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目的 克隆源自临床分离的幽门螺杆菌 (Hp)菌株CagADNA序列 ,比较其C 端氨基酸序列与国外菌株的差异 ,并分析其磷酸化能力和转染胃上皮细胞后的生物学功能。方法 从 39株临床分离的HpDNA基因组中用PCR方法扩增cagAC 端DNA ,经琼脂糖凝胶电泳后割胶回收DNA片段进行测序。分别构建cagA的原核细胞和真核细胞表达载体并进行CagAC 端氨基酸磷酸化能力测定。 结果 有 38株菌株检测到CagADNA片段 ,CagA阳性率为 97.4 % (38/ 39)。测序后发现 ,克隆的 38株阳性菌株CagA蛋白均仅含 1个重复序列和 2～ 4个串联的谷氨酸 脯氨酸 异亮氨酸 酪氨酸 丙氨酸(EPIYA)序列 ,且在重复序列的D2区氨基酸序列均为天冬氨酸 苯丙氨酸 天冬氨酸 (DFD)。来自胃癌和非胃癌患者的CagA蛋白串联的EPIYA序列数目差异无显著性。用原核细胞表达的CagA蛋白进行体外磷酸化试验 ,重复序列中的酪氨酸能被磷酸化 ,其余的在EPIYA中的酪氨酸也能被磷酸化 ,但转染的AGS细胞株中仅重复序列中的酪氨酸能被磷酸化。在转染AGS细胞株后少数细胞 (<10 % )因骨架重构而出现典型的“蜂鸟”样改变 ,与直接用Hp感染AGS细胞时类似 ,且对转染的AGS细胞再用Hp感染后 ,其“蜂鸟”样细胞的百分比也无明显增加。结论 源自国人的HpCagA蛋白结构不同于欧美国家
OBJECTIVE: To clone the CagADNA sequence from clinical isolates of Helicobacter pylori (Hp) and compare its C-terminal amino acid sequence with that of foreign strains and analyze its phosphorylation ability and biological function after transfection into gastric epithelial cells. Methods The cagAC DNA was amplified by PCR from 39 clinically isolated HpDNA genomes. The DNA fragments were sequenced after being gelled by agarose gel electrophoresis. Respectively, to construct cagA prokaryotic cells and eukaryotic expression vector and CagAC terminal amino acid phosphorylation assay. Results CagADNA fragments were detected in 38 strains. The positive rate of CagA was 97.4% (38/39). After sequencing, the cloned 38 strains of positive CagA protein contains only one repeat sequence and 2-4 series of glutamate proline isoleucine tyrosine alanine (EPIYA) sequence, and in the repeat The D2 region amino acid sequence of the sequence is aspartic acid phenylalanine aspartic acid (DFD). There was no significant difference in the number of EPIYA sequences in tandem between CagA proteins from gastric and non-gastric cancer patients. In vitro phosphorylation assays were performed with CagA protein expressed in prokaryotic cells, tyrosine in the repeat was phosphorylated, and the rest of the tyrosine in EPIYA was also phosphorylated but only repeated in the transfected AGS cell line Tyrosine in the sequence can be phosphorylated. A few “cells” (<10%) appeared typical “humming” -like changes after skeleton transfection in AGS cell lines, similar to those obtained when AGS cells were directly infected with Hp, and the transfected AGS cells were infected with Hp , The percentage of “hummingbird” like cells did not increase significantly. Conclusion HpCagA protein structure originating from Chinese is different from that of Europe and the United States