玉米赤霉烯酮降解酶在枯草芽孢杆菌中的表达及其对母猪繁殖性能的影响

Expression of Zearalenone Degrading Enzyme in Bacillus subilis and Its Effects on Reproductive Performance of Sows

本试验旨在建立玉米赤霉烯酮(ZEN)降解酶基因ZEN-jjm在枯草芽孢杆菌中的表达体系,确定其产物的生物降解活性及对母猪繁殖性能的作用。克隆ZEN-jjm基因,经EcoRⅠ和NotⅠ双酶切后连接至p HT01表达载体中,构建重组质粒;转化枯草芽孢杆菌,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)分析ZEN-jjm蛋白表达水平。高效液相色谱法(HPLC)检测表达的ZEN-jjm蛋白降解ZEN的活性。通过对母猪的繁殖性能的评价,确定ZEN降解酶降解ZEN对母猪繁殖性能的影响。双酶切和测序结果表明:ZEN-jjm成功插入p HT01中,SDSPAGE表明获得1株高效表达目的蛋白的重组枯草芽孢杆菌,其大小约为29 ku。HPLC结果表明表达的ZEN-jjm蛋白能有效地降解ZEN;表达的ZEN-jjm蛋白能显著缓解ZEN对繁殖母猪的毒害作用(P<0.05)。综上:1)本研究成功构建了表达ZEN-jjm在枯草芽孢杆菌中的表达载体,并在枯草芽孢杆菌中得到了表达。2)表达的ZEN-jjm蛋白具有降解ZEN的生物活性。3)在饲粮中添加ZEN-jjm蛋白能显著降低ZEN对母猪能繁殖性能的危害。

The purpose of this study w as to establish an expressing system of zearalenone（ ZEN） degrading enzyme gene（ ZEN-jjm） in Bacillus subtilis,and to determine the biodegradation activity and effect of its expression product on reproductive performance of sow s. The ZEN-jjm gene w as cloned and the recombinant expression plasmid w as constructed by ligation cloned ZEN-jjm and p HT01 vector digested w ith EcoRⅠand NotⅠ enzymes,and then transformed into Bacillus subilis. Expression of ZEN-jjm protein in Bacillus subilis was analyzed by dodecyl sulfate polyacrylamide gel electrophoresis（ SDS-PAGE）. And the degrading activity of ZEN-jjm protein w as determined by high performance liquid chromatography（ HPLC）. The effects of ZEN degrading enzyme on reproductive performance of sow s w ere evaluated. The results of double enzyme digestion and DNA sequencing demonstrated that ZEN-jjm w as inserted into p H T01. The SDS-PAGE show ed that one Bacillus subilis strain w ith high-level expression w as obtained,and the size of the expressed protein w as about29 ku. The HPLC result illustrated that the expressed ZEN-jjm protein could effectively degrade ZEN. The evaluation results of reproductive performance of sow s indicated that the expressed ZEN-jjm protein could significantly relieve the toxicity for sow s caused by ZEN（ P〈0.05）. In conclusion： 1） the ZEN-jjm expression vector is successfully constructed and the ZEN-jjm protein is expressed in Bacillus subtilis in this study. 2） The expressed ZEN-jjm protein has the biological activity of degrading ZEN. 3） ZEN-jjm protein can significantly reduce the harm of ZEN on reproductive performance of sow s w hen it is added in the feed.