Tsol18基因密码子优化对其表达及重组蛋白免疫效果的影响  

Effects of Codon Optimization in Tsol18 on Its Expression and Antigenicity of the Recombinant Protein

作　　者：丁军涛[1,2] 骆学农[1] 王颖[1] 张少华[1] 陈晓宇[1] 郑亚东[1] 景志忠[1] 才学鹏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室,兰州730046 [2]中国农业科学院研究生院,北京100081

出　　处：《畜牧兽医学报》2009年第3期409-415,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家高科技研究发展计划“863”项目(2006AA10A207);甘肃省重大科技专项(2GS063-A43-013)

摘　　要：定点突变猪带绦虫六钩蚴(oncosphere)Tsol18基因的4个碱基,并截去其N端16氨基酸信号肽的编码序列,构建pGEX-Tsol18-sp表达载体,IPTG诱导表达后进行产物的SDS-PAGE、Western blot及稳定性分析;取该基因修饰前、后所表达的重组蛋白免疫猪,以ELISA检测抗体水平,并通过屠宰检虫评价、比较免疫效果。结果显示:修饰后的Tsol18-sp基因共有7个碱基发生同义突变,表达产物主要是约38.7 ku的可溶性融合蛋白,在4℃保存2个月后约降解20.1%,能与猪带绦虫六钩蚴阳性血清反应;TSOL18和TSOL18-sp抗原免疫试验猪后均能诱导高水平抗体的产生,但TSOL18-sp抗原的保护率较TSOL18抗原有明显的提高(100%vs 20%)。表明Tsol18经修饰后,其重组表达产物的生物学活性和稳定性都有很大改善,可作为囊虫病免疫预防的候选抗原。The expressive vector of pGEX-Tsol18-sp was constructed using the genetic modified Tsol18 gene of Taenia soliurn onchospere by the site-directed mutation of 4 bases in its coding sequence and removing of the sequence fragment encoding its N-terminal signal peptide. After induced with IPTG, the expressed proteins of TSOl18 and TSOl18-sp were probed by SDS-PAGE, Western blot and its stability was evaluated by storing at 4 ℃. The effects of immuno-protection of these two recombinant proteins were compared by ELISA and counting of Cysticercus in the muscle tissue of experimental pigs. The results revealed that the modified Tsol18-sp, in which 7 bases were synonymously mutated, could be expressed in the recombinant pGEX-Tsol18-sp vector. The soluble recombinant protein with a molecular weight of 38.7 ku can be probed by the specific serum against Taenia solium oncosphere, and only 20.1% was degradated in storage at 4 ℃ for 2 months. Both of the recombinant proteins can induce high titre of antibodies in the vaccinated pigs, but the recombinant protein TSOL18-sp showed a better protection rate （100%） against the challenge of Cysticercus cellulose than TSOL18 revealed（20%） by the reduced counts of Cysticercus in the muscle tissue of vaccinated pigs. It was suggested that the TSOL18-SP is a effective and stabile antigen, and could be used as a potential antigen for the immunoprophylaxis of Cysticercosis cellulosae.

关 键 词：猪带绦虫六钩蚴 Tsol18-sp 基因修饰 抗原性 

分 类 号：S852.734[农业科学—基础兽医学] R383[农业科学—兽医学]