摘要
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。
Objective To evaluate the diversity and biological activity of the Helicobacter pylori(H.pylori) virulence factor CagA isolated in Chinese. Methods The cagA DNA fragments were amplified by PCR from the genomic DNA of 39 isolated strains, 19 of which were from patients with gastric cancer and 20 from chronic gastritis. The DNA fragments were sequenced and cloned respectively, and the ability of phosphorylation in tyrosine in CagA protein was assessed. Results cagA + was found in thirty eight (97.4%) isolated strains. DNA sequencing of all amplified fragments indicated that all cagA + strains contained only one repeated sequence and distinctive tandem five amino acid motif glutamic acid proline isoleucine tyrosine alanine (EPIYA) in the C terminal of the CagA. The number of EPIYA motif was between 2 and 4 in different strains, each of which was an irregular interval of 13 50 amino acids, but there was no significant difference of the number of tandem five amino acid motif EPIYA between gastric cancer strains and gastritis ones. The amino acid sequence (aspartic acid phenylanaline aspartic acid,DFD) in D2 repeated sequence region was also different from that in the Western standard strain (aspartic acid aspartic acid leucine,DDL). Transient expression of CagA in AGS cell was found so that CagA was able to induce the elongation phenotype in small portion of cells (<10%) but to a much lesser extent than Western strains, the finding similar to that of infection of AGS cells with H.pylori bacteria. In addition, transfection of AGS cells followed by infection with the same H.pylori strain did not increase the cellular phenotype. The phosphorylation assay to identify the tyrosine phosphorylation sites in EPIYA was also used. The results demonstrated that tyrosine phosphorylation sites in EPIYA both containing and lacking repeated sequence of CagA protein could be phosphorylated by AGS cell lysate in vitro. However, all these sites can not be phosphorylated in vivo phosphorylation assay. Onl
出处
《中华消化杂志》
CAS
CSCD
北大核心
2004年第5期278-281,共4页
Chinese Journal of Digestion
