石斑鱼抗菌肽Hepcidin基因的克隆、表达与抗菌活性研究
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摘要
本研究根据鲈鱼抗菌肽Hepcidin基因(GenBank登录号AY5472821)的cDNA序列设计了一对特异引物，利用RT-PCR技术从石斑鱼肝脏中扩增到一条基因片段，并采用RACE技术，获得了该基因的全长592bp的cDNA序列(GenBank登录号：DQ177321)。该基因含有一个261bp碱基的阅读框(ORF)，可编码87个氨基酸的多肽，包括24个氨基酸的信号肽、40个氨基酸的优势域和23个氨基酸的成熟肽(内含4个半胱氨酸)；预测成熟肽分子量约为2465．8Da，等电点PI为5．97，是一种阴离子肽。BLAST检索表明，该多肽与其他肽类的同源性较低，但与多种鱼类来源的Hepcidin抗菌肽的同源性较高，推测是鱼类Hepcidin抗菌肽家族的一个新成员。
运用PCR和反向PCR技术扩增获得其基因组DNA序列，与已知Hepcidin基因的结构组成相同，均由3个外显子和2个内含子组成。第一个外显子包含5′UTR、信号肽和部分优势域编码序列，第二个外显子仅编码部分优势域，而第三个外显子编码部分的优势域和成熟肽序列以及3′UTR。分析基因组DNA上游区域，发现含有多个调控序列，包括TATA框和cap帽子结构，确认的转录起始位点与扩增得到的Hepcidin cDNA的5′端起始序列一致；另外还发现了上游调控区内多种转录因子的结合位点。
将不含信号肽的该基因cDNA编码序列与融合表达载体pTrc-CKS连接，转化入E．coli TOP10F′表达菌株，IPTG诱导后得到的表达产物主要以包涵体为主，上清中的部分可溶性蛋白经过金属鏊合层析纯化后，得到较高纯度的融合蛋白，经重组人鼻病毒14亚型P3C蛋白酶酶切，获得了分子量约为14 kDa的融合表达目的蛋白的酶切产物。
研究了融合表达蛋白酶切产物对4种细菌的抗菌作用，发现该蛋白能够选择性抑制部分革兰氏阳性菌(如金黄色葡萄球菌、溶壁微球菌)和革兰氏阴性菌(如大肠杆菌)的生长，但对表皮葡萄球菌无抗菌活性；65℃水浴60 min后仍对溶壁微球菌具有抗菌活性，表明该蛋白具有热稳定性。
综上分析，该基因编码的蛋白质具有显著的Hepcidin抗菌肽的特征，是首次从石斑鱼肝脏中分离到的一条编码Hepcidin抗菌肽的新基因。本研究同时也为基因工程重组生产石斑鱼抗菌肽和该抗菌肽的应用研究奠定基础。
A pair of PCR primer was designed according to the antimicrobial peptide Hepcidin gene of Lateolabrax sp. and a fragment of gene was cloned from the liver of Epinephelus. Awora. To obtained the full-length cDNA sequence of the gene, rapid amplification of cDNA ends (RACE) was used. The full-length cDNA of the gene is 592 nucleotides, which includes a 261 bp ORF encoding a 87-amino acid prepropeptide. the prepropeptide contains a signal peptide (24 amino acids), a prodomain (40 amino acids) and a mature peptide (23 amino acids, with 4 cysteine residues). The mean molecular weight and the theoretical pI of the mature peptide is 2465.8 Da and 5.97 respectively. BLAST in GenBank database reveals that the prepropeptide is different from any other reported antimicrobial peptides. But there is some similarity between the amino acid sequences encoded by the ORF and Hepcidin from some fish,and is predicted as a member of Hepcidin.
Reverse PCR was used for cloning the genomic DNA sequence of the gene. The genomic DNA sequence of this gene is similar to other fish, which contains three exons and two introns. The first exon contains 5'UTR, signal peptide and partial prodomain sequences. The second exon only encodes some part of prodomain, and the third exon encodes 3 'UTR and partial prodomain. Furthermore, general upstream elements of the gene were cloned, which include TATA box and a capping signal. In addition, several consensus-binding motifs for transcription factors were also found in the 5' flanking sequences of this Hepcidin.
The ORF sequence without signal peptide was recombined into pTrc-CKS vector and expressed in Escherichia coli. The expressed protein mainly existed in inclusion body. The fusion proteins were purified by Ni-NTA column. The pure fusion protein was digested with 3C protease.A 14 kDa objective protein was obtained.
Antibacterial activity of the objective protein was tested. The results showed that the protein had obviously antibacterial activity against both Gram-positive and Gram-negative bacterial. Heated under 65℃for 60 min, the protein still has strong antibacterial activity against some bacteria, which indicated that it is a heat-stable protein.
In conclusion, the gene structure and biological function of an antimicrobial peptide belonging to the family of Hepcidin have been characterized. This is the first report on characterization of a novel anionic antimicrobial peptide Hepcidin gene from the liver of E.awora. This study also lays foundation for the study of gene recombinant engeering production of antimicrobial peptide and the application of this antimicrobial peptide.
运用PCR和反向PCR技术扩增获得其基因组DNA序列，与已知Hepcidin基因的结构组成相同，均由3个外显子和2个内含子组成。第一个外显子包含5′UTR、信号肽和部分优势域编码序列，第二个外显子仅编码部分优势域，而第三个外显子编码部分的优势域和成熟肽序列以及3′UTR。分析基因组DNA上游区域，发现含有多个调控序列，包括TATA框和cap帽子结构，确认的转录起始位点与扩增得到的Hepcidin cDNA的5′端起始序列一致；另外还发现了上游调控区内多种转录因子的结合位点。
将不含信号肽的该基因cDNA编码序列与融合表达载体pTrc-CKS连接，转化入E．coli TOP10F′表达菌株，IPTG诱导后得到的表达产物主要以包涵体为主，上清中的部分可溶性蛋白经过金属鏊合层析纯化后，得到较高纯度的融合蛋白，经重组人鼻病毒14亚型P3C蛋白酶酶切，获得了分子量约为14 kDa的融合表达目的蛋白的酶切产物。
研究了融合表达蛋白酶切产物对4种细菌的抗菌作用，发现该蛋白能够选择性抑制部分革兰氏阳性菌(如金黄色葡萄球菌、溶壁微球菌)和革兰氏阴性菌(如大肠杆菌)的生长，但对表皮葡萄球菌无抗菌活性；65℃水浴60 min后仍对溶壁微球菌具有抗菌活性，表明该蛋白具有热稳定性。
综上分析，该基因编码的蛋白质具有显著的Hepcidin抗菌肽的特征，是首次从石斑鱼肝脏中分离到的一条编码Hepcidin抗菌肽的新基因。本研究同时也为基因工程重组生产石斑鱼抗菌肽和该抗菌肽的应用研究奠定基础。
A pair of PCR primer was designed according to the antimicrobial peptide Hepcidin gene of Lateolabrax sp. and a fragment of gene was cloned from the liver of Epinephelus. Awora. To obtained the full-length cDNA sequence of the gene, rapid amplification of cDNA ends (RACE) was used. The full-length cDNA of the gene is 592 nucleotides, which includes a 261 bp ORF encoding a 87-amino acid prepropeptide. the prepropeptide contains a signal peptide (24 amino acids), a prodomain (40 amino acids) and a mature peptide (23 amino acids, with 4 cysteine residues). The mean molecular weight and the theoretical pI of the mature peptide is 2465.8 Da and 5.97 respectively. BLAST in GenBank database reveals that the prepropeptide is different from any other reported antimicrobial peptides. But there is some similarity between the amino acid sequences encoded by the ORF and Hepcidin from some fish,and is predicted as a member of Hepcidin.
Reverse PCR was used for cloning the genomic DNA sequence of the gene. The genomic DNA sequence of this gene is similar to other fish, which contains three exons and two introns. The first exon contains 5'UTR, signal peptide and partial prodomain sequences. The second exon only encodes some part of prodomain, and the third exon encodes 3 'UTR and partial prodomain. Furthermore, general upstream elements of the gene were cloned, which include TATA box and a capping signal. In addition, several consensus-binding motifs for transcription factors were also found in the 5' flanking sequences of this Hepcidin.
The ORF sequence without signal peptide was recombined into pTrc-CKS vector and expressed in Escherichia coli. The expressed protein mainly existed in inclusion body. The fusion proteins were purified by Ni-NTA column. The pure fusion protein was digested with 3C protease.A 14 kDa objective protein was obtained.
Antibacterial activity of the objective protein was tested. The results showed that the protein had obviously antibacterial activity against both Gram-positive and Gram-negative bacterial. Heated under 65℃for 60 min, the protein still has strong antibacterial activity against some bacteria, which indicated that it is a heat-stable protein.
In conclusion, the gene structure and biological function of an antimicrobial peptide belonging to the family of Hepcidin have been characterized. This is the first report on characterization of a novel anionic antimicrobial peptide Hepcidin gene from the liver of E.awora. This study also lays foundation for the study of gene recombinant engeering production of antimicrobial peptide and the application of this antimicrobial peptide.
引文
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