两种分泌型人谷氨酸脱羧酶65片段DNA疫苗的构建与鉴定.pdfVIP

中南大学学报‘ 学版 J Cent South Univ Med Scf ) ) 2007,32(6) CentSouth 997 两种分泌型人谷氨酸脱羧酶65片段DNA疫苗的构建与鉴定 张 松，周鹏程，黄 干，孙 意，周智广 (中南大学湘雅二医院代谢内分泌研究所，中南大学糖尿病中心，长沙41001 1) [摘要] 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GADl90 和 GAD49o 两个片段的 cDNA，分别与hIL一2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，West— ern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。 [关键词] GAD65； 克隆； 融合基因； DNA疫苗； 1型糖尿病 [中图分类号] R587．1 [文献标识码] A [文章编号] 1672—7347(2007)06—0997—05 Construction and identification of 2 secreted human GAD6 5 fragment DNA vaccines ZHANG Song，ZHOU Peng—cheng，HUANG Gan，SUN Yi，ZHOU Zhi—guang (Institute of Metabolism and Endocrinology，Diabetes Cen~r，Second Xiangya Hospital， Central South University，Changsha 41O011，China) Abstract： Objective To construct and identify 2 secreted human GAD65 fragment DNA vac— cines．Methods The GAD 190 GAD490．57o cDNA and hIL一2 signal peptide cDNA were linked ． 315， 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 se— quence，the expression of fusion proteins was detected by Western blot．Results The nucleotide se— quence of the cloned gene was the same as the reported sequence，and their open reading fragment was correct．The products o