幽门螺杆菌omp11基因的克隆及序列分析

Cloning and sequence analysis of the omp11 gene of Helicobacter pylori

目的 对幽门螺杆菌 (Helicobacterpylori,Hp)郑州分离株MEL Hp2 7和NCTC116 37株外膜蛋白基因 (omp11)进行克隆和测序 ;确定不同Hp菌株omp11基因序列的变异性 ,并对该基因编码多肽的化学及免疫学特性进行预测 ,为幽门螺杆菌疫苗抗原的筛选提供数据。方法 提取Hp染色体DNA ,用自行设计的PCR引物 ,从染色体DNA上扩增出omp11基因 ,将其克隆到载体pNEB193中 ,用重组质粒转化大肠杆菌 (E .coliJM10 9)。对重组质粒进行酶切鉴定 ,对插入的omp11基因片段进行测序 ,应用生物信息学软件Omiga2 .0、GeneDoc2 .3和GenBank、Swiss port数据库对 4个Hp菌株 (MEL Hp2 7、NCTC116 37、2 6 6 95、J99)的omp11基因序列进行同源性分析 ,并对该基因编码多肽的主要化学特征和抗原结构域进行预测。结果 MEL Hp2 7和NCTC116 37株omp11基因的核苷酸序列长度均为5 6 1bp ,不同菌株间核苷酸序列的同源性为 96 .6 %～ 98.0 % ,与国内的MEL Hp2 7株同源性最高的菌株是NCTC116 37,二者的同源性为 97.9%。 4株Hpomp11基因编码的氨基酸序列长度均为 186aa ,不同菌株间氨基酸序列的同源性为 98.9%～ 10 0 % ,与MEL Hp2 7株同源性最高的菌株是 2 6 6 95 ,二者的同源性为 99.5 %。预测HpMEL Hp2 7omp11基因编码多肽的相对分子质量 (Mr)

Objective To clone and sequence the omp11 gene of the H.pylori strains NCTC111637 and MEL- Hp 27 isolated from a patient in Zhengzhou city and to determine the diversity of the omp11 genes of H.pylori by sequence analysis of the omp11 genes of 4 H.pylori strains. To investigate the chemical and immunological characteristics of the peptides encoded by the genes for development of H.pylori vaccine. Metholds H.pylori chromosomal DNAs were prepared with H.pylori strains NCTC11637 and MEL- Hp 27 isolated by us. The omp11 gene of H.pylori was amplified from H.pylori chromosomal DNA by PCR. The PCR product was cloned into the vector pNEB193. The recombinant vector was identified by restriction enzyme digestion and sequencing method. The bioinformatics software Omiga 2.0 and GeneDoc 2.3 were employed to analyze the homology of 4 Hp strains (MEL- Hp 27, 11637, 26695, J99) and the chemical and immunological characteristics of the corresponding peptides. Results The lengths of the omp11 genes of the 4 strains were the same as 561bp. The homology of the strains in nucleotide acid was 96.6%～98.0%. The strain MEL- Hp 27 was quite identical to NCTC11637 than the others with homology of 97.9%. The corresponding peptides of the 4 strains showed the same length of 186 aa. Their homogeneity in the amino acids was 98.9%～100%, while the strain MEL- Hp 27 was most identical to 26695 as much as 99.5%. The putative peptide showed a molecular weight of 21.939kDa,a isoelectric point of 9.373 ,and 4 antigenic domains. Conclusions The H.pylori omp11 gene sequence and the putative amino acid sequence were quite conservative and the Chinese strain MEL- Hp 27 showed the highest homology with the oversea strain 26695 in the amino acids sequence. The corresponding peptide of the gene performed the structural characteristics of some typical antigen molecules, which suggest that it might be a novel vaccine candidate.