谷氨酸脱羧酶65片段与IL－10双基因真核表达载体的构建与鉴定

 目的 优化以往构建的以预防 1 型糖尿病为目的的全长谷氨酸脱羧酶（GAD）65 DNA疫苗，尝试构建GAD 65片段与IL-10双基因真核表达载体。方法 从GAD65质粒中扩增出GAD190-315片段和GAD490-570片段的cDNA，以overlap PCR法将之分别与hIL-2信号肽cDNA拼接，得到SGAD190-315、SGAD490-570融合基因。以p43.2-mIL-10质粒为模板，用PCR方法扩增出IL-10基因。将SGAD190-315、SGAD490-570融合基因分别与IL-10基因依次克隆入双启动子真核表达载体pBudCE4.1中，构建出2种双基因重组真核表达载体pBud-SGAD190-315/IL-10和pBud-SGAD490-570/IL-10。2种重组真核表达载体经测序鉴定正确后，用脂质体介导的方法转染COS-7细胞，转染后48和72 h以蛋白质印迹法检测细胞裂解产物及上清液中SGAD190-315和SGAD490-570融合基因的表达，ELISA方法检测细胞上清液中IL-10的表达。结果　核酸序列测定表明克隆的SGAD190-315、SGAD490-570融合基因和IL-10基因序列与报告序列一致。蛋白质印迹法和ELISA方法均检测到SGAD190-315/IL-10和SGAD490-570/IL-10重组真核表达载体在COS-7细胞中的表达。结论　成功构建了2种GAD65片段与IL-10双基因真核表达载体，为1型糖尿病的基因疫苗预防研究提供了实验基础。

Construction and identification of eukaryotic expression vector containing GAD65 fragment and IL-10 gene

Objective To modify an established full-length GAD65 DNA vaccine for prevention of type 1 diabetes, and to construct and identify an eukaryotic expression vectors containing GAD65 fragment and IL-10 gene. Methods The cDNAs of GAD190-315 and GAD490-570 fragments, which were amplified from GAD65 plasmid, were linked with hIL-2 signal peptide cDNA respectively through overlapping PCR. Then, the two fusion genes, SGAD190-315 and SGAD490-570, and IL-10 gene were cloned into an eukaryotic expression vector pBudCE4.1 to establish two recombinant eukaryotic expression vectors, pBud-SGAD190-315/IL-10 and pBud-SGAD490-570/IL-10. After being identified by DNA sequencing, the two recombinant eukaryotic expression vectors were transfected into COS-7 cells respectively under mediation by liposome. Then the expression of the fusion proteins in the COS-7 cells transfected with recombinants were detected using Western blot and the expression of IL-10 was determined by ELISA. Results Nucleotide sequence of the gene cloned into expression vectors were in accordance with the reported sequence, and the products of these recombinant eukaryotic expression vectors were expressed in COS-7 cells as detected by Western blot or ELISA. Conclusions The two eukaryotic expression vectors containing GAD65 fragment and IL-10 gene can be successfully constructed, which is a foundation for further development of gene vaccine against type 1 diabetes.