诱导T细胞免疫反应的日本血吸虫抗原表位的重组表达与免疫原性鉴定  

Recombinant expression and immunogenicity identification of Schistosoma japonicum antigen epitopes inducing T-cell response

作　　者：李健[1] 殷旭仁[1] 余传信[1] 许永良[1] 华万全[1] 何伟[1] 梁幼生[1] 高琪[1] 

机构地区：[1]江苏省血吸虫病防治研究所,卫生部寄生虫病预防与控制技术重点实验室,江苏省寄生虫分子生物学重点实验室,江苏省寄生虫病学重点学科,无锡214064

出　　处：《中国血吸虫病防治杂志》2007年第4期241-246,共6页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(30471515)

摘　　要：目的对预测的日本血吸虫T细胞免疫反应表位进行合成与融合表达,并对其免疫原性进行分析。方法根据3个表位(P7、P17、P18)的基因序列,人工合成正、负链寡核苷酸序列,使正链的5′端带有NcoI酶切位点,负链的3′端带有XhoI酶切位点。将每条表位肽的正、负寡核苷酸链退火形成双链DNA后,定向克隆入表达载体pET32c(+),电转化至E.coliDH5α,经PCR、酶切及DNA序列分析鉴定出分别含有P7、P17、P18序列的重组质粒。提取重组质粒电转化至E.coliBL21(DE3)中,用异丙基硫代-β-D半乳糖苷对表位肽融合蛋白进行诱导表达,经12%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳对表达产物进行分析。表位肽融合蛋白用Ni2+螯合亲和层析胶纯化。同时根据P7、P17及P18的氨基酸序列人工合成这3个表位肽段。用纯化的表位融合蛋白、人工合成的表位肽体外刺激紫外线致弱尾蚴免疫C57BL/6J小鼠脾细胞,3H-TdR掺入法检测其刺激淋巴细胞的增殖效果。结果P7、P17、P183个表位肽基因序列被成功克隆到pET32c(+)中并获得重组质粒,重组质粒均能表达大小约20kDa的融合蛋白。表达产物用Ni2+亲和层析胶纯化后获得纯化的肽融合蛋白。表位P7、P17重组蛋白或合成肽均能有效刺激小鼠淋巴细胞增殖。结论P7、P17肽段是日本血吸虫的T细胞抗原表位。Objective To synthesize and fusion express the predicted T-cell epitopes of Schistosoma japonicum, and analyze their immunogenicities. Methods The plus and minus oligo-nucleic acid strands of epitopes P7, P17, P18 were synthesized following their DNA sequence, respectively. The Nco I restriction enzyme sites were added to the 5＇ end of epitope gene and the Xho I restriction enzyme sites were added to the 3＇ end of epitope gene. The plus and minus strand of each epitope gene was annealed to form double strand DNA fragments. Then the double strand DNA fragments encoding epitope peptide were cloned into the site between Nco I and Xho I of plasmid pET32c（＋） to construct recombinant plasmid which was transformed into E. coli DH5a. The recombinant plasmid containing P7, P17, P18 genes respectively was identified by PCR, restriction digestion and DNA sequencing, and then transformed into E. coli BL21 （DE3） for expressing the fusion protein. The fusion protein of peptide-thioredoxin（Trx） was expressed by inducing with IPTG and analyzed with SDS-PAGE. The fusion proteins were purified with Ni^2＋ column affinity chromatography. Meanwhile, the peptides PT, P17, P18 were synthesized artificially following their amino acid sequence. By using the purified epitope peptide fusion proteins and synthesized epitope peptides, the splenic cells of C57BL/6J mice immunized with ultraviolet-attenuated cercaria of Schistosoma japonicum were stimulated respectively. The stimulation activity of fusion proteins and synthesized peptides were assayed by detecting the incorporation rate of aH-thymidine. Results The double strand DNA fragments of epitopes PT, P17, P18 were successfully cloned to form recombinant plasmids, all of which could express a 20 kDa fusion protein. Both the fusion protein and synthesized epitope peptides of P7 and P17 were able to stimulate the lymphocyte cells to proliferation effectively. Conclusion The peptide P7 and peptide P17 are testified as T-cell epitopes of Schistosoma japonicum.

关 键 词：日本血吸虫 抗原表位 重组表达 淋巴细胞增殖试验 

分 类 号：R383.24[医药卫生—医学寄生虫学]