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

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 7端带有Nco I酶切位点，负链的3‘端带有Xho I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，定向克隆人表达载体pET32c(+)，电转化至E．coli DH5a，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。提取重组质粒转化至E. coli BL21(DE3)中，用异丙基硫代f3-D半乳糖苷对表位肽融合蛋白进行诱导表达，经12%十二烷基硫酸钠一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Niz+螯合亲和层析胶纯化。同时根据P7、P17及P18的氮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、P18 3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20 kDa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。

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

 Objective  To synthesize and fusion express the predicted T-cell epitopes of Schistoso- ma 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 plas- mid 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 N12+ column affinity chromatography. Meanwhile, the peptides P7, P17, P18 were synthesized artificially following their amino acid se- quence. 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 japon- icum were stimulated respectively. The stimulation activity of fusion proteins and synthesized pep- tides were assayed by detecting the incorporation rate of 3H-thymidine. Results  The double strand DNA fragments of epitopes P7, 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. Con- clusion  The peptide P7 and peptide P17 are testified as T-cell epitopes of Schistosoma jaPon,icum.