Molecular characteristics of the omp A gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of >105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.

羊流产衣原体主要外膜蛋白基因的克隆与序列分析

将自行分离、传代培养的内蒙古地区山羊流产衣原体按常规方法分离纯化 ,提取衣原体基因组DNA作为模板 ,按照国外发表的衣原体主要外膜蛋白 (MOMP)基因两端序列设计合成一对引物 ,用PCR方法扩增出一 1 17Kb的DNA片段。利用引物上预先设计的限制性内切酶位点 ,将扩增片段经限制性内切酶切割后连接到 pUC19质粒相应位点上 ,转化大肠杆菌DH5α ,筛选重组子。经PCR检测和内切酶分析鉴定含MOMP基因的重组子质粒。对克隆片段进行全序列分析 ,结果证明得到MOMP全编码序列的基因克隆。本株衣原体MOMP编码区由 1170个核苷酸组成。序列比较发现本株衣原体的MOMP基因与国外的羊流产衣原体S2 6/3株的MOMP基因完全相同 ,与B577株的MOMP基因仅有一个核苷酸的同义变异。

沙眼衣原体多态膜蛋白PmpF基因克隆及生物信息学分析

目的克隆沙眼衣原体的多态膜蛋白PmpF基因,分析其生物学特征。方法以沙眼衣原体L2菌株DNA为模板,PCR扩增PmpF基因,克隆后测序,采用生物信息软件、数据库对其进行同源性比对,并分析其编码蛋白的主要化学特征、结构功能以及B细胞抗原表位。结果 PCR扩增实验株沙眼衣原体PmpF基因核苷酸序列长度为3 099bp,该菌株与同一生物型同源性≥99.97%,与不同生物型同源性<89.32%;与同一生物型氨基酸序列同源性≥99.90%,与不同生物型同源性达<89.25%。编码蛋白的分子质量单位为112.538 2ku,等电点为9.15,该蛋白具有信号肽和2个结构域。对蛋白质的二级结构和柔性区、氨基酸的亲水性、抗原指数及表面可及性预测结果分析,推测该蛋白含有14个优势B细胞抗原表位。结论本研究克隆了沙眼衣原体PmpF基因,推测其表达蛋白含B细胞抗原表位。为研究此基因的生物学功能奠定了基础。

北京地区育龄妇女泌尿生殖道沙眼衣原体感染状况及基因分型初步研究

目的对北京地区育龄妇女沙眼衣原体的感染状况及基因分型进行初步研究。方法利用胶体金免疫法对 177例计划生育门诊及妇科体检的育龄妇女进行筛查，对 9例沙眼衣原体感染阳性者的拭子标本直接进行套式 PCR扩增主要外膜蛋白（ MOMP）基因。将扩增的 DNA进行限制性内切酶长度多态性分析，并对 MOMP基因易变区 VS1、 VS2、 VS3、 VS4进行基因序列分析。结果 177名育龄妇女有 9例沙眼衣原体感染阳性， 9份标本均扩增出 1.1 kb目的 DNA，经限制性内切酶长度多态性分析， 4例确定为 F型， 3例为 E型， 2例为 D型。利用 373A自动序列分析仪，对 MOMP基因易变区 VS1、 VS2、 VS3、 VS4进行基因序列分析，与标准序列比较发现， 2例为 D型， 3例为 E型， 4例为 F型，与限制性内切酶长度多态性分析结果相同，但其中 4例有 MOMP基因变异存在。结论北京地区健康育龄妇女存在一定比例的沙眼衣原体感染，利用对 MOMP基因的多态性分析可以对沙眼衣原体的流行情况作出分析。