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目的:对天花粉蛋白(TCS)的第120-123位氨基酸进行缺失突变,并在原核系统中对其进行表达和纯化。方法:利用重叠延伸PCR的方法得到TCS突变体cDNA,并克隆入原核表达载体pET-28(a+)。酶切和测序鉴定后,将突变体质粒pET-28(a+)-mTCS转化BL21(DE3)表达菌,经IPTG诱导表达后,对表达产物用Ni2+-NTA亲和层析柱进行纯化和Western-blot鉴定。结果:利用重叠延伸PCR成功获得突变天花粉蛋白的编码序列,并构建pET-28(a+)-mTCS原核表达质粒。在BL21(DE3)菌中,突变体蛋白(mTCS)可被IPTG诱导性表达,并被Ni2+-NTA树脂有效纯化。结论:本实验成功获得一种突变体TCS,并建立该突变TCS的原核表达和纯化的实验方法。
Objective: To delete the amino acids 120-123 of trichosanthin (TCS) and express it in prokaryotic system. Methods: TCS mutant cDNA was amplified by overlap extension PCR and cloned into prokaryotic expression vector pET-28 (a +). The recombinant plasmid pET-28 (a +) - mTCS was transformed into BL21 (DE3) expression strain after induced by IPTG. The expressed product was purified by Ni2 + -NTA affinity chromatography and Western- blot identification. Results: The coding sequence of mutant TCS was successfully obtained by overlap extension PCR and the prokaryotic expression plasmid pET - 28 (a +) - mTCS was constructed. In BL21 (DE3) bacteria, the mutant protein (mTCS) is inducibly expressed by IPTG and effectively purified by Ni2 + -NTA resin. Conclusion: This experiment successfully obtained a mutant TCS, and established the mutant TCS prokaryotic expression and purification of experimental methods.