| 摘要: |
| 中国对虾素是从中国对虾血细胞中克隆得到的一种抗菌肽。为了进一步研究中国对虾素(CHP)的功能并为制备特异性抗体作准备，采用大肠杆菌表达外源蛋白的方法,进行了对虾素原核表达的研究。根据从中国对虾血细胞中克隆得到的对虾素基因设计特异性引物扩增中国对虾素成熟肽基因(Chp)，插入原核表达载体pGEX4T1中，在E. coli BL21表达融合蛋白GSTCHP。结果表明，不同表达菌株的融合蛋白表达量为30%—34%，同一菌株在诱导5h后能达到最高表达量。利用GST亲和层析纯化融合蛋白，将融合蛋白用凝血酶裂解以得到CHP，其分子量约为5.6kDa，N端测序结果与期望的成熟对虾肽序列一致。用液体生长抑制方法检测活性，表明重组GST-CHP蛋白及CHP均表现出对大肠杆菌的抑菌活性。 |
| 关键词: 中国对虾 抗菌肽 对虾素 pGEX-4T-1 |
| DOI： |
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| 基金项目:国家重点基础研究发展规划项目,G19990120007号;国家高技术研究发展计划(863计划)项目,2001AA621120号;欧共体第五框架协议项目,ICA4CT200110023;国家自然科学基金项目,30371094号 |
附件 |
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| RECOMBINANT EXPRESSION OF CH-PENAEIDIN FROM CHINESE SHRIMP FENNEROPENAEUS CHINENSIS IN ESCHERICHIA COLi |
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XUE Jian-Feng1, KANG Cui-Jie1, WANG Jin-Xing1, ZHAO Xiao-Fan1, XIANG Jian-Hai2
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1.School of Life Sciences, Shandong University, Jinan, 250100;2.Institute of Oceanology, Chinese Academy of Sciences, Qingdao, 266071
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| Abstract: |
| Penaeidins, a unique antimicrobial peptide family, were first purified from haemocytes of the shrimp Litopenaus vannamei and their genes cloned from the haemocyte shrimp cDNA in 1997. Ch-penaeidin, a new antimicrobial peptide gene of the penaeidin family, was cloned from the hemocytes of Chinese shrimp, Fenneropenaeus chinensis and the gene expression and tissue distribution were studied by RT-PCR, Northern Blot and in situ hybridization.
In order to further study Ch-penaeidin function and to prepare the anti-CHP polyclonal antibody, Escherichia coli was chosen to express the mature Ch-penaeidin (mChp) as a foreign protein. The DNA fragment encoding mature Ch-penaeidin obtained by PCR amplification with the following primers designed from the mChp sequence we cloned by forwarding primer 5′GCGC GAA TTC AAG GGT GGT TAC ACA CGC 3′ containing an EcoR I site (underlined) and reversing primer 5′ GCGC CTC GAG ACT TAC ATC CCA CAT GCA C3′ that contains an Xho I site (underlined). The amplified fragment was digested with EcoR I and Xho I and subcloned into the EcoR I and Xho I sites of pGEX-4T-1 expression vector. The plasmid constructed was denoted as mChp-pGEX-4T-1 and transformed into competent cells of E. coli BL21 for fusion expression. First, small-scale expression (3ml) was tested and the mChp-pGEX recombinants were screened for fusion protein expression on 12.5% SDS-PAGE. Then large-scale expression and purification were performed according to the following steps to obtain mCHP. A single colony of BL21 containing mChp-pGEX plasmid was grown overnight at 37°C in 5ml of LB medium containing 100μg/ml ampicillin. Cultures were then diluted (1:100) in fresh 2YT medium and grown at 37°C for about 4h until the OD600 reached approximately 0.8–1.0. The expression of GST-mCHP was initiated by adding isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1mmol/L and the cultures grown for additional 4h. Cells were pelleted by centrifugation at 7000r/min for 10min and resuspended in 5% of the original volume of PBS, then lysed by ultra-sonication at 4°C. Triton X-100 was added to a final concentration of 1% and the suspension was mixed gently for 30min to facilitate solubilization of proteins. Fusion protein was purified from the supernatant by one-step affinity chromatography on glutathione-Sepharose 4B, which was run according to the manufacturer’s instructions for the GST purification column (Pharmacia). Purified fusion protein of GST-mCHP were digested with thrombin and was run on 15% SDS-PAGE gels and transferred to PVDF membranes (Perkin-Elmer, Foster City, CA) by semi-dry electrophoretic transfer. Protein bands were stained with Ponceau 3R, excised, and sequenced directly by the Edman method on Procise Sequencer (Perkin-Elmer). Antimicrobial activity was tested by liquid growth inhibition assay on Gram-negative E. coli. Amp (ampicillin, 0.25mg/ml). Elution solution GSH (Reduced form of glutathione, 3mg/ml) were used as controls.
Results show that the mature CHP was highly expressed in E. coli BL21 with IPTG induction in percentage of 30% to 34% of total cell proteins. The expression of fusion protein GST-CHP reached the peak level 5 hours later after induction. The expressed proteins fused to GST were purified by Glutathione Sepharose 4B affinity chromatography and cleaved by thrombin for getting CHP. The CHP were identified by analysis of amino acid sequence and molecular mass. Amino acid sequence of the expressed CHP is GSPEFKGGYTRPI, the first five residues are from glutathione S-transferase and the following 9 residues are identical to the mature Ch-penaeidin. Comparison of amino acid sequences of native CHP and rCHP are as follows:
Native mCHP KGGYTRPISRPPYGGGYGNVCTSCHVLTTSQARSCCSRFGRCCVPRRGYSG
rmCHP GSPEFKGGYTRPISRPPYGGGYGNVCTSCHVLTTSQARSCCSRFGRCCVPRRGYSG
The underlined amino acid sequence was derived by automated Edman degradation and the amino acid sequence after Ile8 was deduced from cDNA sequence. The molecular mass of CHP from expressed fusion protein cleaved by thrombin was determined to be about 6kDa on SDS-PAGE consistent with the expected value. The antibacterial activity of CHP was tested by a liquid growth inhibit ion assay on E. coli. Results indicated that the recombinant GST-CHP and CHP has low activity against E. coli. |
| Key words: Fenneropenaeus chinensis, Antimicrobial peptide, Penaedin, pGEX-4T-1 |
