胰岛自身抗原GAD65片段和胰岛素B链基因共表达DNA疫苗的构建与表达

构建两种胰岛自身抗原谷氨酸脱羧酶（ glutamic acid decarboxylase, GAD） 65片段与胰岛素B链基因共表达DNA疫苗，并检测其在体外COS-7细胞中的表达。PCR方法从GAD65质粒中扩增出GAD190-385和GAD490-570两个片段的cDNA，overlap法再分别与信号肽基因进行拼接，将拼接后的融合基因SGAD190-515和SGAD490-570分别与胰岛素B链基因依次克隆入双启动子真核表达载体pBudCFA．1中。重组质粒经酶切和测序鉴定后，脂质体体外转染COS-7细胞，Western blot方法检测目的基因在该细胞中的表达。结果表明核酸序列测定克隆的融合基因和胰岛素B链基因序列与报告序列一致，开放阅读框正确，Western blot显示转染了DNA疫苗的COS-7细胞中均可检测两个目的基因的表达。两种GAD65片段与胰岛素B链基因共表达DNA疫苗均成功构建，为自身免疫糖尿病的免疫干预研究奠定了实验基础。

Construction and Expression of Co-expression DNA Vaccines Containing GAD65 Fragment and Insulin B Chain Gene

To construct two co-expression DNA vaccines containing GAD65 fragment and Insulin B Chain gene, and determine the transient expression products in COS-7 cells in vitro, the GAD190-315, GAD490-570 cDNA were amplified from GAD65 plasmid, and were linked with signal peptide cDNA through overlap PCR, respectively. Then, the two fusion gene and Insulin B Chain gene were cloned into eukaryotic expression vector pBudCE4.1 including double promoters, respectively. Recombinants were identified by restriction enzymes digestion and DNA sequencing of target gene, and were transfected into COS-7 cells with liposome reagents, the expression products were detected in COS-7 cells transfected with recombinants using Western blot. The results showed that nucleotide sequence of the gene cloned into expression vectors were in accordance with the report sequence, and their open reading fragment is correct. The expression products of these DNA vaccines are expressed successfully in COS -7 cells by Western blot. The two co-expression.DNA vaccines containing GAD65 fragment and Insulin B Chain gene are successfully constructed, which provide a foundation for further development of immunotherapy against autoimmune diabetes.