重组铜绿假单胞菌外毒素结构域Ia片段的佐剂功效研究

Study on recombinant domain Ia of Pseudomonas aeruginosa exotoxin A (rEPA) as an adjuvant

采用分子克隆技术,将铜绿假单胞菌PA103株编码的外毒素结构域Ia(DomainIa)的基因重组于原核表达载体pET42b(+)上,构建了pETEPA103蛋白表达载体。转化感受态大肠杆菌DE3。经IPTG诱导表达,初步纯化表达蛋白,用以免疫BALB/c纯系小鼠。制备小鼠脾淋巴细胞悬液,经刀豆素A(ConA)刺激后,用MTT比色法检测特异性淋巴细胞增殖反应。通过rEPA皮下注射BALB/c小鼠耳廓,诱导小鼠迟发型过敏反应(DTH)。采用特异性淋巴细胞增殖反应和DTH试验来检测pETEPA103表达蛋白所引起的小鼠细胞免疫应答水平,淋巴细胞增殖情况与DTH均可间接反映细胞免疫应答水平,进而评价重组铜绿假单胞菌外毒素(rEPA)DomainIa蛋白片段的佐剂功效。

By using molecular cloning technique, the Pseudomonas aeruginosa exotoxin A (EPA)domain Ia gene was cloned into the prokaryotic expression vector pET 42b(+), then pET EPA103 was constructed. The pET EPA103 was transformed into E.coli DE 3 and expressed by IDTG induction. The expressed protein was crudely purified. BALB/c mice were immunized with this protein at intervals of 0, 2 and 4 weeks respectively. After immunization, the suspension of mice spleen lymphocytes were prepared and stimulated by ConA . Specific lymphoproliferation was detected by MTT colorimetric assay, and DTH was induced by subcutaneous injecting rEPA into BALB/c mice. Detection of specific lymphoproliferation and DTH can reflect the response level cell mediated immunity. The study results showed that the expression protein of rEPA domain Ia could have the function of adjuvant.