DNA vaccine construction and expression of encoding gene of 18 000 outer membrane protein of human Helicobacter pylori / 中国免疫学杂志

ABSTRACT

Objective:To construct a recombinant eukaryotic expression vector containing gene encoding 18 000 outer membrane protein from human Helicobacter pylori(H.pylori) and be expressed in COS-7 cell,and lay the foundation for the exploiting DNA vaccine of Hp.Methods:The target genes encoding 18 000 outer membrane protein were acquired from the prokaryotic expression vector pET32a(+)/528 by XholⅠ,KpnⅠ digestion simultaneously,in the same way,the pcDNA3.1 was digested by XhoⅠ,KpnⅠ digestion simultaneously,and the objective genes and pcDNA3.1 were extracted out of agarose electrophoresis with gel kit,and connected by T4 ligase.The recombinant vector pcDNA3.1/528 was used to select and transform,meanwhile express in COS-7 cell.The expressions of recombinant eukaryotic vector in COS-7 cell were investigated by reverse transcriptive-polymerase chain reaction and Western.Results:The gene had inserted into eukaryotic vector was the target gene encoding 18 000 outer membrane protein by enzyme digestion analysis,and objective gene was amplified from COS-7 cell transfected with pcDNA3.1/528 by RT-PCR,the Western showed that recombinant eukaryotic vector could be expressed in COS-7 cell.Conclusion:The recombinant eukaryotic vector pcDNA3.1/528 was constructed and expressed in COS-7 cell successfully.The results obtained lay the foundation for research on development of H.pylori DNA vaccine.