人精子特异性乳酸脱氢酶在大肠杆菌中的表达及其初步应用  

Expression and preliminary use of human sperm-specific lactate dehydrogenase in Escherichia coli

作　　者：辛娜[2] 陈平[1] 黄梁浒[1] 涂向东[1] 吴玉水[1] 兰风华[1] 

机构地区：[1]南京军区福州总医院分子医学研究中心,350014 [2]福建省肿瘤医院检验科,350014

出　　处：《中华检验医学杂志》2008年第8期933-936,共4页Chinese Journal of Laboratory Medicine

基　　金：基金项目：福建省重点科技攻关项目（2002Y032）;全军医药卫生“十五”基金项目（01MA032）

摘　　要：目的构建人精子特异性乳酸脱氢酶（hLDH-C4）的原核表达载体，在大肠杆菌中进行表达，将重组hLDH-C4应用于抗精子抗体（ASA）的检测。方法以人睾丸TriplEx cDNA文库为模板，PCR扩增hLDH-C4编码序列。PCR产物经Hind Ⅲ-XhoⅠ酶切后，克隆至表达载体pET-28a（＋）中，在E-coli BL21（DE3）中诱导His-Tag融合的重组蛋白表达。用免疫印迹、酶活性测定等方法鉴定表达产物。以纯化的重组hLDH-C4为基质，建立检测ASA的ELISA方法。结果构建了hLDH-C4原核表达载体pET-28a（＋）-hLDHC。在IPTG的诱导下，重组菌可高效表达相对分子质量35000的产物，与预期大小相符。免疫印迹显示，重组蛋白可被抗His-Tag单克隆抗体和兔抗人LDH—C4抗体识别。重组菌在IPTG诱导后，其裂菌液的乳酸脱氢酶活性是诱导前的11，2倍。用基于hLDH-C4抗原的间接ELISA法，在一组不育症患者中检测血清抗hLDH-C4抗体，阳性率达30．51％。结论成功地克隆了hLDH-C4编码序列，并在E-coli BL21（DE3）中获得高效的表达，重组hLDH-C4在ASA检测中得到初步应用。Objective To construct a recombinant vector of sperm-specific human lactate dehydrogenase （hLDH-C4） , express it in Escherichia coli （E-coli） BL21 （DE3） and utilize it in the detection of anti-sperm antibody. Methods The coding sequence of hLDH-C4 was amplified from human testis λTripIEx cDNA library, and inserted into pET-28a（＋） after restriction enzyme digestion with Hind Ⅲ and XhoⅠ. The resultant recombinant vector was used to transform E-coli BL21 （DE3） and the His-Tag fused hLDH-C4 was expressed after induction with IPTG. Western blot was used to analyzed the recombinant protein and LDH activity of bacterial lysates was determined. An indirect ELISA method for the detection of anti-sperm antibody was established by using the recombinant hLDH-C4 as antigen matrix. Results pET-28a（＋）-hLDHC was successfully established. The protein with size of 35kD could be induced by IPTG when the recombinant plasmid was transfected into E. coli BL21 （DE3）. Western blot showed that the recombinant protein could be specifically recognized both by anti-His tag monoclonal antibody and by rabbit anti-human LDH-C4 antibody. In addition, the recombinant protein showed high-level LDH activity when the bacterial lysate after IPTG induction was used to check LDH activity. The recombinant hLDH-C4 was confirmed when it was used in indirect ELISA to detect anti-hLDH-C4 antibody. Conclusions The coding sequence of hLDH-C4 is cloned into the vector pET-28a（＋） and recombinant hLDH-C4 was expressed at a high level in E-coil The recombinant hLDH-C4 is useful in the detection of anti-sperm antibody.

关 键 词：L-乳酸脱氢酶 同工酶类 精子 自身抗体 大肠杆菌 重组蛋白质类 

分 类 号：R686[医药卫生—骨科学]