The Construction of the Eukaryotic Expression Vector of Glycerophosphodiester Phosphodiesterase Gene from Treponema pallidum and its Expression in Hela Cells  

作　　者：赵飞骏 吴移谋 刘双全 张晓红 余敏君 

机构地区：[1]Institute of Pathogenic Biology,College of Medicine,Nanhua University,Hengyang 421001,China

出　　处：《Chinese Journal of Sexually Transmitted Infections》2005年第1期24-29,共6页中华性传播感染杂志（英文版）

基　　金：Focal point financial assistance entry(002A046)Department of Education, Hunan Province Scientific Research Foundation(B2003-085) Department of Public Health, Hunan province.

摘　　要：Objective: To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase (Gpd) gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods: The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction (PCR) and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1(+) vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes. The expressed protein was identified by immunocytochemistry and Western blot. Results: The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1(+) vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp. pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1(+) vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion: The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.Objective： To construct the recombinant plasmid containing Glycerophosphodiester phosphodiesterase （Gpd） gene from Treponema pallidum and transfect it into Hela cells to express the encoded outer membrane protein. Methods： The Gpd gene was amplified from the genomic DNA of T.pallidum by polymerase chain reaction （PCR） and inserted into cloning vector pUCm-T. The inserted Gpd gene was subcloned into the appropriate site of pcDNA3.1（＋） vector. After identification by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposomes.The expressed protein was identified by immunocytochemistry and Western blot. Results： The target Gpd gene segment was approximately 1059bp. The DNA sequence of the Gpd gene contained in the pcDNA3.1（＋） vector was consistent with the published nucleotide sequence. The homology of the nucleotide and putative amino acid sequences of the Gpd gene between T. pallidum subsp, pallidum Nichols and various pathogenic treponemal strains ranged from 98% to 100%. Immunocytochemistry and Western blot analysis showed that the constructed Gpd-pcDNA3.1（＋） vector expressed a fusion protein with a calculated molecular mass of 41KDa in Hela cells and that the expressed protein reacted with the sera from syphilis patients. Conclusion： The successful construction and expression of the eukaryotic expression plasmid of the Gpd gene from T.pallidum provide a promising tool to further study the biological activity of T.pallidum and develop a DNA vaccine for syphilis.

关 键 词：Treponema pallidum DNA vaccine Gpdgene Eukaryotic Expression 

分 类 号：R737.33[医药卫生—肿瘤]