两种GAD65片段与IL-4基因共表达DNA疫苗的构建与鉴定

Construction and identification of co-expression DNA vaccine containing GAD65 fragment gene and IL-4 gene

目的构建两种谷氨酸脱羧酶（GAD）65片段与白细胞介素4（IL-4）基因共表达DNA疫苗,并在体外COS-7细胞中对表达产物进行检测。方法从GAD65质粒中扩增出GAD190～315和GAD490～570两个片段的cDNA,Overlap法分别与hIL-2信号肽cDNA基因拼接,将拼接后的融合基因与IL-4基因依次克隆入双启动子真核表达载体pBudCE4.1中。重组子经酶切和测序鉴定后,脂质体转染COS-7细胞,Western blot法检测融合基因的表达,ELISA法检测IL-4表达。结果核酸序列测定表明克隆的融合基因和IL-4基因序列与报告序列一致,开放阅读框正确,Western blot和ELISA显示在COS-7细胞中均可检测两个目的基因的表达。结论两种GAD65片段与IL-4共表达DNA疫苗均成功构建,为1型糖尿病的预防研究提供了实验基础。

Objective To construct co-expression DNA vaccine containing GAD65 fragment gene and I-L4 gene, and identify the transient expression products in COS-7 cells. Methods The GAD190-315 and GAD490-570 cDNA and hIL-2 signal peptide eDNA were linked together through overlap PCR, respect tively. Then,the two fusion gene and IL-4 gene were cloned into eukaryotic expression vector pBudCE4. 1 in order. Recombinants were identified by restriction enzymes digestion and nueleotide sequencing of target gene. The expression of fusion protein was detected in COS-7 cells transfected with recombinants using Western blot and the expression of IL- 4 was determined by ELISA. Results The size of the fusion gene and IL- 4 gene amplified were in accordance with that we expected,Nucleotide sequence of the cloned gene was the same as the report sequence, and their open reading fragment was correct. Western blot or ELISA showed that products of these DNA vaccines were expressed successfully in COS-7 cells. Conclusions The co expression DNA vaccines containing GAD65 fragment gene and IL-4 gene were successfully constructed, which is a foundation for further development of DNA vaccine against type 1 diabetes.