人胶源神经营养因子基因的克隆及在大肠杆菌中的表达

胶源神经营养因子（Ｇｌｉａｌｃｅｌｌｉｎｅｄｅｒｉｖｅｄｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ，ＧＤＮＦ)是大鼠B49细胞系中分离纯化得到的一种蛋白质^[1],由于其对多巴胺神经元的专一性的神经营养作用而被发现。GDNF成熟蛋白由134个氨基酸组成，具有两个N-糖基化位点。它属于TGF-β基因家族但与该家族其它成员的氨基酸序列同源性仅为20%，可能是一个新的亚家族。最近研究表明，它对发育中的运动神经元也有很强的神经营养作用^[1]。对啮齿类^[3,4],灵长类的弥猴^[5]的活体试验表明，胶源神经营养因子是一种治疗神经退化发夹知病如帕金森氏症、肌萎缩性脊髓索硬化症等的非常有效的潜在药物。由于GDNF在体内含量极低而且公在发育早期表达，因而只有通过基因工程方法才能获得大量的GDNF。本文报道采用PCR方法从中国入基因组DNA中扩增出编码GDNF的基因，并实现在大肠杆菌中的高效表达。这为进一步研究GDNF的结构和生物学功能打下了坚实的基础。

Cloning the Gene Coding for Human Glial Cell Line derived Neurotrophic Factor and Its Expression in Escherichia coli

By using PCR method,the hGDNF gene was cloned from Chinese genomic DNA.The hGDNF gene was subcloned into the expression vector pET3a which is under the control of T7 promoter and then transfected into E.coli BL21(DE3).The constructed strain was designated as pET3aTT/BL21(DE3).The sequence data of cloned hGDNF gene showed that it was identical to that of natural one.Four hours after IPTG induction,a 16kDa extra protein which is about 16.5% out of total bacteria proteins in pET3aTT/DE3(BL21) was appeared in the gel of SDS PAGE.The expression products existed in the form of inclusion body.The protein was purified viaion exchange chromatography.The results of amino acid sequencing proved that the sequence of 15 amino acids at the N terminal of the expressed hGDNF was the same as that deduced from DNA sequence.