钩端螺旋体外膜脂蛋白LipL32的重组表达及纯化

目的：构建L32-pQE32重组质粒，诱导表达重组钩端螺旋体外膜脂蛋白LipL32，对重组蛋白进行纯化。方法：采用PCR法从钩端螺旋体DNA中获取编码LipL32的基因片段，构建重组克隆载体pGEM-T／L32和表达载体L32-pQE32，转化受体茵E.coli DH5α和E．coli M15，IPTG诱导表达重组LipL32蛋白。Ni-NTA亲和层析纯化重组LipL32蛋白。结果：扩增出约750bp的LipL32成熟蛋白基因，LipL32基因插入pQE32表达载体，表达产物6个组氨酸与LipL32蛋白的融合蛋白相对分子质量约为31000，与预期大小一致。Western印迹显示能与钩体抗血清特异结合。结论：LipL32蛋白能在大肠杆菌中表达，Ni-NTA亲和层析可有效纯化重组LipL32蛋白，重组LipL32蛋白具有结合活性。

Cloning,expression and purification of leptospiral outer membrane lipoprotein LipL32

Objective:To construct L32-pQE32 recombinant expres sion vectors, and to induce the expression of recombinant leptospiral outer membrane lipoprote in LipL32 and purify the recombinant protein. Methods: Ge ne coding leptospiral LipL32 protein was amplified by PCR,then recombinant cloning vector pGEM-T/L32 and expression vector L32-pQE32 were constructed. The recombinant expression vect or was transformed into the competent host E.coli M15. Recombinant leptospiral Li pL32 protein was expressed by IPTG induced method and purified by Ni-NTA affinity chromatography. Results: Mature leptospiral LipL32 gene fragment about 750 bp was amplified by PCR. LipL32 gene was inserted into expression vector pQE32. The molecular weight of fusion protein was corresponding to the estimated molecular size of mature leptospiral LipL32 protein. Results of Western-blot demonstrated intense LipL32 reactivity with anti-Leptospira sera. Conclusion: LipL32 can be expressed in E.coli. Recombinant leptospiral LipL32 protein can be purified by Ni-NTA affinity chromatography. This protein has high immunoreactivity.