中文摘要：

松弛素作为胰岛素超家族中的一种多肽激素，在生殖机能和很多非生殖生理过程中发挥重要的调控作用，而血中松弛素被看成是犬（Canis lupus）和部分其他动物妊娠的标志。为了简便高效获取犬松弛素，以及开发具有临床应用价值的犬松弛素生物制剂，本研究进行犬松弛素原基因的原核表达及制备其多克隆抗体的研究，即以包含犬前松弛素原基因cDNA的质粒pMD19-T-Cpreprorelaxin1为材料，PCR扩增犬松弛素原基因cDNA，与表达载体pET28a连接，转化大肠杆菌（Escherichia coli） BL21（DE3），异丙基-β-d-硫代半乳糖苷 （isopropyl-β-d-thiogalactoside, IPTG）诱导重组蛋白表达，对表达产物以及纯化后重组蛋白用十二烷基硫酸钠聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE）和Western blot实验进行鉴定；以纯化犬松弛素原重组蛋白为免疫原，免疫家兔（Oryctolagus cuniculus）制备多克隆抗体，酶联免疫吸附实验（enzyme linked immunosorbent assay, ELISA）检测抗血清效价，Western blot实验分析所制备抗体的特异性。结果显示，所构建原核表达载体高表达分子量约为22 kD的融合蛋白，与预期犬松弛素原蛋白的分子量一致，主要以包涵体形式存在，Western blot结果显示纯化前后的重组蛋白均与兔抗组氨酸（histidine, His）标记抗体特异性杂交；犬松弛素原重组蛋白免疫家兔后，ELISA检测抗血清效价大于1∶80 000，Western blot结果证实制备的多克隆抗体与犬松弛素原重组蛋白特异结合。本研究为犬松弛素原的功能研究和临床应用提供了理论依据。

英文摘要：

Relaxin, as a polypeptide hormone of insulin superfamily, plays importantly regulatory roles in the reproductive and non-reproductive physiology processes. Besides, relaxin appeared in peripheral blood, which had been seen as a sign of pregnancy in canine （Canis lupus） and other some animal. To prepare a polyclonal antibody against canine prorelaxin, the cDNA fragment of canine prorelaxin gene was amplified from the recombined plasmid pMD19-T-Cpreprorelaxin1 containing cDNA fragment of canine preprorelaxin gene by PCR, and cloned into the prokaryotic expression plasmid pET28a. Secondly, the recombined expression plasmid of canine prorelaxin was transformed into Escherichia coli BL21 （DE3） and induced by isopropyl-β-d- thiogalactoside （IPTG）, to harvest the fusion protein which were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot. Finally, the polyclonal antibodies were harvested after injecting the rabbits （Oryctolagus cuniculus） with the purified fusion protein of canine prorelaxin emulsified with the Freund＇s complete adjuvant as the immune antigens, and analyzed by enzyme linked immunosorbent assay （ELISA） and Western blot. The results demonstrated that the fusion protein with the consistent molecular weight of 22 kD indicated by SDS-PAGE, was highly expressed as the form of inclusion bodies and positively reacted with the His-labeled antibody by Western Blot. The harvested anti-serum with more than 1∶80 000 titered by ELISA, reacted specifically with the purified fusion protein of canine prorelaxin. This study would lay a potential foundation for further study and clinical application of canine prorelaxin.