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目的 构建弓形虫RH株致密颗粒抗原GRA8的原核和真核重组表达质粒。方法 参照GRA8序列分别设计引物 ,采用PCR从弓形虫RH株基因组DNA中分别扩增出编码GRA8的基因片段 ,克隆至 pMD18-T载体 ;菌落PCR鉴定阳性克隆并测序分析 ;各组阳性克隆的质粒分别亚克隆至原核表达质粒pGEX - 4T - 2和真核表达载体 pVAX1,分别转化大肠杆菌BL2 1和JM10 9,PCR和酶切鉴定转化菌落的插入序列 ;将构建的原核表达菌株经IPTG诱导 ,SDS -PAGE和免疫印迹分析融合蛋白的表达 ;将构建的真核重组表达质粒免疫小鼠 ,观察其诱导的抗体应答。结果 PCR扩增出GRA8基因的特异片段 ,各组阳性克隆的序列正确 ,并分别被亚克隆到原核表达质粒 pGEX - 4T - 2和真核表达载体pVAX1上 ,构建了弓形虫致密颗粒抗原GRA8的原核和真核重组表达质粒 ;原核表达质粒在大肠杆菌中表达了GRA8的融合蛋白 ;真核重组表达质粒诱导小鼠产生了抗弓形虫抗原的抗体。结论 以pGEX - 4T - 2和 pVAX1为载体 ,分别成功构建了GRA8的原核和真核重组表达质粒。
Objective To construct prokaryotic and eukaryotic recombinant plasmids of GRA8, a Toxoplasma gondii RH strain compact particle antigen. Methods According to the sequence of GRA8, primers were designed respectively. The gene encoding GRA8 was amplified from the genomic DNA of RH strain Toxoplasma gondii by PCR and cloned into pMD18-T vector. The positive clones were identified by colony PCR and sequenced. The positive clones The recombinant plasmid pGEX - 4T - 2 was subcloned into prokaryotic expression vector pGEX - 4T - 2 and transformed into eukaryotic expression vector pVAX1. The recombinant plasmids were transformed into E. coli BL21 and JM109 respectively. The inserted sequences were identified by PCR and restriction endonuclease digestion. The prokaryotic expression strains were induced by IPTG, SDS-PAGE and Western blot analysis of fusion protein expression; the eukaryotic recombinant plasmid was immunized mice to observe the induced antibody response. Results The specific fragments of GRA8 gene were amplified by PCR, and the positive clones of each group were correctly cloned into the prokaryotic expression vector pGEX - 4T - 2 and the eukaryotic expression vector pVAX1. The recombinant plasmids of GRA8 Prokaryotic and eukaryotic recombinant plasmids; prokaryotic expression plasmid expressed GRA8 fusion protein in E. coli; eukaryotic recombinant plasmid induced mouse anti-Toxoplasma antigen-producing antibodies. Conclusion The prokaryotic and eukaryotic recombinant plasmids of GRA8 were successfully constructed using pGEX - 4T - 2 and pVAX1 as vectors respectively.