人mBAFF在原核系统的可溶性表达及功能鉴定

Soluble expression of human mBAFF in Escherichia coli and its characterization

目的利用含重组质粒pQE80L-mBAFF的DH5α大肠杆菌,优化表达B细胞活化因子的突变蛋白(mBAFF)。方法研究mBAFF的体外诱导条件,包括诱导时间I、PTG浓度、诱导温度对表达量的影响。对表达条件进行优化后进行大量表达,经Ni2+NTA亲和层析和SepharcryL S200凝胶过滤层析纯化。流式检测突变蛋白与细胞结合的能力以及MTT检测突变蛋白的生物学活性。结果最佳表达条件是诱导时间4 h,诱导温度37℃,IPTG终浓度0.2 mmol/L。mBAFF占菌体总蛋白的35%,蛋白纯度达98%以上,所获突变蛋白能与淋巴细胞表面受体结合,但不能刺激淋巴细胞增殖。结论优化可溶性表达后能提高mBAFF蛋白质在DH5α大肠杆菌表达,并经Ni2+NTA亲和层析和Sepharcryl S200凝胶过滤层析得到纯度高的表达产物,所获结果为进一步研究创造了条件。

Objective To optimized expression conditions of human mBAFF by using recombinant vector pQE80L-mBAFF, and Escherichia coli DH5α. Methods The effects of inducing time, IPTG concentration, and temperature on mBAFF expression were assayed. After optimization of expression conditions, mBAFF were expressed and the proteins were purified by Ni^2＋-NTA chromatography and SepharcryL S200 chromatography. The binding activity of mBAFF to cell was assayed by flow cytometry; the biological activity of mBAFF was detected by MTT assay. Results Optimum inducing time, temperature, IPTG concentration for mBAFF expression were found, which were 4h, 37℃, and 0.2 mmol/L, respectivelg. The mBAFF protein is about 35% of total protein of the host. After purification, the purity of the expressed protein reached above 98%. The mutant protein ccould bind the surface receptors of lymphocytes but could not stimulate proliferation of lymphocytes. Conclusion Optimum conditions for mBAFF protein expression are obtained, which provides a basis for gene engineering technology.