畜牧兽医学报 ›› 2009, Vol. 40 ›› Issue (3): 409-415.doi:

• 预防兽医 • 上一篇    下一篇

Tsol18基因密码子优化对其表达及重组蛋白免疫效果的影响

丁军涛1,2,骆学农1,王颖1,张少华1,陈晓宇1,3,郑亚东1,景志忠1,才学鹏1*   

  1. 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;3.甘肃农业大学,兰州 730070
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-03-24 发布日期:2009-03-24
  • 通讯作者: 才学鹏

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

DING Jun-tao1,2, LUO Xue-nong1, WANG Ying1, ZHANG Shao-hua1, CHEN Xiao-yu1,3, ZHENG Ya-dong1, JING Zhi-zhong1, CAI Xue-peng1*   

  1. 1.Key Laboratory of Animal Parasitology of Gansu Province, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046,China; 2.Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081,China; 3.Gansu Agricultural University, Lanzhou 730070,China
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-03-24 Published:2009-03-24

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。

Abstract: The expressive vector of pGEX-Tsol18-sp was constructed using the genetic modified Tsol18 gene of Taenia solium 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.