Feng Bai-sui, Yao Xue-hua, Zhao Guo-qiang
Department of Digestive Medicine, Institute of Clinical Medical Research of Universities Henan China, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, China.
Zhonghua Yi Xue Za Zhi. 2007 Feb 27;87(8):570-2.
To construct a eukaryotic green fluorescent protein expressing vector containing the fragment of cytotoxin associated gene A (CagA) of Helicobacter pylori (Hp) so as to lay a foundation for the research of gene vaccine of gastric cancer. pEGFP-C3-CagA.
The fragment of gene CagA was amplified from Hp using PCR. The amplified product was examined by electrophoresis and sequence determination. This fragment was inserted into pGEM-T plasmid and pEGFP-C3 fluorescent expression vector. The recombined plasmid pEGFP-C3-CagA was transfected into the gastric carcinoma cells of the strain BGC823 by lipoplasty method. Fluorescence microscopy was used to observe the expression of pEGFP-C3-CagA under.
CagA was inserted in the plasmid correctly. It was verified by DNA sequencing and restriction enzyme. The enhanced green fluorescent protein eukaryotic expression vector carrying CagA of Helicobacter pylori (pEGFP-C3-CagA) was recombined correctly and transfected in gastric carcinoma cell strain BGC823. Green fluorescence was observed in transfected gastric carcinoma cell.
Construction of the enhanced green fluorescent protein eukaryotic expression vector carrying CagA was successful.
构建含幽门螺杆菌(Hp)细胞毒素相关基因A(CagA)片段的真核绿色荧光蛋白表达载体,为胃癌基因疫苗的研究奠定基础。即pEGFP-C3-CagA。
采用PCR从Hp中扩增CagA基因片段。扩增产物经电泳及测序检测。将该片段插入pGEM-T质粒和pEGFP-C3荧光表达载体。通过脂质体法将重组质粒pEGFP-C3-CagA转染至BGC823株胃癌细胞。利用荧光显微镜观察pEGFP-C3-CagA的表达情况。
CagA正确插入质粒,经DNA测序和限制性内切酶验证。携带幽门螺杆菌CagA的增强型绿色荧光蛋白真核表达载体(pEGFP-C3-CagA)重组正确并转染至胃癌细胞株BGC823。在转染的胃癌细胞中观察到绿色荧光。
携带CagA的增强型绿色荧光蛋白真核表达载体构建成功。