【摘要】 构建1型糖尿病关键自身抗原胰岛素原(Proinsulin)的DNA疫苗,并在体外对其表达产物进行鉴定。从人胰腺cDNA文库用半巢式PCR扩增出编码Proinsulin的cDNA,克隆入载体PGEM-T中,再将其亚克隆至真核表达载体pcDNA3.1(+)中,构建重组载体pcDNA3.1(+)/Proinsulin,测序后在真核细胞COS-7中进行体外短暂表达,Western印迹法对其表达产物进行鉴定。结果克隆的人Proinsulin基因,测序表明其cDNA序列与Genbank公布的序列基本相同,仅在第63位氨基酸(C肽区)有一CAG(Glu)→CGG(Arg)的突变,Western免疫印迹法证实DNA疫苗在COS-7细胞中有效地获得了表达。结果说明运用基因克隆方法构建的Proinsulin DNA疫苗,在真核细胞内能有效地表达Proinsulin重组抗原。该疫苗的构建成功,为1型糖尿病的免疫干预提供了实验基础。更多还原

【Abstract】 To construct the DNA vaccine of human Proinsulin, which is recognized as the crucial and specific autoantigen oftype 1 diabetes, and confirm the transient expression product in eukaryotic cells. Human Proinsulin complete cDNA was ampli-fied with half net-PCR method from human pancreas cDNA library, and cloned into PGEM-T vector. Then the target gene wassubcloned into eukaryotic expression vector pcDNA3. 1 (+) to generate DNA vaccine-recombinant plasmid pcDNA3. 1 (+)/Proinsulin. Afer the DNA vaccine being confirmed to contain the correct target gene sequence with fluorescence sequencing,COS-7 was transfected with it in vitro by DOTAP liposome, and the expression of protein was detected with Western blot. Thesequencing results for cloned human Proinsulin gene showed that the insertion of cDNA is correct and the sequence is similar tothat of human Proinsulin cDNA in Cenbank, except for the site 63 amino acid (C peptide region)where there is a mutation[ CAG (Glu ) → CGG (Arg) ]. Western blot confirmed that the products of DNA vaccine pcDNA3. 1 (+) / Proinsulin was efficient -ly expressed in eukaryotic cells in vitro. It is conclused that eukaryotic cells transfected with human Proinsulin DNA vaccineobtained by the method of gene engineering can express recomhinant Proinsulin antigen efficiently. An experimental basis forimmune intervention of Type 1 diabetes is provided.更多还原