柔嫩艾美耳球虫沉默信息调节因子2真核表达质粒的构建及在细胞中的表达

CONSTRUCTION AND EXPRESSION OF EUKARYOTIC EXPRESSION PLASMID CONTAINING SILENT INFORMATION REGULATOR 2 GENE OF EIMERIA TENELLA

为构建柔嫩艾美耳球虫沉默信息调节因子2(Eimeria tenella silent information regulator 2,Et SIR2)的真核表达质粒,以柔嫩艾美耳球虫第二代裂殖子c DNA为模板,PCR扩增出Et SIR2基因,PCR产物经酶切回收纯化目的片段后与经相应酶切的真核表达载体p CAGGs连接。连接产物经PCR和酶切鉴定,再经测序鉴定正确后,分别转染DF-1细胞和BHK细胞进行表达,用Western blot和间接免疫荧光鉴定Et SIR2基因的表达情况。结果表明:成功扩增了Et SIR2基因,长度为909 bp;Western blot可见大小约为37 k Da的表达蛋白条带;间接免疫荧光可以检测到特异性绿色荧光,表明成功构建了Et SIR2的真核表达质粒p CAGGsEt SIR2,并能在哺乳动物细胞和禽类细胞中表达。该研究结果为深入研究Et SIR2的生物学特性和球虫DNA疫苗的研制打下了基础。

The full-length cDNA of silent information regulator 2 of Eimeria tenella （EtSIR2） was amplified in PCR from the cDNA of the second-generation merozoites in order to construct the eukaryotic expression plasmid. The PCR products and pCAGGs vectors were digested with the same restriction enzymes and ligated. The recombinant EtSIR2 plasmid was confirmed through PCR, enzyme digestion and sequencing and then transfected into DF-1 cells and BHK cells, respectively. The expression of recombinant EtSIR2 in the transfected cells was examined in indirect immunofluorescence assay （IFA） and Western blot. The results showed that the EtSIR2 gene was 909 bp in length. Western blot also indicated that the antiserum to recombinant EtSIR2 strongly recognized a protein with molecular mass at 37 kDa in the transfected cells. Specific green fluorescence was observed in IFA. Construction and eukaryotic expression of EtSIR2 has laid the foundation for future research on biological functions and DNA vaccine.