摘要
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 ISA72 0和 Al(OH) 3佐剂乳化后 ,免疫 C5 7BL/ 6和昆明小鼠 ,通过尾蚴膜反应和 EL ISA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。EL ISA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 ISA72 0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 .0 1 ) ,昆明鼠的抗血清滴度远高于 C5 7BL/ 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- 97f 3在毕氏酵母中获得高效表达 。
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.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2004年第1期29-33,共5页
Academic Journal of Second Military Medical University
基金
世界卫生组织专项基金 ( IDA 10 9915 )
