锯缘青蟹阴离子抗菌肽SCY2基因克隆、表达特性及其抗菌活性的研究
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摘要
本研究根据实验室获得的SCY2部分cDNA序列,设计特异性引物,以雄性锯缘青蟹的性腺组织RNA为模板,利用RT—PCR及5',3'-RACE等方法,克隆获得了抗菌肽SCY2的全长cDNA序列947bp,编码100个氨基酸,预测分子量约为10925.4Da,等电点PI为4.84,与已发表的锯缘青蟹抗菌肽Scygonadin基因的氨基酸序列相似性为65.08%,因而认为SCY2是在锯缘青蟹中发现的又一个新的阴离子抗菌肽。主要结果如下:
采用PCR及反向PCR等方法,对基因组DNA及其侧翼序列进行扩增,获得了SCY2基因组DNA的全长序列,该基因组全长为2607bp,包含3个外显子和2个内含子。与Scygonadin具有相似的基因结构组成;获得了SCY2基因的上下游侧翼序列,5'上游序列含有转录起始位点以及TATA box等启动子所具有的特征序列。
用RT-PCR和Northern-blot等方法,分别检测了SCY2基因在锯缘青蟹(雄性和雌性)不同组织器官(鳃、甲壳、眼、血细胞、肝胰脏、心脏、肌肉、甲壳下皮层、胃和生殖系统等)中的转录表达情况,发现该基因仅在雄性生殖系统中表达。该表达特性与Scygonadin相似。检测细菌感染条件下锯缘青蟹各组织SCY2及Scygonadin表达情况,发现在感染后48 h内,SCY2和Scygonadin mRNA在性腺组织中的表达水平均无明显变化,说明外部细菌感染不是SCY2的直接诱导表达因素。
为确定SCY2的抗菌功能,根据SCY2编码序列,构建了pTrc-CKS/SCY2融合基因表达载体。将表达载体质粒转化大肠杆菌,以IPTG诱导表达,表达产物主要以包涵体形式存在,通过变性复性后获得融合蛋白SCY2-CKS。利用金属鏊合层析纯化获得SCY2融合蛋白,并以3C蛋白酶酶切获得重组表达的SCY2。用0.4mg/ml重组SCY2在11种海洋动物病原菌和耐药性细菌进行抗菌活性检测(细菌杀伤指数(KI)),结果显示:SCY2能够选择性的抑制革兰氏阴性菌和阳性菌的生长,对水产动物病原菌嗜水气单胞菌(Aeromonas hydrophia A.h)有显著的抑菌活性,对金黄色葡萄球菌,溶壁微球菌,谷棒杆菌等也具有较强的抑菌作用。
A novel antimierobial peptide SCY2 cDNA was cloned from the seminal plasma of the mud crab,Scylla serrata(Forsk(?)l,1775) by using a degenerated reverse transcriptase(RT)-PCR and rapid amplification of cDNA ends(RACE).The full-length cDNA of SCY2 consisted of 947 nucleotides,encoding a polypeptide of 100 amino acids.The mean molecular weight of mature peptide is 10925.4 Da.The identity of amino acid sequences between the homologous cDNA and seygonadin cDNA was 65.08%and the theoretical pI of the mature peptide is 4.84,which suggests that it is also an anionic molecule as scygonadin(pI 6.09).The amino acid sequence of it is different from any other reported antimicrobial peptide,indicating that it is a new gene.
Genomic DNA sequence of SCY2 was determined by PCR and inverse PCR,consisting of 2607 bp including two introns and three exons similar to the genomic organization of scygonadin.A TATA box and a cap was found at the location where 5' site started for transcription.In addition,several consensus-binding motifs for transcription factors were also found.
Using the semi-quantitative RT-PCR and northern blot methods,SCY2 transcripts were investigated in various tissues including exoskeleton,subcuticular epithelia,gills, heart,hepatopancreas,stomach,muscle,eyes,female and male reproductive apparatus. The result showed the SCY2 gene was only expressed in the male reproductive apparatus.
While testing on mRNA transcripts of either SCY2 or scygonadin,respectively in bacterially challenged mud crab,it was found that there was no significant change of SCY2 and scygonadin mRNA abundance in all of the tested tissues,suggesting that bacterial infection can not induce the transcription of both SCY2 and scygonadin genes.
The antibacterial activity of SCY2 was evaluated on ten strains of bacteria using the recombinant fusion SCY2 expressed in Escherichia coli system.The SCY2 recombinant pTrc-CKS vector was constructed,the fusion protein was expressed in Escherichia coli with IPTG induction.The expressed protein existed in the inclusion body which was denaturalized,dissolved and recovered.The fusion proteins were purified by Ni-NTA column,The recombinant SCY2 after cleavage with 3C protease exhibited obvious antibacterial activities against several bacteria tested including Aeromonas hydrophia,Staphylococcus aureus,Corynebacterium glutamicum.Thus, SCY2 is a male-specific anionic antimicrobial pepetide transcribed by a new gene and belongs to a family of scygonadin but might play a different role in innate immunity in Scylla serrata.
采用PCR及反向PCR等方法,对基因组DNA及其侧翼序列进行扩增,获得了SCY2基因组DNA的全长序列,该基因组全长为2607bp,包含3个外显子和2个内含子。与Scygonadin具有相似的基因结构组成;获得了SCY2基因的上下游侧翼序列,5'上游序列含有转录起始位点以及TATA box等启动子所具有的特征序列。
用RT-PCR和Northern-blot等方法,分别检测了SCY2基因在锯缘青蟹(雄性和雌性)不同组织器官(鳃、甲壳、眼、血细胞、肝胰脏、心脏、肌肉、甲壳下皮层、胃和生殖系统等)中的转录表达情况,发现该基因仅在雄性生殖系统中表达。该表达特性与Scygonadin相似。检测细菌感染条件下锯缘青蟹各组织SCY2及Scygonadin表达情况,发现在感染后48 h内,SCY2和Scygonadin mRNA在性腺组织中的表达水平均无明显变化,说明外部细菌感染不是SCY2的直接诱导表达因素。
为确定SCY2的抗菌功能,根据SCY2编码序列,构建了pTrc-CKS/SCY2融合基因表达载体。将表达载体质粒转化大肠杆菌,以IPTG诱导表达,表达产物主要以包涵体形式存在,通过变性复性后获得融合蛋白SCY2-CKS。利用金属鏊合层析纯化获得SCY2融合蛋白,并以3C蛋白酶酶切获得重组表达的SCY2。用0.4mg/ml重组SCY2在11种海洋动物病原菌和耐药性细菌进行抗菌活性检测(细菌杀伤指数(KI)),结果显示:SCY2能够选择性的抑制革兰氏阴性菌和阳性菌的生长,对水产动物病原菌嗜水气单胞菌(Aeromonas hydrophia A.h)有显著的抑菌活性,对金黄色葡萄球菌,溶壁微球菌,谷棒杆菌等也具有较强的抑菌作用。
A novel antimierobial peptide SCY2 cDNA was cloned from the seminal plasma of the mud crab,Scylla serrata(Forsk(?)l,1775) by using a degenerated reverse transcriptase(RT)-PCR and rapid amplification of cDNA ends(RACE).The full-length cDNA of SCY2 consisted of 947 nucleotides,encoding a polypeptide of 100 amino acids.The mean molecular weight of mature peptide is 10925.4 Da.The identity of amino acid sequences between the homologous cDNA and seygonadin cDNA was 65.08%and the theoretical pI of the mature peptide is 4.84,which suggests that it is also an anionic molecule as scygonadin(pI 6.09).The amino acid sequence of it is different from any other reported antimicrobial peptide,indicating that it is a new gene.
Genomic DNA sequence of SCY2 was determined by PCR and inverse PCR,consisting of 2607 bp including two introns and three exons similar to the genomic organization of scygonadin.A TATA box and a cap was found at the location where 5' site started for transcription.In addition,several consensus-binding motifs for transcription factors were also found.
Using the semi-quantitative RT-PCR and northern blot methods,SCY2 transcripts were investigated in various tissues including exoskeleton,subcuticular epithelia,gills, heart,hepatopancreas,stomach,muscle,eyes,female and male reproductive apparatus. The result showed the SCY2 gene was only expressed in the male reproductive apparatus.
While testing on mRNA transcripts of either SCY2 or scygonadin,respectively in bacterially challenged mud crab,it was found that there was no significant change of SCY2 and scygonadin mRNA abundance in all of the tested tissues,suggesting that bacterial infection can not induce the transcription of both SCY2 and scygonadin genes.
The antibacterial activity of SCY2 was evaluated on ten strains of bacteria using the recombinant fusion SCY2 expressed in Escherichia coli system.The SCY2 recombinant pTrc-CKS vector was constructed,the fusion protein was expressed in Escherichia coli with IPTG induction.The expressed protein existed in the inclusion body which was denaturalized,dissolved and recovered.The fusion proteins were purified by Ni-NTA column,The recombinant SCY2 after cleavage with 3C protease exhibited obvious antibacterial activities against several bacteria tested including Aeromonas hydrophia,Staphylococcus aureus,Corynebacterium glutamicum.Thus, SCY2 is a male-specific anionic antimicrobial pepetide transcribed by a new gene and belongs to a family of scygonadin but might play a different role in innate immunity in Scylla serrata.
引文
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