人类α型叶酸受体基因的克隆及其真核表达载体的构建

Cloning of gene of human folate receptor alpha and its construction of eukaryotic expression vector

目的 探讨如何以人类α型叶酸受体 (FOLR - 1)基因为基础构建核酸疫苗。方法 应用RT -PCR技术 ,从人类卵巢癌细胞系———SKOV3细胞中扩增FOLR - 1基因 ,插入克隆载体 pGEM -TEasy ,经DNA自动测序仪测序证实后 ,以亚克隆法构建于真核表达载体 pcDNA3.1(+) ,并使用限制性内切酶酶切鉴定。结果 从卵巢癌细胞系SKOV3中成功扩增出FOLR - 1基因 ,并克隆入 pcDNA3.1(+)载体。结论 成功构FOLR - 1的重组克隆及真核表达载体 ,为今后利用FOLR - 1进行卵巢上皮性肿瘤的免疫及导向治疗研究打下了基础。

Objective To study how to construct polynucleotide vaccine based 013.the gene of human folate receptor alpha(FOLR-1).Methods FOLR4 gene was amplified frcrn human ovarian carcinoma cell SKOV3 by RT-PCR.The product of PCR was ligated into pGEM-T Easy vector after restriction endonuclease digestion.Gene fragment encoding FOLR-1 was correctly ins^ted into the vector,which Was determined by an automated DNA sequencer and restriction endonuclease digestion,and then Was subdoned tO corresponding siteS cut with EcoR I plus Xho I of eukaryotic expression vector pcDNA3.1(+).Results FOLR-1 gene(774 bp)Was successfully oanstructed.And the gene sequence cloned into pcDNA3.1(+)was consistent with the known sequence after determination.Conclusion The recombinant human FOLR-1 gene clone has been established successfully and thus provides a basis for further research of the immunotherapy or targeting therapy for ovarian carcinoma.