亚克隆至VR1012真核表达载体,构建真核表达质粒VR1012-17m。分别转染COS-7、HeLa和Lewis细胞,并进行检测。结果酶切鉴定和序列分析证实,构建的重组质粒含17m序列,经转染的COS-7、HeLa和Lewis细胞,RT-PCR检测均有17m mRNA转录,Western blot检测均有17m蛋白的表达,并与MUC1 VNTR单抗产生特异性反应。结论已成功构建17m肿瘤抗原真核表达载体,并在COS-7、HeLa和Lewis细胞中得到表达,可用于MUC1疫苗和肿瘤生物学治疗的进一步研究。
Objective To construct the eukaryotic expression vector of gene encoding 17 tandem repeats of human mucin 1 （ 17MUC1 VNTRs） ,and express the gene in COS-7 ,HeLa and Lewis cells. Methods Synthesize 1MUC1 VNTR by PCR using the designed primer and clone into pGEM-T easy vector. Digest the constructed recombinant plasmid with Sal I and Xho I , and ligate the di- gested product in turn to construct 17MUC1 VNTRs which was subcloned to eukaryotic expression vector VR1012. Transfect COS-7, HeLa and Lewis cells with the constructed recombinant VR1012-17m,then test for the transcription of mRNA by RT-PCR and the expression of 17m antigen protein by Western blot. Results Both restriction analysis and sequencing proved that the constructed recombinant plasmid VR1012-17m contained 17m gene. RT-PCR showed transcription of 17m mRNA in transfected COS-7, HeLa and Lewis cells. 17m protein was expressed in all the transfected cells and showed specific reaction with McAb against MUC1 VNTR,as proved by Western blot. Conclusion The eukaryotic expression vector for 17m tumor antigen was successfully constructed and expressed in COS-7, HeLa and Lewis cells,which laid a foundation of further study on MUC1 vaccine and biotherapy of tumors.
出处
《中国生物制品学杂志》
CAS
CSCD
2007年第9期642-645,共4页
Chinese Journal of Biologicals
