两种分泌型人谷氨酸脱羧酶65片段DNA疫苗的构建与鉴定

目的:构建和鉴定分泌型人谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)65片段DNA疫苗.方法:从人GAD65质粒中扩增出GAD190-315和GAD490-570两个片段的cDNA,分别与hIL-2信号肽cDNA基因拼接,将拼接后的片段克隆入pBudCE4.1真核表达载体,测序鉴定,并用Western印迹检测融合基因的表达.结果:核酸序列测定表明克隆的融合基因序列与报告序列一致,开放阅读框正确,Western印迹检测显示这两种人GAD65片段分泌表达.结论:两种分泌型人GAD65片段DNA疫苗均成功构建,为1型糖尿病的预防提供了实验基础.

Construction and identification of 2 secreted humanGAD65 fragment DNA vaccines

Objective To construct and identify 2 secreted human GAD65 fragment DNA vaccines.Methods The GAD190-315,GAD490-570 cDNA and hIL-2 signal peptide cDNA were linked through overlapping PCR,respectively.The fusion gene was cloned into eukaryotic expression vector pBudCE4.1.After the DNA vaccine being determined to contain the correct target nucleotide sequence,the expression of fusion proteins was detected by Western blot.Results The nucleotide sequence of the cloned gene was the same as the reported sequence,and their open reading fragment was correct.The products of these DNA vaccines were expressed and secreted in eukaryotic cell using Western blot.Conclusion The pBudCE4.1/SGAD190-315 and pBudCE4.1/SGAD490-570 secreted human GAD65 fragment DNA vaccines were successfully constructed,which is a foundation for immune prevention of type 1 diabetes.