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【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。
【Objective】 To study the refolding of Yorkshire pig SLA-3 (SLA-3-YDY) protein in vitro with foot-and-mouth disease virus polypeptide. 【Method】 The SLA-3-YDY extracellular region was designed and the target gene was amplified by PCR. The fragment was cloned into pMD19-T Simple Vector. The positive clones were screened by double enzyme digestion and sequenced. The correct fragment was sequenced and ligated to Expression vector p ET-21a (+), transformed E. coli competent BL21 cells induced by IPTG, SDS-PAGE detection of the target protein. The inclusion body protein was extracted by sonication, and the heavy chain SLA-3-YDY, light chain sβ2m and foot-and-mouth disease virus Hu52 were added into the refolding solution at a molar ratio of 1: 1: 1 by dilution dilution method and purified by molecular sieve chromatography And detect whether the compound renaturation. 【Result】 The target gene of SLA-3-YDY was obtained by PCR amplification. The positive cloned sequence was consistent with the original sequence. The recombinant plasmid pET-21a (+) was successfully identified by restriction endonuclease digestion and expressed by IPTG. SDS-PAGE analysis showed that the target gene was about 33 kD in size and the inclusion body protein was proved by SDS-PAGE. Inclusion body protein size and bacterial protein size. Molecular sieves and SDS-PAGE analysis showed that renaturation of heavy chain, light chain and polypeptide was achieved by dilution-refolding, and the protein complex SLA-3-Hu52-sβ2m (45 kD) was obtained. 【Conclusion】 The prokaryotic expression vector of Yorkshire pig SLA-3 was constructed and the target protein was obtained. The renaturation of the heavy chain and the light chain of Hu52 and SLA-3 of the foot-and-mouth disease virus was realized. The structure and function of SLA-3 were further studied in the future basis.