大肠杆菌表达SLA-3蛋白与口蹄疫病毒多肽的复性研究

Expression of SLA-3 in Escherichia coli and refolding the protein with peptides derived from foot-and-mouth disease virus

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pMD19-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pET-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pET-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 kD，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 kD)。【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 refolding between SLA-3-YDY protein derived from Yorkshire swine and peptides derived from foot-and-mouth disease virus in vitro. [Methods] A pair of primers was designed to amplify the extracellular domain of SLA-3-YDY and then the PCR product was cloned into pMD19-T Simple Vector. After cleaved by Nde I and Xho I, the positive clones were selected to be sequenced. Analyzed by biological soft, the cloned product with correct sequences was selected to be inserted into pET-21a(+) and transformed into BL21. After induction with IPTG, the interest of protein was detected by SDS-PAGE. The bacteria were broken ultrasonically, then inclusion body from the bacteria was isolated. The heavy chain of SLA-3-YDY, light chain sβ2m and the epitope-peptides Hu52 from foot-and-mouth disease virus (FMDV), were refolded at a ratio of 1:1:1 in diluted refolding buffer followed by purification in molecular sieve of superdex 200 to detect whether the complex was refolded. [Results] The PCR result shows that the SLA-3-YDY gene was amplified successfully. After sequencing and analysis, the sequence of the positive clone of SLA-3-YDY was consistent with the primary sequence. By cleavage, the interest of gene was proved to be successfully inserted into pET-21a(+) Vector. After induction with IPTG and SDS-PAGE detection, the interest of gene was expressed and the molecular weight was about 33 kD. The isolated inclusion body was also detected by SDS-PAGE, and it was shown that the molecular weight of the inclusion body was consistent with the interest of protein. By refolding in a dilution system, the heavy chains of SLA-3, peptides and light chain succeed to be refolded in vitro. Then, by using the molecular sieve column to separate and purify the refolded proteins and detection by SDS-PAGE, it was shown that the complex protein of SLA-3-Hu52-sβ2m were obtained (45 kD) finally. [Conclusion] The prokaryotic expressing vector of SLA-3 derived from Yorkshire swine was constructed successfully in this research, and the interest of protein was obtained so as to the SLA-3-Hu52-sβ2m complex was refolded finally, which will lay a base to study the structure and function of the SLA-3-YDY in future.