人睫状神经营养因子突变体基因克隆及高效表达

为了提高人睫状神经营养因子（ＣＮＴＦ）的生物学活性，用ＰＣＲ方法获取Ｎ端缺失１４个氨基酸的ＣＮＴＦ基因片段，经酶切鉴定、核酸测序证实突变体的核苷酸序列，将其重组至表达质粒ｐＢＶ２２０，构建了ＣＮＴＦ突变体表达载体ｐＢＶ－ＣＮＴＦΔ．用ＳＤＳ－ＰＡＧＥ测定其表达水平，鸡胚背根节无血清培养法检测表达蛋白的生物学活性．结果表明，ｐＢＶ－ＣＮＴＦΔ能表达生物学活性高于天然ＣＮＴＦ的约２６ｋＤ蛋白质，表达水平达３０％．为今后通过基因工程方法获得ＣＮＴＦ突变体，从而制备高效的ＣＮＴＦ制剂创造了条件．

Cloning and High Expression of Mutated RecombinantCiliary Neurotrophic Factor

To construct a engineering bacteria which can express higher biological activity ciliary neurotrophic factor(CNTF) mutated protein.The CNTF mutated gene fragment which was truncated out 14 amino acid in N terminal was obtained with PCR.This mutated gene was sequenced,recombined into an expression vector pBV220 and expressed in E.coli DH5 α induced at 42℃.The expression level was measured by SDS PAGE.The biological actitity of expressed product was measured by chick dorsal ganglion culture.The mutation expression level was about 30% in total bacteria protein.The biological activity of inclusion body after renaturation was about 3 times higher than that of natural CNTF in chick dorsal ganglion culture.The results of the investigation will be a basis to obtain CNTF mutation with genne engineering technology and to create a new way for producing ideal CNTF.