胶质细胞源神经营养因子cDNA全序列的克隆及其生物学功能的研究

The Cloning of Glial Cell Line derived Neurotrophic Factor cDNA Holosequence and the Studying of Its Biological Activities

为了研究ＧＤＮＦ在神经系统中的生物学功能，通过ＲＴ－ＰＣＲ方法从大鼠睾丸总ＲＮＡ中扩增出ＧＤＮＦｃＤＮＡ全序列，序列分析表明与ＧｅｎＢａｎｋ中的顺序完全相同．将ＧＤＮＦｃＤＮＡ以非融合方式连接在真核表达载体ｐＥＧＦＰ－ＮⅠ的绿色荧光蛋白的上游，在ＣＭＶ启动子控制下表达．通过绿色荧光蛋白报告基因的表达表明ＧＤＮＦｃＤＮＡ能在真核细胞ＨｅＬａ中很好表达．采用裸ＤＮＡ转染方法研究ＧＤＮＦ对损伤的坐骨神经的修复作用，在雏鸡出生后３ｈ切断其右侧坐骨神经，将ｐＥＧＦＰ－ＧＤＮＦ与Ｌｉｐｏｆｅｃｔｉｎ的混合物注射到坐骨神经切断位点附近肌肉内，５ｄ后追补一次．２０ｄ后进行实验检测，观察到ＧＤＮＦｃＤＮＡ的转染阻止了切断神经侧的腰脊髓内［Ｌ４－Ｌ６］运动神经元的大量死亡，并显著促进了切断坐骨神经的再生．

GDNF cDNA holosequence was cloned from rat testis by RT PCR method to clarify the biological activities of GDNF in the nervous system.Sequencing analysis showed that it was completely the same as what reported in GenBank.Then GDNF cDNA was cloned into eucaryotic expression vector pEGFP N1 at 5′ end of EGFP gene encoding green fluorescence protein mutant 1 with human codon usage preferences in the form of nonfused proteins and made it expressed under the control of CMV promoter.The expression of EGFP exhibits that GDNF cDNA can be well expressed in the HeLa cell,a commonly used eucaryotic host cell.To study the effects of GDNF on injured sciatic nerve by plasmid DNA transfection the posthatched chicken of either sex was anesthetized by ether and the right sciatic nerve was transected.Then the mixture of GDNF cDNA recombinant plasmid and lipofectin was injected into the muscles around cut side.The additional injection was performed afeter 5 days.Twenty days later a detecting experiment was carried out.It was observed that the GDNF cDNA transfection could increase 30% motoneuron survivals in lumbar spinal cord(L4-L6) and promote the regeneration of cut sciatic nerves to distal ends.