噬藻体PaV-LD主要衣壳蛋白、穿孔素和内肽酶基因的克隆及表达分析  

CLONING AND EXPRESSION ANALYSIS OF MAJOR CAPSID PROTEIN GENE, ENDOPEPTIDASE AND HOLIN GENE OF CYANOPHAGE PAV-LD

作　　者：李三华[1,2] 高恶斌[1] 欧铜[1,2] 张奇亚[1,2] 

机构地区：[1]中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉430072 [2]中国科学院大学,北京100049

出　　处：《水生生物学报》2013年第2期252-260,共9页Acta Hydrobiologica Sinica

基　　金：中国科学院先导专项项目(KSCX2-EW-Z-3);国家自然科学基金项目(31072239);淡水生态与生物技术国家重点实验室基金(2011FBZ12)资助

摘　　要：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。he 142 open reading frame（ORF） of the genome of cyanophage,PaV-LD（Planktothrix agardhii virus isolated from the Lake Donghu）,was elucidated recently.However,the characteristic of genomic,phylogenetic position,and mechanism of infection remained to be examined on PaV-LD,a novel tailless caynophage.With the relatively stable structure and evolutionarily conserved sequence,the major capsid protein（MCP） gene has been an important reference in the classification of these viruses.Endopeptidase and holin were considered to play an important role in the progress of invasion and release of viruses.The MCP gene（073R）,endopeptidase gene（123L）,and holin gene（124L） of cyanophage PaV-LD were cloned and the expressions were analysed.PaV-LD 073R was amplified and cloned,and the prokaryotic expression plasmid pET-32a-073R was constructed.The prokaryotic expression products of PaV-LD 073R was analyzed by SDS-PAGE after being induced with isopropyl beta-D-thiogalactopyranoside（IPTG）.The fusion protein was purified via affinity chromatography and was then used to immunize BALB/C mice to prepare the antiserum against PaV-LD 073R.Next the antiserum was used for Western blotting analysis and the temporal expression pattern of PaV-LD 073R was characterized during PaV-LD infection at different times.The results of western blotting indicated that the MCP was initially expressed at 48h post-infection（p.i.） and developed into particularly enhanced from 60h p.i.to 84h p.i.The data also demonstrated that 073R was a late expression gene of the tailless cyanophage genome.To further investigate the phylogenetic position of PaV-LD,a phylogenetic tree was constructed using the Neighbor-Joining method with 34 tailed cyanophages and 2 phycoviruses（viruses that infect eukaryotic algae）.The analysis of the phylogenetic tree revealed that the amino acid sequences in PaV-LD 073R grouped more closely with tailless phycoviruses instead of the tailed cyanophages,indicating that PaV-LD had the morphological characteristic

关 键 词：水华蓝藻噬藻体PaV-LD 主要衣壳蛋白基因(073R) 内肽酶基因(123L) 穿孔素基因(124L) 

分 类 号：X179[环境科学与工程—环境科学]