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
最近阐明了水华蓝藻噬藻体PaV-LD(Planktothrix agardhii Virus isolated from Lake Donghu)的全基因组序列,这是一个含有142个ORF的双链DNA噬藻体。在此,我们对其主要衣壳蛋白基因073R,内肽酶和穿孔素基因123L-124L(PaV-LD基因组中两...最近阐明了水华蓝藻噬藻体PaV-LD(Planktothrix agardhii Virus isolated from Lake Donghu)的全基因组序列,这是一个含有142个ORF的双链DNA噬藻体。在此,我们对其主要衣壳蛋白基因073R,内肽酶和穿孔素基因123L-124L(PaV-LD基因组中两个相邻的ORF)进行了基因克隆与表达分析。将073R克隆后构建原核表达质粒pET-32a-073R,并用IPTG进行诱导表达,073R融合蛋白经纯化后,进行免疫小鼠制备抗体;通过Western blot检测经噬藻体感染宿主细胞后073R的表达时序,结果显示在宿主细胞裂解之初,即PaV-LD感染48h以后073R开始表达,表明073R是一个晚期基因;同时073R推导的氨基酸序列与34株噬藻(菌)体及2株藻病毒(感染真核藻的病毒)的主要衣壳蛋白的氨基酸序列进行序列比对,显示073R与无尾的藻病毒衣壳蛋白亲缘关系更近。PCR扩增内肽酶和穿孔素基因123L-124L,并构建质粒pOP123L-124L,将其转入模式藻集胞藻PCC6803细胞中,质粒pOP123L-124L与藻集胞藻PCC6803基因组发生重组,形成重组藻;测定了重组藻与野生藻的生长速率,并绘制生长曲线;制备超薄切片,进一步比较和观察重组藻与野生藻的超微结构的变化。结果显示重组藻与野生藻存在生长速率与超微形态的显著差异。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.
文摘最近阐明了水华蓝藻噬藻体PaV-LD(Planktothrix agardhii Virus isolated from Lake Donghu)的全基因组序列,这是一个含有142个ORF的双链DNA噬藻体。在此,我们对其主要衣壳蛋白基因073R,内肽酶和穿孔素基因123L-124L(PaV-LD基因组中两个相邻的ORF)进行了基因克隆与表达分析。将073R克隆后构建原核表达质粒pET-32a-073R,并用IPTG进行诱导表达,073R融合蛋白经纯化后,进行免疫小鼠制备抗体;通过Western blot检测经噬藻体感染宿主细胞后073R的表达时序,结果显示在宿主细胞裂解之初,即PaV-LD感染48h以后073R开始表达,表明073R是一个晚期基因;同时073R推导的氨基酸序列与34株噬藻(菌)体及2株藻病毒(感染真核藻的病毒)的主要衣壳蛋白的氨基酸序列进行序列比对,显示073R与无尾的藻病毒衣壳蛋白亲缘关系更近。PCR扩增内肽酶和穿孔素基因123L-124L,并构建质粒pOP123L-124L,将其转入模式藻集胞藻PCC6803细胞中,质粒pOP123L-124L与藻集胞藻PCC6803基因组发生重组,形成重组藻;测定了重组藻与野生藻的生长速率,并绘制生长曲线;制备超薄切片,进一步比较和观察重组藻与野生藻的超微结构的变化。结果显示重组藻与野生藻存在生长速率与超微形态的显著差异。
