恶性疟原虫动合子表面蛋白单链抗体与按蚊Cecropin A融合基因的构建、原核表达及生物活性的初步评价

Construction and prokaryotic expression of the fusion gene Cep&m4B7,and preliminary evaluation of its bioactivity

目的构建、原核表达恶性疟原虫动合子表面蛋白单链抗体与按蚊Cecropin A融合基因并对其表达产物的生物学活性进行评价。方法根据按蚊对编码同种氨基酸不同密码子的偏嗜性,对鼠源性恶性疟原虫动合子表面蛋白单链抗体4B7编码基因进行改造,并与按蚊抗菌肽Cecropin A基因融合;将融合基因Cep&m4B7克隆入原核表达载体pET32a(+)并在大肠埃希菌中进行表达;对表达产物进行SDS-PAGE和Western blot分析,体外抑菌试验鉴定表达蛋白的抑菌活性。结果构建了融合基因Cep&m4B7和重组表达质粒pET32a(+)-Cep&m4B7,融合基因在大肠埃希菌中以包涵体形式高效表达融合蛋白,Western blot显示重组蛋白能被抗6-His抗体识别;琼脂糖扩散法显示纯化后的目的蛋白无抑菌活性。结论成功构建了融合基因Cep&m4B7,该基因在大肠埃希菌中高效表达,表达产物纯化后无体外抑菌活性。

Objective To construct and express the fusion gene Cep＆m4B7 and preliminarily evaluate the biologic activity of the expression product. Methods The coding sequence of the single chain antibody 4B7 from mice was modified according to Anopheles gambiae preferred codons, and the modified gene （m4B7） was then fused with the coding sequence of Cecropin A. The fusion gene Cep＆m4B7 was subcloned into the prokaryotic expression vector pET32a（＋） and expressed in Escherichia coli after the inducement of IPTG. The expressed product was analyzed by SDS-PAGE and Western blot, and its biologic activity was evaluated with agar diffusion method. Results The fusion gene Cep＆m4B7 was correctly constructed and the recombinant plasmid pET32a（＋）-Cep＆m4B7 was expressed effectively in E. coli in the form of inclusion bodies. The fusion protein reacted with 6-his-tag antibody specifically. The outcome of agar diffusion method showed that the recombinant protein has no bacteriostatic activity, Conclusion The fusion gene Cep＆m4B7 was constructed successfully and highly expressed in E. coll. However, the purified recombinant protein showed no bacteriostatic activity in preliminary test.