一种减毒肺炎链球菌溶血素突变体的构建与表达

Construction and Over-expression of A Pneumolysin Mutant Derived from Streptococcus Pneumoniae D39

目的使用重叠PCR方法构建构建△Al46Ply突变体，原核可溶性表达△Al46Ply蛋白，并明确其毒力变化情况；分析肺炎链球菌溶血素（pneumolysin，Ply）在不同血清型肺炎链球菌（streptococcus pneumoniae，SPN）中的表达情况。方法以SPND39型基因组DNA为模板设计合成构建突变体pry基因所需引物；利用重叠PCR方法扩增合成△Al46ply突变体。通过溶血实验分析其溶血活性，利用中和试验验证△A146Ply诱导产生的特异性抗体中和野生Ply毒素溶血能力，并利用Western印迹检测5株不同血清型肺炎链球菌流行菌株中Ply蛋白表达情况。结果突变体基因测序结果显示，Plyl46位密码子GCT3个碱基被缺失，△Al46ply突变体构建成功，并实现了△Al46Ply的可溶性表达，得到纯度〉90％的重组蛋白。△A146Ply蛋白浓度为100000ng／ml亦未表现出溶血活性。△Al46Ply蛋白诱导产生的特异性抗体能够中和野生Ply毒素的溶血活性。Western印迹结果显示，△Al46Ply诱导产生的多克隆抗体可与国内临床常见4株肺炎链球菌有交叉反应。结论△Al46Ply蛋白是一种安全的肺炎链球菌疫苗候选分子，可刺激机体产生具有中和作用的特异性抗体。

Objective To construct △A146ply mutant by the method of overlap PCR and produce soluble △A146Ply protein. Methods Template DNA was isolated from cultured Streptococcus pneumoniae D39, and upstream and downstream fragments of the target site were amplified with their specific primers, followed by an overlap extension with the upstream and downstream fragments as template DNA molecules. The ensuing full-length PCR product was then cloned into pW28 ex- pression vector, followed by sequencing. Protein △ A146Ply was induced with IPTG and further putiffed. It was then analyzed by SDS-PAGE. Results Oligonucleotides GCT, which determines the synthesis of the 146th Alanine, was successfully deleted from ply gene. △A146ply gene was successfully cloned into pW28 vector, which was confirmed by gene sequencing. △A146Ply fusion protein was successfully expressed in soluble manner under the condition of 0. 3 M IPTG at 23 ℃. △ A146Ply nearly lost its hemolytic effectgiven that no red blood cells were lysed even at the concentration of 100 000 ng/ml. Additionally, anti-△A146Ply serum was able to block the hemolyisis effeet caused by pneumolysin. Western-blot analysis revealed that anti-△A146Ply serum cross-reac- ted with the four most common clinical isolates used in this experiment. Conclusion A safer △A146Ply protein is suceessfully obtained, whieh can be used to further evaluate its effeetiveness against pneumocoocal infections in animals.