人CⅡTA基因不同单倍型cDNA真核表达载体的构建

目的 构建人MHCⅡ类反式激活因子（CⅡTA）基因3种不同单倍型cDNA的真核表达载体。方法 以野生型重组质粒EBS-NPL-CⅡTA cDNA为模板,用重叠延伸PCR定点突变技术对CⅡTA基因编码区的两个非同义单个核苷酸多态性（SNP）位点进行突变,制备CⅡTA基因另外3种单倍型CDNA的部分片段。将其分别克隆至EBS-NPL-CⅡTA酶切后的线性化载体上,通过菌落PCR及酶切鉴定获得阳性克隆,并对其进行测序鉴定。然后将4种单倍型的真核表达载体和空载体EBS-NPL分别转染至HepG2细胞,用间接细胞免疫荧光技术检测其HLA-DR分子的表达。结果 成功制备人CⅡTA基因3种单倍型CDNA的部分片段,并获得含有CⅡTA基因3种不同单倍型cDNA完整序列的真核表达载体。证实未经转染和转染空载体的HepG2细胞无HLA-DR分子的表达,转染4种真核表达载体的HepG2细胞均可表达HLA-DR分子。结论 成功构建人CⅡTA基因不同单倍型的真核表达载体,为研究CⅡTA基因不同单倍型功能之间的差异奠定基础。

Construction of eukaryotic expression vectors containing different haplotype cDNA of human CⅡTA gene

Objective To construct eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene. Method cDNA fragments of three different CIITA haplotypes were obtained by inducing one or two single nucleotide mutations of wild type recombinant plasmid EBS-NPL-CIITA cDNA, which correspond to two non-homonymy single nucleotide polymorphism (SNP) sites in the coding region of human CIITA gene, using overlap extension PCR site-directed mutagenesis technology. The above-mentioned three haplotype cDNAs were respectively cloned to EBS-NPL-CIITA linearized vectors. Positive clones were identified by colonial PCR and restriction endonuclease digestion and were sent to be sequenced. Then eukaryotic expression vectors containing four different haplotypes and an empty vector EBS-NPL were transfected into HepG2 cells respectively. HLA-DR was detected by indirect cell immunof-luorescence technique. Results The cDNA fragments of three different human CIITA haplotypes were successfully constructed, and the eukaryotic expression vectors containing three different haplotype cDNAs of human CIITA gene were obtained. No expression of HLA-DR was observed in the original HepG2 cells and empty vector transfected HepG2 cells and the expression of HLA-DR emerged in the HepG2 cells transfected with four eukaryotic expression vectors. Conclusion The eukaryotic expression vectors containing three different haplotype cDN As of human CIITA gene were successfully constructed, and they are essential for our further study of the functional differences of them.