睫状神经营养因子cDNA的克隆、表达、纯化及生物活性测定

Studies on expression and purification of human ciliary neurotrophic factor

目的 :利用基因工程技术获得有较高生物学活性的睫状神经营养因子 (CNTF)蛋白。方法 :应用反转录 PCR技术直接扩增出人 CNTF c DNA,克隆入 p UC19载体中 ,用双脱氧法进行手工序列分析 ,构建表达菌株 p BV2 2 0 - CNTF/ DH5α。表达的蛋白质得到纯化 ,用鸡胚背根节无血清培养法检测表达蛋白生物学活性。结果 :所克隆的 CNTF基因序列与文献报道完全一致 ,p BV2 2 0 - CNTF/ DH5α能特异表达分子量大约为 2 6 0 0 0的蛋白质 ,表达量约为菌体总蛋白量的 35 %。 CNTF蛋白质经纯化后纯度约 95 % ,而且表达蛋白有很高的生物学活性。结论 :该研究结果为

Objective:In order to obtain high biological activities of CNTF.Methods:CNTF cDNA was obtained by RT PCR. The product was cloned into pBV220 and transformed into E.coli DH5α after sequenced.CNTF was purified and the biological activity was defined by chicken embryonic ganglion neurons culture. Results :The PCR sequence was identical to these reported.E.coli DH5α/pBV CNTF can specially express about 26 000 protein.The purity of rhCNTF was about 95% after purified;CNTF protein can significantly promote growth of chick embryonic ganglion.Conclusion:The present study is benegicial to the research of CNTF structure and function and lays a foundation for developing gene engineering medicine.