修饰后日本血吸虫Sjc-97基因的真核表达及其免疫原性

目的:研究日本血吸虫大陆株副肌球蛋白基因第3区域片段(Sjc-97f3)毕氏酵母表达产物的免疫原性.方法:根据毕氏酵母密码子使用频率重新设计Sjc-97核苷酸序列,依据不对称PCR原理人工合成Sjc-97f3,并在毕氏酵母中表达.表达产物通过Ni-NTA亲和层析和凝胶过滤层析方法纯化,再分别与ISA720和Al(OH)3佐剂乳化后,免疫C57BL/6和昆明小鼠,通过尾蚴膜反应和ELISA反应检测特异性抗体.结果:Sjc-97f3基因在毕氏酵母中获得了高水平分泌表达,其表达产物能与日本血吸虫免疫血清进行特异性免疫反应.经与佐剂乳化后,该蛋白具有良好的免疫原性.ELISA结果显示重组蛋白在小鼠体内可激发明显的抗体反应,但2种佐剂所诱导的抗体水平以ISA720佐剂为高; 该抗原在不同品系小鼠中的抗体水平存在显著差异(P<0.01),昆明鼠的抗血清滴度远高于C57BL/6小鼠.小鼠免疫血清能识别日本血吸虫表面抗原,产生特异的尾蚴膜反应. 结论:密码子优化的Sjc-97f3在毕氏酵母中获得高效表达,其产物具有良好的免疫原性.

Expression and immunogenicity of redesigned Schistosoma japonicum Sjc-97 gene in Pichia pastoris

Objective:To determine the immunogenicity of re combinant Schistosoma japonicum Sjc 97f3 protein expressed in Pichia pa storis . Methods: The DNA sequence of Sjc 97f3 was redesig ned by yeast Pichia preferential codon usage, synthesized by asymmetric PCR based method, and expressed in Pichia pastoris . The recombinant protein was purified by N i NTA affinity chromatography and gel filtration chromatography. The inbred C57 BL/6 strains and outbred KM strains of mice were subcutaneously immunized with t he protein formulated by either adjuvant ISA720 or aluminium hydroxide. The spe cific antibodies were detected by cercarian huellen reaction and ELISA analysis. Results: The Sjc 97 gene was successfully expressed in Pichia pastoris and the product reacted with anti Sj 97 serum. It was s hown that the protein was highly immunogenic after reaction with adjuvants in mi ce. Moreover, the antibody level induced by rSjc 97f3 ISA720 was higher than t ha t by rSjc 97f3 aluminium hydroxide. KM mice generated significantly hi gher level of the antibody compared to C57BL/6 strain of mice ( P <0.01). Cer carian huellen reaction indicated that the immune sera recognized the antig en on the surface of parasite. Conclusion: The codon optimised Sjc 97f3 can be successfully expressed in Pichia pas toris and the recombinant protein is highly immunogenic in mice.