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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。
Objective To construct the eukaryotic expression vector pcDNA3.0-GroEL of Helicobacter pylori heat shock protein GroEL gene and provide references for the gene vaccine of Helicobacter pylori. Methods Genomic DNA of Helicobacter pylori was extracted by PCR. The GroEL gene was amplified by PCR and cloned into pMD19-T vector. After identification by PCR, restriction enzyme digestion and sequencing, the GroEL gene fragment was cut with restriction enzyme and cloned into eukaryotic expression vector pcDNA3.0 to construct recombinant plasmid pcDNA3.0-GroEL; using PCR, digested recombinant plasmid pcDNA3.0-GroEL identified. As a result, a fragment of GroEL gene of Helicobacter pylori was amplified by about 1 640 bp. The recombinant plasmid pcDNA3.0-GroEL was double digested with XhoⅠ and EcoRⅠ to generate a small fragment of the same size as the GroEL gene PCR product and a different fragment from pcDNA3. Large fragment of the 0-GroEL recombinant plasmid, indicating that the GroEL gene has been successfully inserted into the pcDNA3.0 plasmid. Conclusion The Helicobacter pylori GroEL gene eukaryotic expression vector pcDNA3.0-GroEL was successfully constructed.