幽门螺杆菌GroEL基因真核表达载体构建及鉴定

目的构建幽门螺杆菌热休克蛋白GroEL基因的真核表达载体pcDNA3.0-GroEL,为幽门螺杆菌的基因疫苗研制提供参考依据。方法提取幽门螺杆菌基因组DNA,PCR扩增GroEL基因,克隆至pMD19-T载体,通过PCR、酶切及测序鉴定后,将GroEL基因片段用限制性内切酶切下,克隆至真核表达载体pcDNA3.0,构建pcDNA3.0-GroEL重组质粒;采用PCR、酶切对重组质粒pcDNA3.0-GroEL进行鉴定。结果扩增出幽门螺杆菌GroEL基因片段约1 640 bp;pcDNA3.0-GroEL重组质粒经XhoⅠ和EcoRⅠ双酶切,产生1个与GroEL基因PCR产物大小一致的小片段和1个不同于pcDNA3.0-GroEL重组质粒的大片段,表明GroEL基因已成功插入pcDNA3.0质粒中。结论成功构建幽门螺杆菌GroEL基因真核表达载体pcDNA3.0-GroEL。

Construction and identification of a Helicobacter pylori GroEL gene

Objective To construct and identify a Helicobacter pylori(H.pylori) GroEL eukaryotic expression vector pcDNA3.0-GroEL for the development of gene vaccine of H.pylori.Methods Genomic DNA of H.pylori was extracted from H.pylori ATCC 26695.The GroEL gene fragment was amplified from the genomic DNA of H.pylori by PCR.The purified PCR fragment was ligated into the pMD19-T simple vector.The positive clones were screened and identified by PCR from bacteria directly and the PCR products were digested via double enzymes following sequencing of the recombinant plasmid.The GroEL gene fragments from TA clones were cloned into the eukaryotic expression vector pcDNA3.0 using restriction enzymes.The recombinant plasmid pcDNA3.0-GroEL was identified by PCR,enzyme digestion and sequencing.Results The GroEL gene fragment was amplified correctly,with a gene of about 1 640 bp,and a H.pylori GroEL eukaryotic expression vector pcDNA3.0-GroEL was successfully constructed.This indicated that GroEL gene was successfully inserted into pcDNA3.0 plasmid.Conclusion Construction of the H.pylori GroEL eukaryotic expression vector pcDNA3.0-GroEL lays a foundation for the development of H.pylori gene vaccine.