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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 study the expression and purification of Schistosoma japonicum (SjPrx), inoculate mice and in vitro stimulation of spleen cells with SjPrx I in vitro and in vivo. Methods Gen Bank SjPrx coding sequence was designed, primers were designed for PCR amplification. The plasmid was constructed and transformed into competent cells by transformation. Then a small amount of amplified strains were obtained. The clones were successfully detected by electrophoresis. The fusion protein was verified by western blot (WB). The recombinant SjPrx Ⅰ was injected into BALB / c mice via intraperitoneal injection, and the cytokines secreted by splenocytes were detected by the BMI and flow cytometry (FCM). Results SjPrx Ip ET28a plasmid was successfully constructed, and soluble SjPrx I was induced after induced by isopropyl β-D-thiogalactoside (IPTG). The purified SjPrxⅠ-immunized mice, The lymphocytes expressed 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) secreted by Th2 cells at a high level. Conclusion Recombinant SjPrx I was preliminarily considered to have Th2-type immunomodulatory function, but it did not significantly inhibit the pre-existing Th1 shift in vivo.