To investigate the DNA sequence polymorphism of Chlamydia trachomatis omp1 gene, urogenital samples were collected from 4 different cities in South China, DNA was extracted, and an approximately 980-bp-long fragment o...To investigate the DNA sequence polymorphism of Chlamydia trachomatis omp1 gene, urogenital samples were collected from 4 different cities in South China, DNA was extracted, and an approximately 980-bp-long fragment of the omp1 gene was amplified by nested polymerase chain reaction (nPCR). DNA sequence was determined, genotyping was performed by BLAST similarity search, and multiple alignment was performed with CLUSTAL X. Then a phylogenetic tree was constructed by Mega 3 software to illustrate the evolutionary relationships between clinical isolates and reference strains. Ninety-six specimens were sequenced, and 28 genetic variants were detected, among which E was the most prevalent genotype. The omp1 gene was highly conserved for genotypes E and F, but appeared slightly less conserved for other genotypes, where the sequences displayed one to several nucleotide substitutions relative to the reference sequence. Phylogenetic tree showed that C.trachomatis serotypes were mainly divided into three clusters, according to previous grouping in the B, F-G, and C complexes, and the clinical isolates were highly related to the corresponding reference strains. It concluded that the omp1 gene of the isolated C.trachomatis strains exhibited remarkable DNA sequence polymorphism, which can encourage for vaccine design and infection control.展开更多
Objective:To directionally clone the ompl gene from Chlamydia trachomatis(Ct)F Genotype onto a plasmid vector for constructing a rudimentary DNA vaccine.Methods:The complete ompl gene from genomic DNA of Ct F genotype...Objective:To directionally clone the ompl gene from Chlamydia trachomatis(Ct)F Genotype onto a plasmid vector for constructing a rudimentary DNA vaccine.Methods:The complete ompl gene from genomic DNA of Ct F genotype wild species was amplified with primers designed by computer.The recombinant gene was obtained by restriction enzyme cutting,linking the gene with the plasmid vector in vitro,transforming the recombinant gene into bacteria,and extracting the DNA from the bacteria.Results:DNA extracting from the bacteria was composed of the impl gene and plasmid,which is identified by three methods of singular restrictive enzyme cutting,double restrictive enzyme cutting and PCR.Conclusion:Cloning of the ompl gene from the Ct F genotype means that a rudimentary DNA vaccine was successfully constructed.展开更多
To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was ampli...To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.展开更多
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 seque...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.展开更多
文摘To investigate the DNA sequence polymorphism of Chlamydia trachomatis omp1 gene, urogenital samples were collected from 4 different cities in South China, DNA was extracted, and an approximately 980-bp-long fragment of the omp1 gene was amplified by nested polymerase chain reaction (nPCR). DNA sequence was determined, genotyping was performed by BLAST similarity search, and multiple alignment was performed with CLUSTAL X. Then a phylogenetic tree was constructed by Mega 3 software to illustrate the evolutionary relationships between clinical isolates and reference strains. Ninety-six specimens were sequenced, and 28 genetic variants were detected, among which E was the most prevalent genotype. The omp1 gene was highly conserved for genotypes E and F, but appeared slightly less conserved for other genotypes, where the sequences displayed one to several nucleotide substitutions relative to the reference sequence. Phylogenetic tree showed that C.trachomatis serotypes were mainly divided into three clusters, according to previous grouping in the B, F-G, and C complexes, and the clinical isolates were highly related to the corresponding reference strains. It concluded that the omp1 gene of the isolated C.trachomatis strains exhibited remarkable DNA sequence polymorphism, which can encourage for vaccine design and infection control.
文摘Objective:To directionally clone the ompl gene from Chlamydia trachomatis(Ct)F Genotype onto a plasmid vector for constructing a rudimentary DNA vaccine.Methods:The complete ompl gene from genomic DNA of Ct F genotype wild species was amplified with primers designed by computer.The recombinant gene was obtained by restriction enzyme cutting,linking the gene with the plasmid vector in vitro,transforming the recombinant gene into bacteria,and extracting the DNA from the bacteria.Results:DNA extracting from the bacteria was composed of the impl gene and plasmid,which is identified by three methods of singular restrictive enzyme cutting,double restrictive enzyme cutting and PCR.Conclusion:Cloning of the ompl gene from the Ct F genotype means that a rudimentary DNA vaccine was successfully constructed.
文摘To clone and construct the recombinant plasmid containing the major outer membrane protein (MOMP) gene of Chlamydia trachomatis (C.trachomatis) and to express the fusion protein in E.coli BL21, the MOMP gene was amplified by polymerase chain reaction (PCR) from genome of C.trachomatis serovar D. The fragment was cloned into the prokaryotic expression vector pET-22b(+) after digestion with BamHⅠ and NotⅠ and transformed into E.coli XL1-Blue. Recombinants were selected by enzyme digestion and sequencing and the recombinant plasmid with MOMP gene was then transformed into E.coli BL21 with IPTG to express the target gene. The expression recombinant proteins were purified by Ni-NTA affinity chromatography, and identified by SDS-PAGE and Western blot. It was found that a 1.2?kb MOMP gene was isolated. The DNA sequence of MOMP was found to be just the same as the sequence published by GenBank. A recombinant plasmid containing MOMP gene was constructed to express the fusion proteins in E.coli. SDS-PAGE analysis showed that the relative molecular weight of the recombinant protein was about 47?kDa that was consistent with the theoretical predicted value, and the specificity of the expressed protein was conformed by Western blot. It concluded that the MOMP gene could be expressed in the prokaryotic system, by which it provided the foundation for the future studies on the biological activities of C.trachomatis and for the development of vaccine against this pathogen.
文摘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.
