金黄色葡萄球菌α-溶血素的克隆及可溶性融合表达

Clone and soluble fusion expression of α-HL of Staphylococcus aureus

目的:表达纯化可溶性金黄色葡萄球菌α-溶血素(α-HL)作为抗原,用以后期制备全人源抗α-HL抗体,为金黄色葡萄球菌感染提供新的治疗手段。方法:提取金黄色葡萄球菌总RNA,经反转录聚合酶链反应(RT-PCR)技术,扩增α-HL cDNA,将cDNA双酶切后与载体pCold-TF连接,转化E.coli TOPO 10,菌落PCR及测序验证插入基因的正确性。将测序正确的α-HL/pCold-TF重组质粒转化E.coli BL21进行低温诱导表达,SDS-PAGE和Western blot验证表达产物的正确性。结果:PCR扩增得到的α-HL基因大小为900 bp左右;将重组质粒α-HL/pCold-TF转化E.coli BL21,经15℃低温诱导表达24 h,诱导得到可溶性的α-HL融合蛋白,融合蛋白大小为90 kD左右(载体上的TF分子伴侣大小为45 kD左右);经Western blot验证表达纯化的蛋白为α-HL融合蛋白。将纯化的α-HL蛋白注射BABL/c小鼠制备抗血清,结果显示抗血清与金黄色葡萄球菌结合良好,效价大于10 000倍。结论:成功对α-HL进行了基因克隆,并成功表达纯化到可溶性的α-HL融合蛋白。

Objective： Expression and purification of the α-HL of Staphylococcus aureus as antigen for making full human anti-α-HL antibody later,providing of new treatment for Staphylococcus aureus infection. Methods： The total RNA of Staphylococcus aureus was extracted and the cDNA of α-HL was amplified by RT-PCR. The DNA of α-HL and p Cold-TF plasmid was digested and ligated by T4 ligase and then transformed into E. coli TOPO 10. The recombinant plasmid α-HL / pCold-TF which verified by sequencing was transformed into E. coli BL21 for expression. The expression products was identified by SDS-PAGE and Western blot. Results： The size of amplified cDNA of α-HL was about 900 bp and the expressed soluble fusion protein of α-HL was about 90 kD（ including the molecular chaperone in the vector） after inducing expression for 24 h at 15℃. The Western blot results showed that the expressed protein was the fusion protein of α-HL. The purified α-HL was injected into BABL / c mice for making antiserum. The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10 000 times. Conclusion： The α-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of α-HL was successfully expressed.