裂解基因E和核酸酶基因串联表达载体的构建及大肠杆菌菌影的制备

目的构建噬菌体裂解基因E和核酸酶基因串联表达载体,制备高质量的大肠杆菌菌影。方法自行设计一对引物,PCR扩增PhiX174噬菌体裂解基因E,将该基因亚克隆至原核表达载体pGEX-6P-1,构建大肠杆菌菌影的表达载体pGEX-E。在E基因基础上与辅助基因葡萄球菌核酸酶A(SN)基因串联,并插入pGEX-6P-1载体,构建双基因串联高效表达载体pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,采用CaCl2法将其转入大肠杆菌BL21(DE3),经IPTG诱导,制备大肠杆菌菌影。结果裂解基因E单基因及串联基因已成功插入融合表达载体pGEX-6P-1,所构建的大肠杆菌菌影表达载体pGEX-E、pGEX-E-5aaLinker-SN和pGEX-E-15aaLinker-SN,诱导后经透射电镜及活菌计数显示大肠杆菌已发生不同程度裂解,制成了大肠杆菌菌影。电镜下菌影形态完整,内容物已释放到胞外。结论已成功构建了噬菌体裂解基因E单基因及裂解基因和核酸酶基因串联基因表达载体,并制成了大肠杆菌菌影,为进一步研究菌影这一新型的疫苗及佐剂形式奠定了基础。

Construction of Prokaryotic Coexpression Vector for Lysis Gene E and Staphylococcus Nuclease Gene and Preparation of E.coli Bacterial Ghost

Objective To construct the prokaryotic coexpression vector for lysis gene E and Staphylococcus nuclease gene and prepare highly purified E.coli bacterial ghost.Methods Amplify lysis gene E from plasmid PhiX174 RFI by PCR using a pair of designed primers and subclone to recombinant plasmid pGEX-6P-1 to construct expression vector pGEX-E for E.coli bacterial ghost.Link JP2]Staphylococcus nuclease A(SN) gene to the amplified E gene,then insert the tandem gene into recombinant plasmid pGEX-6P-1.JP]Transform the constructed high expression vector pGEX-E-5aaLinker-SN and pGEX-E-15aaLinker-SN to E.coli BL21(DE3) by calcium chloride method for expression of bacterial ghost under induction of IPTG.Results Both lysis gene E and tandem gene were successfully inserted into coexpression vector pGEX-6P-1. Transmission electron microscopy and viable counting proved that the bacteria of E.coli transformed with pGEX-E,pGEX-E-5aaLinker-SN and pGEX-E-15aaLinker-SN were lyzed at different degrees,and bacterial ghosts were produced.The bacterial ghosts were in intact forms under transmission electron microscope,and their contents were released to extracellular region.Conclusion The prokaryotic coexpression vector for lysis gene E and Staphylococcus nuclease gene was successfully constructed,and E.coli bacterial ghosts were prepared,which laid a foundation of study on bacterial ghost as a novel vaccine and adjuvant.