花鼠乳酸脱氢酶C基因cDNA的克隆、分析及原核表达

Cloning,characterization and prokaryotic expression of lactate dehydrogenase C cDNA in Tamias sibiricus

为研究花鼠乳酸脱氢酶C(lactate dehydrogenase C,LDH-C)对花鼠免疫不育控制的影响,以花鼠睾丸c DNA为模板,通过RT-PCR技术得到花鼠LDH-C基因c DNA编码区,并进行序列分析,构建花鼠LDH-C的原核表达载体,导入到大肠杆菌BL21(DE3)中诱导表达,并采用聚丙烯酰胺凝胶电泳和免疫印迹法对表达产物进行鉴定。结果显示:扩增出的c DNA片段为999 bp,编码332个氨基酸,含有完整的开放阅读框;负电荷残基与正电荷残基均为36个;预测蛋白质分子量为37k D,理论等电点为7.04,无信号肽和跨膜区,推测其是一种非分泌、疏水性蛋白。α螺旋、无规则卷曲以及延伸链是s LDH-C蛋白二级结构的主要成分。重组菌在IPTG诱导下获得了约37 k D带有His-Tag的目的蛋白。

In order to investigate the effects of lactate dehydrogenase C (LDH-C) on chipmunks,Tamias sibiricus,immune infertility control,the cDNA of chipmunks LDH-C was cloned from the chipmunks testis by RT-PCR,and its sequence was analyzed.The LDH-C gene was constructed into the prokaryotic expression vector,and this vector was transformed into E.coli BL21 (DE3) and was induced by IPTG.The SDS-PAGE and western-blot were conducted to identify the expression products.The results showed that the cDNA was 999 bp and encoded for a polypeptide of 332 amino acids,which contained complete open reading frame.The amount of negatively charged residues and positively charged residues were both 36 and the predicted molecular mass was around 37 kD.The theoretical isoelectric point (pI) was 7.04,and there was no signal peptide or transmembrane region.The LDH-C protein was predicted as non-secreted and hydrophobicity protein.Alpha helix,random coil and extended strand were the main components of the secondary structure of LDH-C.A 37 kD target protein with His-Tag was obtained from prokaryotic expression induced by IPTG.