【作者】 凡复；

【导师】 吴跃明； 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 PR-39与Protegrin-1(PG-1)都是猪源抗菌肽。PR-39具有广谱的抗细菌、真菌作用；能诱导嗜中性粒细胞的定向迁移,促进伤口恢复,减缓病理损伤,并且具有抗癌作用。PG-1具有很强的抗细菌活性和中等程度的抗真菌作用,还具有快速杀菌的活性；能够抵抗有囊膜的病毒,具有不同程度的抗HIV病毒的活性。本研究利用了大肠杆菌和紫花苜蓿作为生物反应器,串联表达PR-39与PG-1。1.抗菌肽Protegrin-1、PR-39基因在大肠杆菌中的融合表达为了使猪源抗菌肽Protegrin-1(PG-1)、PR-39在原核表达载体中获得表达,在不改变抗菌肽氨基酸顺序的基础上,根据大肠杆菌偏爱密码子表,替换部分密码子,通过重叠PCR化学合成PG-1、PR-39成熟肽基因片段,重组至融合表达载体pGEX-4T-1中,分别构建融合表达载体pGEX-PG-1、pGEX-PR-39,转化至大肠杆菌BL21(DE3)中,筛选得到的阳性克隆用IPTG进行诱导。SDS-PAGE电泳和Western-blotting检测表明融合蛋白已经成功表达。纯化得到的融合蛋白用肠激酶切割后,经亲合层析得到目的蛋白。重组抗菌肽PG-1和PR-39分别为1.38mg/1、1.11mg/l。抑菌实验结果表明重组抗菌肽具有生物活性。2.抗菌肽PR-39和Protegrin-1基因在大肠杆菌中的串联表达为了在大肠杆菌中串联表达抗菌肽PR-39和PG-1,设计了四条短核苷酸链,重叠PCR技术扩增得到PR-39和PG-1的串联基因。PR-39和PG-1的基因之间由盐酸羟胺切割位点(Asn-Gly)连接。串联基因片段插入到融合表达载体pGEX-4T-1中,构建融合表达载体pGEX-PR39-PG1,转化至大肠杆菌BL21(DE3)中,阳性克隆用IPTG进行诱导表达。SDS-PAGE电泳分析和Western-blotting检测表明串联基因已经成功表达。融合蛋白GST-PR39-PG1先经盐酸羟胺切割释放出重组抗菌肽PG-1,再经肠激酶切割得到重组抗菌肽PR-39。两种抗菌肽的产量分别为1.5mg/1、1.9mg/l。重组抗菌肽经抑菌试验,表现出生物活性,且抗菌肽PR-39与PG-1混合的抑菌活性强于PR-39与PG-1单个的抑菌活性。3.抗菌肽Protegrin-1和PR-39基因在紫花苜蓿中的串联表达为了在紫花苜蓿中实现抗菌肽PR-39和PG-1的共表达,用大豆多聚半乳糖醛酸内切酶抑制蛋白(Endopolygalacturonase-inhibiting protein, PGIP)引导肽作为信号肽,用在植物体内可被特异性切割的短肽作为连接肽,通过化学合成PR-39和PG-1的串联基因。串联基因片段插入到本实验室前期构建好的植物表达载体pC-1301-PMI中,同时构建包含单个抗菌肽的植物表达载体,导入农杆菌对紫花苜蓿遗传转化。由于紫花苜蓿的遗传转化周期长效率低,实验只获得To代转PR-39和PG-1串联基因紫花苜蓿2株,转PR-39基因的紫花苜蓿4株,转PG-1基因的紫花苜蓿5株。转基因植株经研磨体外抑菌试验表明表达的抗菌肽具有抑菌活性。更多还原

【Abstract】 Antimicrobial peptide PR-39 is from swine, and has played a multifunction role in host innate immunity. PR-39 shows a broad spectrum of antimicrobial activity, and is chemotactic for neutrophils and capable of regulating vascular cell-cell interaction. It can inhibit invasion and metastasis of cancer cells and apoptosis of hypoxic endothelial cells. There is a significant interest in developing this peptide for pharmaceutical applications. Protegrin-1 (PG-1) was first discovered in porcine leukocytes. It kills microorganism by forming ion channels in cellular membranes. Due to the broad range of antimicrobial activity of PG-1, it is considered as a potential pharmaceutical agent too. There is no report about the heterologous expression of mature antimicrobial peptides PR-39 and PG-1. In this study, PR-39 and PG-1 were co-expressed in E. coli and alfalfa.1. Fusion expression of Protegrin-1, PR-39 gene from swine in Escherichia Coli To express swine antimicrobial peptide PG-1 and PR-39 in prokaryotic expression system, the part of codon of gene was replaced by codon preference of E. coli. Complementary primers were designed and the integrity of the PG-1 or PR-39 fragment was amplified by SOE-PCR. The amplified fragments were inserted into pGEX-4T-1, and the fusion expression plasmid pGEX4T-PG-1 and pGEX4T-PR-39 were constructed. The plasmids were transferred into E. coli BL21(DE3) and induced by IPTG SDS-PAGE and Western-blotting analysis showed that the recombinants had expressed the specific proteins. By Glutathione Sepharase 4B affinity chromatography, the fusion proteins GST-PG-1 and GST-PR-39 were obtained and purified. After cleaved by enterokinase, recombinant antimicrobial peptides, PG-1 and PR-39, were obtained. The yields of recombinant PG-1 and PR-39 were 1.38 mg/1 and 1.11 mg/1 respectively. The recombinant peptides showed antibacterial activities by growth inhibition method.2. Tandem expression of PR-39 and Protegrin-1 gene in Escherichia Coli To implement co-expression of antimicrobial peptide PR-39 and Protegrin-1 (PG-1) in prokaryotic expression system, a tandem gene fragment encoding PR-39 and PG-1 has been synthesized chemically. The cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. The fragment was inserted into vector pGEX-4T-1 and expressed in Escherichia coli. The fusion protein GST-PR39-PG1, purified by affinity chromatography, was cleaved first by hydroxylamine hydrochloride to release recombinant PG-1 and then by enterokinase to release PR-39. Purification of recombinant PR-39 and PG-1 was achieved, and 1.5mg PG-1 and 1.9mg PR-39 were obtained respectively from 11 culture. The recombinant antimicrobial peptides showed antibacterial activity.3. Tandem expression of PR-39 and Protegrin-1 gene in Alfalfa To implement co-expression of antimicrobial peptide PR-39 and Protegrin-1 (PG-1) in alfalfa, a tandem gene fragment encoding PR-39 and PG-1, upstream of which the signal peptide geng was added, has been synthesized chemically. An intervening sequence (linker peptide) was introduced between PR-39 and PG-1. The fragment was inserted into plant express vector pC-1301-PMI which was constructed by our lab previously. At the same time, the plant express vectors containing single peptides were constructed. The express vectors plasmids were transformed into agrobacterium tumefaciens. Alfalfa was transformed by agrobacterium tumefaciens. After PCR detection,2 of alfalfa transfected with tandem genes,4 of alfalfa transfected with PR-39 and 5 of alfalfa transfected with PG-1 were obtained. The recombinant antimicrobial peptides showed antibacterial activity by growth inhibition method in vitro.更多还原