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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 outer membrane protein gene (omp11) of MEL Hp2 7 and NCTC116 from Helicobacter pylori (Hp) isolates in Zhengzhou City. The variability of the omp11 gene sequences of different Hp strains was determined. Chemical and immunological characteristics of the prediction for H. pylori vaccine antigen screening data. Methods Hp chromosomal DNA was extracted and the omp11 gene was amplified from the chromosomal DNA by using the designed PCR primers. The omp11 gene was cloned into the vector pNEB193 and transformed into E. coli (E. coli JM109) using the recombinant plasmid. The recombinant plasmids were identified by restriction enzyme digestion and sequencing. The inserted omp11 gene fragments were sequenced. Four strains of Hp (MEL Hp27, NCTC116 37, 2 6) were sequenced using bioinformatics software Omiga2 .0, GeneDoc2 .3 and GenBank, Swiss port database 695, J99) homology analysis of the omp11 gene sequence, and the gene encoding the main chemical characteristics of the polypeptide and the antigen domain prediction. Results The nucleotide sequences of the omp11 gene in 37 strains of MEL Hp2 7 and NCTC116 were all 56 bp in length and 96.6% to 98.0% in the nucleotide sequences of different strains. The most-derived strain was NCTC11637, with a homology of 97.9%. The four Hpomp11 genes encoded amino acid sequences were all 186aa, the amino acid sequence homology between different strains was 98.9% ~ 100%, and the strain with the highest homology with MEL Hp27 was 2 6 6 95 The homology is 99.5%. Prediction of relative molecular mass of HpMEL Hp2 7omp11 gene encoding polypeptide (Mr)