皱纹盘鲍和栉孔扇贝抗菌肽的研究
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摘要
皱纹盘鲍(Haliotis discus hannai)和栉孔扇贝(Chlamys farreri)是我国黄渤海区的重要自然资源,也是我国北方的重要养殖品种。九十年代中期以来,病害频繁发生,造成的经济损失达百亿元,直接威胁到现有产业的生存。为了促进我国贝类养殖业的复兴和健康发展,实现我国蓝色农业的可持续化,海水养殖动物的免疫抗病机制成为最突出和亟待解决的问题。
抗菌肽(antibacterial peptide)是基因编码的肽类抗菌分子,它们广泛存在于生物体内,是脊椎动物、无脊椎动物和植物的先天性免疫关键因子。长期以来,国内外对抗菌肽的研究主要集中在高等动物和一些低等昆虫方面,海洋抗菌肽研究是10年来发展起来的一个热点。本论文以我国重要海水养殖动物——栉孔扇贝和皱纹盘鲍为研究对象,开展抗菌肽的研究,取得了以下结果:
(1)本文利用固相提取和HPLC技术,结合灵敏的酶标抗微生物活性检测法,从栉孔扇贝血液中纯化出1种抗菌肽。经测定其分子量为2000Da,部分氨基酸序列为GQPGHTGNAH……。
(2)从作者所在实验室构建的皱纹盘鲍肝肾cDNA文库中,筛选得到了鲍防御素基因EST。通过序列分析,发现该基因cDNA序列编码66个氨基酸残基,其前体由信号肽、前导肽和成熟肽组成。该前体的成熟肽含42个氨基酸,推测分子量为4323Da、等电点为8.02。氨基酸序列同源性分析表明,该多肽与昆虫防御素的相似性最高、达到了70%。因成熟肽二级结构具有典型的昆虫防御素结构特征,作者认为该多肽应属于抗菌肽中的昆虫防御素亚家族,是一种新型抗菌肽,并将其命名为鲍防御素(hd-def)。
(3)用鳗弧菌和金黄色葡萄球菌刺激皱纹盘鲍,能诱导hd-def的表达,且鳗弧菌刺激引起的表达量比金黄色葡萄球菌诱导的高,说明该防御素基因属于诱导型表达。在被检测的5种组织中,hd-def基因仅在肝胰腺中表达,而在其它组织(性腺、鳃、肌肉和外套膜)中均未检测到表达。说明该基因具有明显的组织表达特异。
人工感染刺激后,不同时间皱纹盘鲍的hd-def表达量有很大差异。感染早期(2hr)表达非常微弱,4hr时表达量明显增大,感染中晚期(12hr)表达量最大。该结果提示,hd-def基因可能参与皱纹盘鲍的抗细菌感染。
(4)采用基因组步移法,获得了4032bp的全长基因组序列。分析表明,该基因由3个内含子和4个外显子编码组成;3个内含子大小分别为497、2357和528bp,其中两个内含子存在于编码信号肽的序列中,这种是一种防御素基因的新结构模式,在其他昆虫防御素均未报道。
(5)本文利用酵母表达系统,成功构建鲍hd-def基因的重组表达载体pPIC9k-hddef。重组酵母体外表达4.3kd蛋白,具有抗鳗弧菌和金黄色葡萄球菌的活性。
Abalone Haliotis discus hannai and scallop Chlamys farreri are important natural resources in Huanghai and Bohai Sea area of China, but also the importance of culture in northern China. Since the 1990s, the frequent occurrence of diseases, causing economic losses of 10 billion yuan, a direct threat to the survival of existing industries. In order to promote China's shellfish aquaculture and the healthy development, to pursue durative of China's blue agriculture, the immune resistance mechanisms of aquaculture animals become the most prominent and requiring urgent solution.
The antibacterial peptides are the gene encoding peptide antibacterial molecules, which are widespread in the organism. They are key congenital immune factor for vertebrates, invertebrates and plants. For a long time, the antimicrobial peptide research was focused on the higher animals and some insects, marine antimicrobial peptide is a hot topic in recent 10 years. To study Haliotis discus hannai and Chlamys farreri as a research objective, This paper carried out antimicrobial peptides research. The results made the following findings:
(1) By using solid-phase extraction and HPLC technology, combined with sensitive anti-microbial enzymic activity test, one kind of antimicrobial peptides was purified from Chlamys farreri blood. It has a molecular weight of 2000 Da, some amino acid sequence was determined to GQPGHTGNAH…….
(2) Via analysis, 322 nucleotides from the defensin-related EST were identified in the liver and kidney cDNA library of Haliotis discus hannai Ino. Sequence analysis showed that the tailing signal AATAAA located at 3 'poly (A), 5' with ATG initiation codon, which has won the full-length cDNA sequences. In the ORF (open reading frame) of cDNA sequence, full -length 201 nucleotides encoding 66 amino acid residues of the prepropeptide. Analyzing the signal peptide and enzymatic digestion of the coding protein, found that its precursor consist of signal peptide (18 AA), the leader peptide (6 AA) and the mature peptide (42 AA). Submitted mature peptide amino acid sequence analysis proved the amino acid sequence of defensin has high similarity to other insects defensin, and similarity of 70% to the beetles Anomala cuprea.
(3) The bacterial stimulation treatment showed that the gene expression is inducible. Under normal circumstances (control group) the defensin gene was not expressed. Gram-positive and Gram-negative bacteria infection can cause the gene expression, but V.anguillarum induced high expression than Staphylococcus aureus.
Under artificial infection in the stimulation of different time stages, the hd-def gene expression exist great differences. In the early stage of infection (2 hr) expression is very weak, in process of 4 hr the expression increased significantly, late in the infection (12 hr) hd-def expression is up to the largest volume, but in 0,6,8 and 24 hr the hd-def gene transcriptional expression was not detected.
(4) The 4032 bp of the full-length genome sequence was obtained using genome walking. The analysis results showed that the gene consist of three introns and four exons coding, which three intron size were 497, 2357 and 528 bp, two of them were in the presence of the coded signal peptide sequence. This is a new structure model for defense gene. It was not reported in other insects defense.
5) The recombinant gene expression vector pPIC9k-hddef of the hd-def gene was successfully constructed by using yeast expression system. In vitro, the expressed 4.3 kd protein of the recombinant yeast has anti-V. anguillarum and anti-E.coli activity.
抗菌肽(antibacterial peptide)是基因编码的肽类抗菌分子,它们广泛存在于生物体内,是脊椎动物、无脊椎动物和植物的先天性免疫关键因子。长期以来,国内外对抗菌肽的研究主要集中在高等动物和一些低等昆虫方面,海洋抗菌肽研究是10年来发展起来的一个热点。本论文以我国重要海水养殖动物——栉孔扇贝和皱纹盘鲍为研究对象,开展抗菌肽的研究,取得了以下结果:
(1)本文利用固相提取和HPLC技术,结合灵敏的酶标抗微生物活性检测法,从栉孔扇贝血液中纯化出1种抗菌肽。经测定其分子量为2000Da,部分氨基酸序列为GQPGHTGNAH……。
(2)从作者所在实验室构建的皱纹盘鲍肝肾cDNA文库中,筛选得到了鲍防御素基因EST。通过序列分析,发现该基因cDNA序列编码66个氨基酸残基,其前体由信号肽、前导肽和成熟肽组成。该前体的成熟肽含42个氨基酸,推测分子量为4323Da、等电点为8.02。氨基酸序列同源性分析表明,该多肽与昆虫防御素的相似性最高、达到了70%。因成熟肽二级结构具有典型的昆虫防御素结构特征,作者认为该多肽应属于抗菌肽中的昆虫防御素亚家族,是一种新型抗菌肽,并将其命名为鲍防御素(hd-def)。
(3)用鳗弧菌和金黄色葡萄球菌刺激皱纹盘鲍,能诱导hd-def的表达,且鳗弧菌刺激引起的表达量比金黄色葡萄球菌诱导的高,说明该防御素基因属于诱导型表达。在被检测的5种组织中,hd-def基因仅在肝胰腺中表达,而在其它组织(性腺、鳃、肌肉和外套膜)中均未检测到表达。说明该基因具有明显的组织表达特异。
人工感染刺激后,不同时间皱纹盘鲍的hd-def表达量有很大差异。感染早期(2hr)表达非常微弱,4hr时表达量明显增大,感染中晚期(12hr)表达量最大。该结果提示,hd-def基因可能参与皱纹盘鲍的抗细菌感染。
(4)采用基因组步移法,获得了4032bp的全长基因组序列。分析表明,该基因由3个内含子和4个外显子编码组成;3个内含子大小分别为497、2357和528bp,其中两个内含子存在于编码信号肽的序列中,这种是一种防御素基因的新结构模式,在其他昆虫防御素均未报道。
(5)本文利用酵母表达系统,成功构建鲍hd-def基因的重组表达载体pPIC9k-hddef。重组酵母体外表达4.3kd蛋白,具有抗鳗弧菌和金黄色葡萄球菌的活性。
Abalone Haliotis discus hannai and scallop Chlamys farreri are important natural resources in Huanghai and Bohai Sea area of China, but also the importance of culture in northern China. Since the 1990s, the frequent occurrence of diseases, causing economic losses of 10 billion yuan, a direct threat to the survival of existing industries. In order to promote China's shellfish aquaculture and the healthy development, to pursue durative of China's blue agriculture, the immune resistance mechanisms of aquaculture animals become the most prominent and requiring urgent solution.
The antibacterial peptides are the gene encoding peptide antibacterial molecules, which are widespread in the organism. They are key congenital immune factor for vertebrates, invertebrates and plants. For a long time, the antimicrobial peptide research was focused on the higher animals and some insects, marine antimicrobial peptide is a hot topic in recent 10 years. To study Haliotis discus hannai and Chlamys farreri as a research objective, This paper carried out antimicrobial peptides research. The results made the following findings:
(1) By using solid-phase extraction and HPLC technology, combined with sensitive anti-microbial enzymic activity test, one kind of antimicrobial peptides was purified from Chlamys farreri blood. It has a molecular weight of 2000 Da, some amino acid sequence was determined to GQPGHTGNAH…….
(2) Via analysis, 322 nucleotides from the defensin-related EST were identified in the liver and kidney cDNA library of Haliotis discus hannai Ino. Sequence analysis showed that the tailing signal AATAAA located at 3 'poly (A), 5' with ATG initiation codon, which has won the full-length cDNA sequences. In the ORF (open reading frame) of cDNA sequence, full -length 201 nucleotides encoding 66 amino acid residues of the prepropeptide. Analyzing the signal peptide and enzymatic digestion of the coding protein, found that its precursor consist of signal peptide (18 AA), the leader peptide (6 AA) and the mature peptide (42 AA). Submitted mature peptide amino acid sequence analysis proved the amino acid sequence of defensin has high similarity to other insects defensin, and similarity of 70% to the beetles Anomala cuprea.
(3) The bacterial stimulation treatment showed that the gene expression is inducible. Under normal circumstances (control group) the defensin gene was not expressed. Gram-positive and Gram-negative bacteria infection can cause the gene expression, but V.anguillarum induced high expression than Staphylococcus aureus.
Under artificial infection in the stimulation of different time stages, the hd-def gene expression exist great differences. In the early stage of infection (2 hr) expression is very weak, in process of 4 hr the expression increased significantly, late in the infection (12 hr) hd-def expression is up to the largest volume, but in 0,6,8 and 24 hr the hd-def gene transcriptional expression was not detected.
(4) The 4032 bp of the full-length genome sequence was obtained using genome walking. The analysis results showed that the gene consist of three introns and four exons coding, which three intron size were 497, 2357 and 528 bp, two of them were in the presence of the coded signal peptide sequence. This is a new structure model for defense gene. It was not reported in other insects defense.
5) The recombinant gene expression vector pPIC9k-hddef of the hd-def gene was successfully constructed by using yeast expression system. In vitro, the expressed 4.3 kd protein of the recombinant yeast has anti-V. anguillarum and anti-E.coli activity.
引文
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