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目的研究大肠杆菌中表达的炭疽毒素致死因子氨基端与前列腺干细胞抗原(PSCA)融合蛋白的生物活性。方法分别扩增炭疽毒素致死因子LF(lethal factor)N端254个氨基酸(LFn)的基因片段和PSCA氨基酸29～100的基因片段,将两片段先后克隆进分泌型载体pAS22中,构建成表达质粒pAS-LFn-PSCA,在大肠杆菌中表达,并对融合蛋白进行纯化和活性检测。结果实现了融合蛋白LFn-PSCA的分泌型表达。纯化后纯度可达90％以上。体外试验显示LFn-PSCA对小鼠巨噬细胞无毒性,并保留了LFn与保护性抗原结合的活性。结论在大肠杆菌中实现了LFn-PSCA的分泌型表达,为继续进行以炭疽毒素为载体增强机体免疫应答的研究打下基础。
Objective To study the biological activity of the anthrax toxin lethal factor amino terminal and prostatic stem cell antigen (PSCA) fusion protein expressed in Escherichia coli. Methods A gene fragment of 254 amino acids (LFn) at the N terminal of lethal factor and a gene fragment of 29-100 of PSCA amino acids were amplified by PCR and cloned into secretory vector pAS22, respectively, to construct the expression plasmid pAS-LFn-PSCA was expressed in E. coli and the fusion protein was purified and its activity assayed. As a result, the secretory expression of the fusion protein LFn-PSCA was achieved. Purity up to 90% purity. In vitro tests showed that LFn-PSCA is non-toxic to mouse macrophages and retains the activity of LFn in binding to protective antigens. Conclusion The secretory expression of LFn-PSCA was achieved in Escherichia coli, which lays a foundation for the further research on anthrax toxin-enhanced immune response.