A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplif...A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR.The target fragment was inserted into the linearized pGEM-T easy vector.After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software.The analysis results showed that the cloned DNA fragment had a longest open reading frame(ORF) of 1035 nt,it predicted to be encoded a 344-aa protein with the molecular weight of 36 kD.Hydrophobicity analysis suggested that the protein was highly hydrophilic,especialy at the first 24 amino-acid,this region could function as signal peptide.The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene,and the alignment of the amino acid sequence was 98%.The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E.coli BL21(DE3)by BamH and Sal I.The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GST-MOMP,furthermore,the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AHML316.Considering all these together,it proved that the cloned gene represented the major outer membrane protein gene of AHML316,and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.展开更多
以军团菌DNA为模板,PCR扩增获得军团菌主要外膜蛋白基因(M a jor ou ter m em brane prote in gene,om pS),与原核表达质粒pUC 18定向重组,构建重组质粒,转化大肠杆菌BL 21,并用限制性酶酶切分析、聚合酶链式反应、核酸序列分析、十二...以军团菌DNA为模板,PCR扩增获得军团菌主要外膜蛋白基因(M a jor ou ter m em brane prote in gene,om pS),与原核表达质粒pUC 18定向重组,构建重组质粒,转化大肠杆菌BL 21,并用限制性酶酶切分析、聚合酶链式反应、核酸序列分析、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、W estern印迹进行鉴定。实验结果表明我们扩增出了军团菌914 bp的om pS基因,成功构建了重组质粒pLPom pS,并在原核系统中得到了表达。展开更多
文摘A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene(omp) of Aeromonas hydrophila.With the specific primers,a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR.The target fragment was inserted into the linearized pGEM-T easy vector.After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software.The analysis results showed that the cloned DNA fragment had a longest open reading frame(ORF) of 1035 nt,it predicted to be encoded a 344-aa protein with the molecular weight of 36 kD.Hydrophobicity analysis suggested that the protein was highly hydrophilic,especialy at the first 24 amino-acid,this region could function as signal peptide.The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene,and the alignment of the amino acid sequence was 98%.The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E.coli BL21(DE3)by BamH and Sal I.The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GST-MOMP,furthermore,the fusion protein was specifically recognized by anti-serum which raised against the major outer membrane protein of AHML316.Considering all these together,it proved that the cloned gene represented the major outer membrane protein gene of AHML316,and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.
文摘以军团菌DNA为模板,PCR扩增获得军团菌主要外膜蛋白基因(M a jor ou ter m em brane prote in gene,om pS),与原核表达质粒pUC 18定向重组,构建重组质粒,转化大肠杆菌BL 21,并用限制性酶酶切分析、聚合酶链式反应、核酸序列分析、十二烷基磺酸钠-聚丙烯酰胺凝胶电泳、W estern印迹进行鉴定。实验结果表明我们扩增出了军团菌914 bp的om pS基因,成功构建了重组质粒pLPom pS,并在原核系统中得到了表达。
