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

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.