摘要
目的构建含目的基因多亮氨酸重复区免疫球蛋白样区1(LRIG1)的重组质粒pEGFP-C1-LRIG1,为胶质瘤等多种上皮源性肿瘤的分子治疗奠定基础。方法采用RT-PCR方法,从人全血总RNA中,扩增出3 282bp的LRIG1cDNA片段,再用XhoⅠ和ClaⅠ双酶切后,定向克隆到真核细胞表达载体pEGFP-C1中,用限制性内切酶酶切分析和DNA序列分析鉴定重组质粒;免疫细胞化学法检测LRIG1基因的表达情况。结果人LRIG1基因的cDNA已克隆到真核细胞表达载体pEGFP-C1质粒中;经脂质体转染SHG44细胞后,G418进行筛选,可见转染细胞胞膜上有大量LRIG1蛋白表达。结论成功构建了pEGFP-C1-LRIG1的真核表达载体,为研究LRIG1基因在胶质瘤等多种上皮源性肿瘤中的作用和其治疗奠定一定的实验基础。
Objective To generate eukaryotic expression vector of pEGFP-C1-lecocine-rich repeats and immunoglobulin-like domain 1（LRIG1） and obtain its stable expression in SHG44 cells so as to lay the foundation for molecular therapy of glioma and other tumors of epithelial origin.Methods A 3 282 bp cDNA fragment was amplified from the total RNA of the human whole blood cells by RT-PCR method and cloned into the plasmid pEGFP-C1.The vector was identified by the double digestion with restriction enzymes XhoⅠ and ClaⅠ,and was sequenced by the Sanger-dideoxy-mediated chain termination.The expression of the LRIG1 gene was detected by immunocytochemistry.Results The results showed that the cDNA fragment included 3 282 bp total coding region.The recombinant eukaryotic cell expression vector of pEGFP-C1-LRIG1 was constructed successfully,and the sequence of insert was identical to the published sequence.The SHG44 cells transfected with the pEGFP-C1-LRIG1 plasmid expressed a high level of the LRIG1 protein in the cytoplasm.Conclusion The recombinant plasmid pEGFP-C1-LRIG1 can provide a very useful tool for research on LRIG1 gene＇s role in glioma and other tumors of epithelial origin and lay the experimental foundation for the treatment.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2013年第4期541-544,共4页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
陕西省科技计划项目(No.2009K12-02)~~
