日本血吸虫超氧化物歧化酶的表达与免疫活性鉴定

Expression and identification of immune activity of cytosolic superoxide dismutase of Schistosoma japonicum

目的表达日本血吸虫胞浆内超氧化物歧化酶(superoxide dismutase,SOD),并研究其免疫原性。方法采用反转录PCR方法从血吸虫成虫mRNA中扩增出SOD基因序列,再亚克隆到表达载体pGEX-4T-3中构建重组表达质粒并转化到E.coli BL21(DE3)中。用IPTG对含有重组质粒的转化子细菌进行诱导表达重组的GST-SOD蛋白。用重组GST-SOD蛋白免疫小鼠制备免疫血清,并用免疫印迹试验观察重组GST-SOD融合蛋白的免疫原性。结果日本血吸虫胞浆内SOD基因被成功地扩增,并构建成功含此基因的重组表达质粒Sj SOD-pGEX-4T-3。含重组质粒的E.coli在IPTG的诱导下能表达一大小约43kDa的GST-SOD重组蛋白。用此蛋白免疫小鼠产生的特异性抗体水平最高能达1∶12800,该重组蛋白可以被血吸虫重感染兔血清所识别。结论日本血吸虫中国大陆株胞浆内SOD表达成功,并具有良好的免疫原性。

Objective To express the cytosolic superoxide dismutase （SOD） of Schistosoma japonicurn and analyze its antigenicity. Methods The DNA sequence of Schistosorna japonicurn gene of cytosolic SOD was amplified by reverse transcription polymerase chain reaction （RT-PCR） and subcloned into the expression vector pGEX-4T-3 to construct a recombinant plasmid Sj SOD- pGEX-4T-3. This recombinant plasmid was transformed into component cell of E. coli BL21 （DE3）. The fusion protein of GST-SOD was expressed by inducing with IPTG and purification by affinity chromatography. The specific antiserum was prepared by immunizing the BALB/c mouse with GST-SOD fusion protein, and the immnuogenicity of GST-SOD was investigated by Western blot analysis. Results The gene of cytosolic SOD was amplified successfully and subcloned into expression vector pGEX-4T-3 forming the recombinant expression plasmid Sj SOD-pGEX-4T-3. The fusion protein GST-SOD was expressed after the recombinant containing Sj SOD-pGEX-4T-3 being induced by IPTG. Immunizing the BALB/c mouse with the fusion protein GST-SOD produced high titer specific antiserum of 1 ： 12 800 and the fusion protein GST-SOD was recognized by sera of severe infection rabbits. Conclusions The cytosolic SOD has been expressed successfully and has a preferable immunogenicity.