摘要
目的构建梅毒螺旋体(Tp)外膜蛋白甘油磷酸二酯磷酸二酯酶基因(Gpd)的真核表达载体pcDNA3.1(+)-Gpd,鉴定其在Hela细胞的表达,为开展Tp DNA疫苗动物实验打下基础。方法用PCR从Tp Nichols株基因组中扩增Gpd基因,构建真核表达重组质粒pcDNA3.1(+)-Gpd,脂质体介导转染入Hela细胞,以免疫组化及Western-blot检测在Hela细胞中的表达。结果双酶切及DNA测序显示重组质粒含有1 059 bp的目的基因片段,读码框架正确,无突变。该重组质粒在Hela细胞能有效表达分子量为41 kDa的目的蛋白,重组蛋白能与梅毒螺旋体阳性血清反应。结论构建的质粒pcD-NA3.1(+)-Gpd成功地在体外真核细胞中表达。
Objective To develop the DNA vaccine to Treponema pallidum （Tp）, a pcDNA3.1 （ ＋ ） - Gpd plasmid was constructed and transfected into Hela cells to express the outer membrane protein Glad. Methods Gpd Gene was cloned from the genomic DNA of Tp by polymerase chain reaction （PCR）, and subcloned into pcD- NA3.1 （ ＋ ） vector. After identified by sequencing and restrictive enzymes digestion, the recombinant plasmid was transfected into Hela cells using liposome. The expressed protein was identified by immunohistochemical technique and western- blot. Results The target gene Gpd fragment about 1059bp was obtained. In Hela cells,the pcD- NA3.1 （ ＋ ） - Gpd plasmid expressed a fusion protein with molecular weight of 41 kDa, and the protein could respond to positive serum from syphilis patient. Conclusion Gpd gene was expressed in eukaryotic cell system, which makes for studying the biological activities and the development of the syphilis DNA vaccine.
出处
《南华大学学报（医学版）》
2005年第4期476-478,494,共4页
Journal of Nanhua University(Medical Edition)
基金
湖南省教育厅重点资助项目(002A008)
湖南省卫生厅科研基金(B2003-085)
