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目的研究重组人睫状神经因子(rh-CNTF)的表达、纯化及聚乙二醇化修饰、纯化方法。方法重组人睫状神经因子(rh-CNTF)工程菌经37℃培养至对数生长期,加入IPTG诱导4h,经QSepharose F.F凝胶柱纯化和复性后得到纯化的rh-CNTF。以体外培养鸡胚背根神经节(DRG)方法测定重组蛋白rh-CNTF的神经营养活性。用单甲氧基聚乙二醇丁醛(mPEG-ButyrALD-20k)对rh-CNTF进行修饰,所得修饰产物经DEAESepharose F.F和Superdex 75凝胶分离纯化后获得mPEG-rh-CNTF偶联物。结果目的蛋白占总蛋白30%左右,为包涵体,经QSepharose F.F凝胶柱纯化和复性,获得纯度达90%的rh-CNTF。表达的rh-CNTF能促进培养的鸡胚背根神经节神经突起的生长。修饰后的单聚PEG-rhCNTF经DEAESepharose F.F和Superdex 75凝胶纯化后纯度达到95%。结论确立了rh-CNTF蛋白表达纯化及PEG修饰的方法。
Objective To study the expression, purification and modification of recombinant human ciliary nerve factor (rh-CNTF). Methods Recombinant human ciliary neurogenic factor (rh-CNTF) engineered bacteria were cultured in logarithmic growth phase at 37 ℃ and induced by IPTG for 4 h. The purified rh-CNTF was purified and renatured by QSepharose F. F gel column. The neurotrophic activity of recombinant protein rh-CNTF was assayed in vitro by chick embryo dorsal root ganglion (DRG). The rh-CNTF was modified with monomethoxy polyethylene glycol butyraldehyde (mPEG-ButyrALD-20k), and the obtained modified product was isolated and purified by DEAE Sepharose F. F and Superdex 75 gel to obtain mPEG-rh-CNTF conjugate. Results The target protein accounted for about 30% of the total protein. The inclusion body was purified and refolded by QSepharose F. F gel column to obtain rh-CNTF with purity of 90%. The expressed rh-CNTF promoted the growth of cultured chick embryo dorsal root ganglion neurites. The modified monopoly PEG-rhCNTF was purified to 95% purity by DEAE Sepharose F. F and Superdex 75 gel. Conclusion The method of rh-CNTF protein expression and purification and PEG modification was established.