大肠杆菌表达SLA-3蛋白与口蹄疫病毒多肽的复性研究

发布时间：2018-05-08 17:18

本文选题：大约克猪 + SLA-　；参考：《微生物学通报》2017年02期

【摘要】：【目的】研究大约克猪SLA-3(SLA-3-YDY)蛋白在体外与口蹄疫病毒多肽的复性。【方法】设计SLA-3-YDY胞外区引物,PCR扩增目的基因,并将此片段克隆至p MD19-T Simple Vector上,双酶切筛选出阳性克隆并测序,测序正确的片段连接至表达载体p ET-21a(+)上,转化大肠杆菌感受态BL21细胞中,IPTG诱导,SDS-PAGE检测目的蛋白的表达。超声破碎菌体,提取包涵体蛋白,通过稀释复性法将重链SLA-3-YDY、轻链sβ2m和口蹄疫病毒多肽Hu52按摩尔比1:1:1加入复性液,经分子筛层析纯化并检测复合物是否复性。【结果】PCR扩增获得SLA-3-YDY目的基因,经测序阳性克隆序列与原序列一致。经酶切鉴定,证明目的基因与p ET-21a(+)载体成功连接,经IPTG诱导表达、SDS-PAGE检测,显示目的基因表达,大小约33 k D,SDS-PAGE检测包涵体蛋白,证明包涵体蛋白大小与菌体蛋白大小一致。分子筛及SDS-PAGE检测发现,通过稀释复性法实现了重链、轻链和多肽的复性,并得到蛋白复合物SLA-3-Hu52-sβ2m(45 k D)。【结论】构建了大约克猪SLA-3原核表达载体,获得目的蛋白,实现了口蹄疫病毒多肽Hu52与SLA-3重链及轻链的复性,为今后进一步研究SLA-3的结构和功能奠定基础。
[Abstract]:[objective] to study the renaturation of SLA-3 SLA-3-YDYY protein from Yorkshire pigs with foot-and-mouth disease virus polypeptide in vitro. [methods] the target gene was amplified by designing primers for SLA-3-YDY extracellular region and cloned into p MD19-T Simple Vector. The positive clones were screened by double enzyme digestion and sequenced. The correct sequence was ligated to the expression vector pET-21a (), and the expression of the target protein was detected by SDS-PAGE induced by IPTG in Escherichia coli receptive BL21 cells. The inclusion body protein was extracted by ultrasonic fragmentation. The heavy chain SLA-3-YDY, the light chain s 尾 2m and the foot-and-mouth disease virus polypeptide Hu52 were added into the refolding solution at 1:1:1 molar ratio by dilution renaturation. The target gene of SLA-3-YDY was amplified by PCR, and the positive clone sequence was consistent with the original sequence. Restriction endonuclease analysis showed that the target gene was successfully linked to pET-21a () vector and detected by SDS-PAGE induced by IPTG. The target gene was detected by SDS-PAGE. The size of inclusion body protein was detected by 33 kD SDS-PAGE. It was proved that the size of inclusion body protein was the same as that of bacterial protein. Molecular sieve and SDS-PAGE detection showed that the heavy chain, light chain and polypeptide were renatured by dilution renaturation method, and the protein complex SLA-3-Hu52-s 尾 2m(45 kD was obtained. [conclusion] the prokaryotic expression vector of Yorkshire porcine SLA-3 was constructed and the target protein was obtained. The refolding of FMDV polypeptide Hu52 and SLA-3 heavy chain and light chain was realized, which laid a foundation for further study on the structure and function of SLA-3.
【作者单位】：大连大学生命科学与技术学院;中国疾病预防控制中心病毒病预防控制所;
【基金】：国家自然科学基金项目(No.31172304) 辽宁省教育厅重点实验室项目(No.LZ2015003)~~
【分类号】：S852.65

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