LFn-PSCA融合蛋白的表达和生物活性研究  

Expression and bioactivity of the fusion protein LFn-PSCA

作　　者：于少洋[1] 易绍琼[1] 徐俊杰[1] 葛猛[1] 付玲[1] 于长明[1] 李冠霖[1] 陈薇[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《中华微生物学和免疫学杂志》2006年第10期929-932,共4页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金(30400393)

摘　　要：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 express the fusion protein of LFn-PSCA and test its bioactivity in vitro. Methods Anthrax toxin is a tripartite toxin secreted by Bacillus anthracis that consists of protective antigen （PA）, lethal factor （LF） and oedema factor （EF）. The fragment including N-terminal 254 amino acids of LF （LFn） and the fragment of prostate stem cell antigen （PSCA） gene were amplified by PCR respectively. The two fragments were inserted into the secreted expressed vector pAS22 in tandem. The plasmid carrying the fusion gene was then transformed into E. coli DH5α and the expressed fusion protein was purified by chromatography. The cytotoxic activity and the binding activity of the LFn-PSCA were tested in macmphage cell line J774A. 1 cells. Resuits The fusion protein LFn-PSCA was purified over 90% homogeneity. The bioactivity assay suggests it was non-toxic to J774A. 1 macmphage cells in combination with PA and retained the ability to compete with LF for binding to PA when incubated with J774A. 1 macmphage cells. Conclusion Secreted expression of LFn-PSCA was obtained in E. co/i DH5α. The results provide the basis for exploring the possibility of using an anthrax toxin system in inducing cellular immtmity.

关 键 词：致死因子 前列腺干细胞抗原 融合蛋白 

分 类 号：R392[医药卫生—免疫学]