外源性重组激活基因在人类细胞中的表达研究

Study on expression of exogenous recombination activated genes in human cells

目的:通过恢复RAG-1、RAG-2分子的表达挽救功能性淋巴细胞的分化、发育,探讨从基因水平上根治RAG-免疫缺陷病的技术方法,为进一步进行基因治疗打下基础。方法:(1)采用RT-PCR方法克隆人类RAG-1、RAG-2编码基因,同时构建RAG-1、RAG-2真核表达载体;(2)采用转染方法得到转染细胞体,使目的基因在人类细胞得到表达。结果:阳性克隆的测序结果与人类RAG-1、RAG-2基因完全相同,并且分别在获得重组细胞中检测到782 bp的人类RAG-1 mRNA转录产物和378 bp的人类RAG-2 mRNA转录产物,而pcDNA3.1空白对照未检测到任何hRAG-1 mRNA。结论:成功克隆了人类RAG-1、RAG-2基因,并在转染的人类细胞中获得了外源RAG-1、RAG-2的mRNA表达。

Objective:Rescuing differentiation and development of functional lymphocytes by restoring expressions of RAG-1 and RAG-2 molecules,and discussing the technical methods to radically cure RAG-immunodeficiency at the genetic level,lay the foundation for further gene therapy.Methods:(1) Human RAG-1 and RAG-2 coding genes were cloned by RT-PCR method,and eukaryotic expression vectors for RAG-1 and RAG-2 were constructed at the same time;transfected cells were obtained by transfection,genes were expressed in human cells.Results:Positive clones were sequenced and the results were identical to human RAG-1 and RAG-2 genes,and 782 bp of human RAG-1 mRNA transcripts and 378 bp of human RAG-2 mRNA transcripts were detected in obtained recombinant cells,however,no HRAG-1 mRNA was detected in pcDNA3.1 blank control.Conclusion:Human RAG-1 and RAG-2 genes were successfully cloned,and obtained exogenous RAG-1 and RAG-2 mRNA expression in transfected human cells.