重组日本血吸虫中国大陆株磷酸丙糖异构酶蛋白质表达、纯化及特性分析

EXPRESSION AND CHARACTERIZATION OF RECOMBINANT TRIOSEPHOSPHATE ISOMERASE(RTPI)OF SHISTOSOMA JAPONICUM CHINESE STRAIN

磷酸丙糖异构酶（ＴＰＩ）是已被公认的血吸虫病疫苗候选分子之一，本研究将日本血吸虫中国大陆株ＴＰＩ分子基因ＤＮＡ片段定向克隆到表达载体ｐＧＥＸ－４Ｔ－３（编码２６ｋＤａ的ＧＳＴ蛋白）中，构建重组表达质粒，然后转化大肠杆菌ＢＬ２１感受态细胞，阳性克隆用ＩＰＴＧ诱导，阳性菌株可表达分子量约５５ｋＤａ的融合蛋白（ＧＳＴ－ＴＰＩ），此融合蛋白可与ＧｌｕｔａｔｈｉｏｎｅＳｅｐｈａｒｏｓｅ４Ｂ结合成复合物，用凝血酶切割融合蛋白复合物，获得分子量约２８ｋＤａ的重组表达ＴＰＩ分子。重组ＴＰＩ分子具有磷酸丙糖异构酶活性，并能被日本血吸虫重感染兔血清，血吸虫病人血清所识别。重组ＴＰＩ免疫家兔产生抗体的效价最高可达１∶２５６００，该血清抗体能识别日本血吸虫中国大陆株成虫抗原中分子量约２８ｋＤａ的蛋白条带。

The Triosephosphate Isomerase(TPI) is one of vaccine candidatesi of Schistosomiasis admittedly.The gene of TPI was subcloned into the expression vector pGEX4T3(which encoding 26kDa GST protein)to construct the expression plasmid,the recombinant plasmid were transformed to component cells of Ecol.BL21.The positive strain containing the recombinant plasmid could express a specific fusion protein (GST-TPI,WM55kDa)induced by IPTG,the fusion protein could coupled with Glutathione Sepharose 4B specifically.A pure rTPI protein (MW28kDa) was obtained from the fusion protein by digesting the conjugated body of Glutathione sepharose 4Bfusion protein with the Thrombin.The pure rTPI has the activity of Triosephosphate Isomerase(TPI),and may be recognized by the serum of rabbit infected seriously with Schistosoma japonica and patient suffering from schistosomiasis.The rabbits were vaccinated with rTPI,the highest level of the anti-rTPI antibody was 1256000,the Westernblot test showed that the anti-TPI antibody could recognize the antigen (MW28kDa) of adult worm of Schistosoma japonica Chinese strain.