日本血吸虫过氧化物氧化还原蛋白SjPrxⅠ的原核表达及其免疫调节功能

Protein peroxiredoxin-1 of Schistosoma japonicum: expression,purification and its immunoregulation

目的对日本血吸虫氧化还原蛋白(peroxiredoxin of schistosoma japonicum,SjPrx)的表达、纯化,接种小鼠和体外刺激脾细胞,研究SjPrx I在体内外诱导免疫应答的作用及可能机制。方法 Gen Bank获取SjPrx编码序列,设计合成引物进行PCR扩增;构建质粒,通过转化将构建的重组质粒转入感受态细胞,然后小量扩增菌株,电泳检测克隆成功表达。蛋白质印迹法(western blot,WB)验证融合蛋白后大量表达纯化。将重组SjPrxⅠ通过腹腔注射BALB/c小鼠,本科联免疫吸附实验和流式细胞术(flow cytometry,FCM)检测脾细胞分泌细胞因子。结果成功构建了SjPrx I-p ET28a质粒,经异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达后得到可溶性SjPrx I,纯化后的SjPrxⅠ免疫小鼠,其淋巴细胞高水平表达Th2细胞分泌的IL-4(t=6.69,P<0.001)、IL-10(z=3.18,P=0.001)和IL-13(t=4.21,P=0.001)。结论制备了重组SjPrx I,初步认为其具有Th2型免疫调节功能,但并不能显著抑制体内业已存在的Th1的偏移。

Objective To explore the effects and mechanisms regarding SjPrx I in immune responses both in vivo and in vitro via inoculating mice with SjPrx and stimulating spleen cells,after expressing and purifying SjPrx. Methods The SjPrx coding sequence was obtained from Gen Bank,and primers were designed and synthesized for PCR amplification.To construct SjPrx-p ET28 a plasmids and Escherichia coli was transformed with the recombinant plasmid SjPrx-p ET28 a. Fusion expression was induced,purified and verified by Western blotting,and then inoculation was conducted with SjPrx I into BALB / c mice via intraperitoneal injection. Cytokines secreted from spleen cells were detected by ELISA and flow cytometry（ FCM）. Results Recombinant plasmid SjPrx-p ET28 a was successfully constructed,and soluble SjPrxⅠwas purified and obtained by IPTG induction. Th2 cytockines including IL-4（ t = 6. 69,P〈0. 001）,IL-10（ z = 3. 18,P = 0. 001） and IL-13（ t = 4. 21,P = 0. 001） were detected in high expression levels in supernatants of spleen cells of BALB / c mice with r SjPrxⅠadministration. Conclusions The r SjPrxⅠmight have an enhanced effect on Th2 response rather than on underlying Th1 dominated immunity of host animals.