家蝇抗菌肽Cec Md在大肠杆菌中的高效表达、纯化及其活性的研究
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摘要
抗生素在畜牧业中的广泛应用甚至滥用,降低了动物性产品的品质,威胁到了消费者的健康利益。这不仅危害了我国的公共卫生安全,破坏畜牧产业链的正常运转,而且在很大程度上削弱了我国动物性产品在国际市场上的竞争力。抗菌肽(Antimicrobial peptides,AMPs)为一类阳离子小肽,是生物体固有免疫系统的重要组成成分,具有广谱杭菌活性。AMPs以其独特的生物活性和作用机理,已经成为当前生物学、医药学和农学领域的研究热点。AMPs具有无致病菌耐药性、无残留、耐高温以及具有靶细胞高度特异性等优点,将成为传统抗生素在饲料工业领域理想的替代品。故深化抗菌肽的基础性研究及其系列产品的开发对提升畜禽产品质量、提高畜禽生产性能和疫病抵抗能力乃至推动整个畜牧业的发展具有重要意义。
本研究主要开展了如下几个方面的探索:
(1)Cec Md基因的克隆与生物信息学分析。利用脂多糖(LPS)诱导或非诱导的家蝇幼虫体内抽提总RNA,根据家蝇cecropin基因(GenBank:DQ 232774)设计特异性引物,应用RT-PCR技术扩增出编码Cec Md开放阅读框(ORF)的cDNA序列,将其克隆入pMD18-T载体,经PCR、双酶切和测序鉴定正确后,对此基因及其氨基酸推导序列进行系统的生物信息学分析。结果表明从LPS诱导的家蝇幼虫体内成功的扩增出家蝇抗菌肽cecropin基因,但从未诱导的虫体内未能获得此基因;克隆的家蝇Cec Md基因大小为192 bp,编码由63氨基酸残基组成的多肽,分子量6739.94,其中强碱性氨基酸8个、强酸性氨基酸4个、疏水性氨基酸30个、极性氨基酸14个;Cec Md最可能的裂解位点在A23和G24之间,提示N端1～23氨基酸为Cec Md的信号肽,C端24～63氨基酸构成成熟肽;3-D结构预测结果表明,Cec Md的成熟肽含有2个螺旋结构,不含二硫键;Cec Md与果蝇cecropins的同源性显著高于其他昆虫,达到81.0%以上,最高者为84.1%。
(2)Cec Md基因在E.coli中的表达与产物纯化。将构建好的pMD18-T/Cec Md经双酶切后,获取线性目的基因片段,将其定向重组于表达载体pET32a(+)和pGEX-6P-1,进行PCR、双酶切鉴定和DNA测序分析。确定成功构建重组表达载体pET32a(+)/Cec Md和pGEX-6P-1/Cec Md后,转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE检测目的蛋白的表达。结果表明,在SDS-PAGE胶片中未检测出pET32a(+)/Cec Md重组载体的蛋白表达条带;经SDS-PAGE和Western blot检测,证明pGEX-6P-1/Cec Md在BL21(DE3)获得大量表达,成功建立pGEX-6P-1/Cec Md/BL21(DE3)重组基因工程菌株;重组蛋白以包涵体形式存在,后者用High-Affinity GST Resin亲和层析柱进行纯化,然后用PreScissionTM Protease对融合蛋白进行切割,获得纯化的Cec Md蛋白;测定得出本蛋白样品Cec Md融合蛋白的含为336.83mg/mL,Cec Md在大肠杆菌BL21(DE3)中表达的得率为650mg/L培养液。
(3)重组工程菌表达条件的优化。对重组基因工程菌pGEX-6p-1/Cec Md/BL21(DE3)在不同培养基中的生长曲线进行了测定。结果表明:TB培养基最适合重组基因工程菌的生长;IPTG诱导后,菌体生长曲线发生改变,使抗菌肽重组菌的生长密度低于对照组(非重组基因工程菌)。发现抗菌肽的表达可降低宿主菌的生长密度,此效应也从侧面反应了Cec Md融合蛋白的抑菌活性;通过对Cec Md融合蛋白最佳表达条件的初步摸索,确定最佳的发酵条件为:
①培养基:TB培养基;②IPTG浓度: 0.8mmol/L;③诱导时间: 6h;④温度:37℃。最终确定了pGEX-6p-1/Cec Md/BL21(DE3)菌株的高效表达条件,进而获得了高表达的Cec Md,为其在生物反应器中的高密度培养提供了实验依据。
(4)家蝇抗菌肽Cec Md活性的研究。结果显示,Cec Md和Cec Md融合蛋白(未经PreScissionTM Protease切割的Cec Md融合蛋白)均抑制大肠杆菌、沙门菌和停乳链球菌的生长,二者抑菌圈大小相仿,与氨苄青霉素的抑菌活性接近;Cec Md对大肠杆菌、沙门菌和停乳链球菌三菌株的最小抑菌浓度(MIC)均低于氨苄青霉素组,Cec Md对大肠杆菌和鸡沙门氏菌的最小抑菌浓度是氨苄青霉素组的50%,Cec Md在浓度为5.86μmol/L时,就对停乳链球菌的生长产生抑制作用,但氨苄青霉素只有浓度>750μmol/L时才出现抑菌活性。热稳定性检测说明Cec Md的热稳定性较好,高温20min不能破坏Cec Md的结构和抑菌活性。通过探索Cec Md对SD大鼠和三黄鸡的外周血淋巴细胞转化率的影响,发现Cec Md能显著提高实验动物的外周血淋巴细胞转化率,说明Cec Md对机体的细胞免疫功能有积极的促进作用。
As the extensive application even the abuse of antibiotics, especially antimicrobial growth promoters (AGPs) in animal husbandry over the recent years, more and more antibiotic-resistant bacteria have emerged, and even many serious problems involving food safety and environmental pollution were triggered accordingly, which has ceaselessly plagued the public though antibiotic has been thought as the booster of animal production. Antibiotic abuse in animal husbandry decreases the quality of animal products and threats the health status of general public, which not only jeopardizes the public health security, does detrimental to operation of industry chain of animal husbandry, but also weakens competitiveness of animal products of our country in global market. Antimicrobial peptides (AMPs) are kinds of cationic peptides with broad antimicrobial spectrum and they are essential components of innate immune system. At present, many study fields such as biology, medicine, pharmacy, and agronomy focused AMP for its high-performance and unique action mechanisms. AMPs possess such several merits as non-drug resistance, non-residual, high temperature resistance and high specificity on target cells, which make itself as the substitute of traditional antibiotics in feed industry. Therefore, further doing the basic researches on antimicrobial peptide is significance to improve quality of animal products, enhance performance, increase resistant ability to diseases, even to promote development of the entire animal husbandry. Some works were conducted as following:
(1) Cloning and bioinformic analysis of gene of Cec Md from Musca domestica larvae. Total RNAs were isolated from Musca domestica larvae with or without lipopolysaccharide (LPS) challenging and cDNA encoding the open reading frame (ORF) of Cec Md were amplified by RT-PCR using primers designed according to the known sequence of Musca domestica Cecropin (GenBank: DQ232774). Gene fragments of Cec Md were ligated into pMD-18T vector and, then were identified by PCR, double restriction enzyme digestion and sequencing. Bioinformic analysis on fragments of Cec Md gene and its deduced amino acids were completed. Results indicated that gene of Cec Md was successfully cloned from Musca domestica larvae challenged with LPS but failed to obtain it from the maggots without LPS inducements. The coding region of Cec Md was 192 bp in length which encoded a polypeptide consisted of 63 amino acid residues which consisted of 8 strong basic (+) amino acids, 4 strong acidic (-) amino acids, 30 hydrophobic amino acids and 14 polar amino acids. the most likely cleavage site was between residue 23 and 24, which demonstrated that the part of 1～23 amino acids in Cec Md was signal peptide and of 24～63 amino acids was mature peptide. Results of prediction on three-dimensional (3-D) structure models showed that the complete peptide, a mature peptide plus signaling peptide, of Cec Md contained four helixes whereas the mature peptide only possessed two, both of which were no disulfide bonds. Analysis of homology of sequence indicated that the homologous rates of amino acid sequences of cecropins between Musca domestica and Drosophila were higher than those of other species, reaching 84.1%.
(2) Expression of Cec Md gene in E.Coli and purification of its expression products. Fragment of Cec Md gene from pMD18-T/Cec Md with double digestion were cloned into expression vetor pET32a (+) or pGEX-6P-1 respectively and then identified by PCR, digestion of double restriction enzymes and DNA sequencing. Results showed that two recombinant expression vectors pET32a (+)/Cec Md or pGEX-6P-1/Cec Md were constructed successfully and then the recombinants were transformed into E.coli BL21 (DE3) with induction of IPTG and, finally identified the expression level of target protein using SDS-PAGE. Results demonstrated that bands of protein from pET32a (+)/Cec Md were not detected whereas pGEX-6P-1/Cec Md expressed its protein in E.coli BL21 (DE3) with the identification of SDS-PAGE and Western blot. It indicated that genetic engineering strains was constructed and the proteins were highly expressed as inclusion body. Fusion protein was purified using affinity chromatography column High-Affinity GST Resin, and digested with PreScissionTM Protease. Rtesults indicated that concentration of Cec Md fusion protein in samples was 336.83mg/mL , and the yield rate of Cec Md expressed in E.Coli. was 650mg from per liter medium.
(3) Optimization of expression conditions of recombinants. Growth curves of recombinant pGEX-6p-1/Cec Md/BL21 (DE3) in various cultures were assayed and the results showed that TB medium was the optimum one for the growth of the bacteria. After induction of IPTG, growth curves of E.Coli were altered and density of recombinant cells was lower than those of non-recombinant ones, which indicated that Cec Md has antibacterial activity. Optimization of expression conditions of fusion protein were preliminary explored and the results showed that the optimum combination for fermentation of the cells were: 1) culture: TB; 2) concentration of IPTG: 0.8mmol/L; 3) time of induction: 6h; 4) temperature: 37℃. Optimum expression conditions of fusion protein were determined in this work, which provided experimental basis for the high-density culture of recombinants strain pGEX-6p-1/Cec Md/BL21 (DE3) in fermentation system.
(4) Studies on activity of Cec Md. Results indicated that both Cec Md and Cec Md fusion protein (without digestion of PreScissionTM Protease) inhibited the growth of Escherichia coli, Salmonella gallinarum and Streptococcus dysgalactiae. Inhibition zones among Cec Md, Cec Md fusion protein and ampicillin were in same size. Minimal inhibitory concentration (MIC) of Cec Md on Escherichia coli and Streptococcus dysgalactiae were 50% lower than those of ampicillin, and Cec Md at concentration of 5.86μmol/L inhibited growing of Streptococcus dysgalactiae while ampicillin did not show antibacterial activity on this bacteria until its concentration reached 750μmol/L. Evaluation of thermal stability indicated that Cec Md possessed well thermal stability and its structure was not destroyed and, accordingly its activity was maintained in a high temperature condition. Results demonstrated that lymphocyte transformation rates in peripheral blood in SD rats and Sanhuang chickens were increased by Cec Md, which demonstrated that this peptide played an active part in regulatory function in cellular immunity.
本研究主要开展了如下几个方面的探索:
(1)Cec Md基因的克隆与生物信息学分析。利用脂多糖(LPS)诱导或非诱导的家蝇幼虫体内抽提总RNA,根据家蝇cecropin基因(GenBank:DQ 232774)设计特异性引物,应用RT-PCR技术扩增出编码Cec Md开放阅读框(ORF)的cDNA序列,将其克隆入pMD18-T载体,经PCR、双酶切和测序鉴定正确后,对此基因及其氨基酸推导序列进行系统的生物信息学分析。结果表明从LPS诱导的家蝇幼虫体内成功的扩增出家蝇抗菌肽cecropin基因,但从未诱导的虫体内未能获得此基因;克隆的家蝇Cec Md基因大小为192 bp,编码由63氨基酸残基组成的多肽,分子量6739.94,其中强碱性氨基酸8个、强酸性氨基酸4个、疏水性氨基酸30个、极性氨基酸14个;Cec Md最可能的裂解位点在A23和G24之间,提示N端1～23氨基酸为Cec Md的信号肽,C端24～63氨基酸构成成熟肽;3-D结构预测结果表明,Cec Md的成熟肽含有2个螺旋结构,不含二硫键;Cec Md与果蝇cecropins的同源性显著高于其他昆虫,达到81.0%以上,最高者为84.1%。
(2)Cec Md基因在E.coli中的表达与产物纯化。将构建好的pMD18-T/Cec Md经双酶切后,获取线性目的基因片段,将其定向重组于表达载体pET32a(+)和pGEX-6P-1,进行PCR、双酶切鉴定和DNA测序分析。确定成功构建重组表达载体pET32a(+)/Cec Md和pGEX-6P-1/Cec Md后,转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE检测目的蛋白的表达。结果表明,在SDS-PAGE胶片中未检测出pET32a(+)/Cec Md重组载体的蛋白表达条带;经SDS-PAGE和Western blot检测,证明pGEX-6P-1/Cec Md在BL21(DE3)获得大量表达,成功建立pGEX-6P-1/Cec Md/BL21(DE3)重组基因工程菌株;重组蛋白以包涵体形式存在,后者用High-Affinity GST Resin亲和层析柱进行纯化,然后用PreScissionTM Protease对融合蛋白进行切割,获得纯化的Cec Md蛋白;测定得出本蛋白样品Cec Md融合蛋白的含为336.83mg/mL,Cec Md在大肠杆菌BL21(DE3)中表达的得率为650mg/L培养液。
(3)重组工程菌表达条件的优化。对重组基因工程菌pGEX-6p-1/Cec Md/BL21(DE3)在不同培养基中的生长曲线进行了测定。结果表明:TB培养基最适合重组基因工程菌的生长;IPTG诱导后,菌体生长曲线发生改变,使抗菌肽重组菌的生长密度低于对照组(非重组基因工程菌)。发现抗菌肽的表达可降低宿主菌的生长密度,此效应也从侧面反应了Cec Md融合蛋白的抑菌活性;通过对Cec Md融合蛋白最佳表达条件的初步摸索,确定最佳的发酵条件为:
①培养基:TB培养基;②IPTG浓度: 0.8mmol/L;③诱导时间: 6h;④温度:37℃。最终确定了pGEX-6p-1/Cec Md/BL21(DE3)菌株的高效表达条件,进而获得了高表达的Cec Md,为其在生物反应器中的高密度培养提供了实验依据。
(4)家蝇抗菌肽Cec Md活性的研究。结果显示,Cec Md和Cec Md融合蛋白(未经PreScissionTM Protease切割的Cec Md融合蛋白)均抑制大肠杆菌、沙门菌和停乳链球菌的生长,二者抑菌圈大小相仿,与氨苄青霉素的抑菌活性接近;Cec Md对大肠杆菌、沙门菌和停乳链球菌三菌株的最小抑菌浓度(MIC)均低于氨苄青霉素组,Cec Md对大肠杆菌和鸡沙门氏菌的最小抑菌浓度是氨苄青霉素组的50%,Cec Md在浓度为5.86μmol/L时,就对停乳链球菌的生长产生抑制作用,但氨苄青霉素只有浓度>750μmol/L时才出现抑菌活性。热稳定性检测说明Cec Md的热稳定性较好,高温20min不能破坏Cec Md的结构和抑菌活性。通过探索Cec Md对SD大鼠和三黄鸡的外周血淋巴细胞转化率的影响,发现Cec Md能显著提高实验动物的外周血淋巴细胞转化率,说明Cec Md对机体的细胞免疫功能有积极的促进作用。
As the extensive application even the abuse of antibiotics, especially antimicrobial growth promoters (AGPs) in animal husbandry over the recent years, more and more antibiotic-resistant bacteria have emerged, and even many serious problems involving food safety and environmental pollution were triggered accordingly, which has ceaselessly plagued the public though antibiotic has been thought as the booster of animal production. Antibiotic abuse in animal husbandry decreases the quality of animal products and threats the health status of general public, which not only jeopardizes the public health security, does detrimental to operation of industry chain of animal husbandry, but also weakens competitiveness of animal products of our country in global market. Antimicrobial peptides (AMPs) are kinds of cationic peptides with broad antimicrobial spectrum and they are essential components of innate immune system. At present, many study fields such as biology, medicine, pharmacy, and agronomy focused AMP for its high-performance and unique action mechanisms. AMPs possess such several merits as non-drug resistance, non-residual, high temperature resistance and high specificity on target cells, which make itself as the substitute of traditional antibiotics in feed industry. Therefore, further doing the basic researches on antimicrobial peptide is significance to improve quality of animal products, enhance performance, increase resistant ability to diseases, even to promote development of the entire animal husbandry. Some works were conducted as following:
(1) Cloning and bioinformic analysis of gene of Cec Md from Musca domestica larvae. Total RNAs were isolated from Musca domestica larvae with or without lipopolysaccharide (LPS) challenging and cDNA encoding the open reading frame (ORF) of Cec Md were amplified by RT-PCR using primers designed according to the known sequence of Musca domestica Cecropin (GenBank: DQ232774). Gene fragments of Cec Md were ligated into pMD-18T vector and, then were identified by PCR, double restriction enzyme digestion and sequencing. Bioinformic analysis on fragments of Cec Md gene and its deduced amino acids were completed. Results indicated that gene of Cec Md was successfully cloned from Musca domestica larvae challenged with LPS but failed to obtain it from the maggots without LPS inducements. The coding region of Cec Md was 192 bp in length which encoded a polypeptide consisted of 63 amino acid residues which consisted of 8 strong basic (+) amino acids, 4 strong acidic (-) amino acids, 30 hydrophobic amino acids and 14 polar amino acids. the most likely cleavage site was between residue 23 and 24, which demonstrated that the part of 1～23 amino acids in Cec Md was signal peptide and of 24～63 amino acids was mature peptide. Results of prediction on three-dimensional (3-D) structure models showed that the complete peptide, a mature peptide plus signaling peptide, of Cec Md contained four helixes whereas the mature peptide only possessed two, both of which were no disulfide bonds. Analysis of homology of sequence indicated that the homologous rates of amino acid sequences of cecropins between Musca domestica and Drosophila were higher than those of other species, reaching 84.1%.
(2) Expression of Cec Md gene in E.Coli and purification of its expression products. Fragment of Cec Md gene from pMD18-T/Cec Md with double digestion were cloned into expression vetor pET32a (+) or pGEX-6P-1 respectively and then identified by PCR, digestion of double restriction enzymes and DNA sequencing. Results showed that two recombinant expression vectors pET32a (+)/Cec Md or pGEX-6P-1/Cec Md were constructed successfully and then the recombinants were transformed into E.coli BL21 (DE3) with induction of IPTG and, finally identified the expression level of target protein using SDS-PAGE. Results demonstrated that bands of protein from pET32a (+)/Cec Md were not detected whereas pGEX-6P-1/Cec Md expressed its protein in E.coli BL21 (DE3) with the identification of SDS-PAGE and Western blot. It indicated that genetic engineering strains was constructed and the proteins were highly expressed as inclusion body. Fusion protein was purified using affinity chromatography column High-Affinity GST Resin, and digested with PreScissionTM Protease. Rtesults indicated that concentration of Cec Md fusion protein in samples was 336.83mg/mL , and the yield rate of Cec Md expressed in E.Coli. was 650mg from per liter medium.
(3) Optimization of expression conditions of recombinants. Growth curves of recombinant pGEX-6p-1/Cec Md/BL21 (DE3) in various cultures were assayed and the results showed that TB medium was the optimum one for the growth of the bacteria. After induction of IPTG, growth curves of E.Coli were altered and density of recombinant cells was lower than those of non-recombinant ones, which indicated that Cec Md has antibacterial activity. Optimization of expression conditions of fusion protein were preliminary explored and the results showed that the optimum combination for fermentation of the cells were: 1) culture: TB; 2) concentration of IPTG: 0.8mmol/L; 3) time of induction: 6h; 4) temperature: 37℃. Optimum expression conditions of fusion protein were determined in this work, which provided experimental basis for the high-density culture of recombinants strain pGEX-6p-1/Cec Md/BL21 (DE3) in fermentation system.
(4) Studies on activity of Cec Md. Results indicated that both Cec Md and Cec Md fusion protein (without digestion of PreScissionTM Protease) inhibited the growth of Escherichia coli, Salmonella gallinarum and Streptococcus dysgalactiae. Inhibition zones among Cec Md, Cec Md fusion protein and ampicillin were in same size. Minimal inhibitory concentration (MIC) of Cec Md on Escherichia coli and Streptococcus dysgalactiae were 50% lower than those of ampicillin, and Cec Md at concentration of 5.86μmol/L inhibited growing of Streptococcus dysgalactiae while ampicillin did not show antibacterial activity on this bacteria until its concentration reached 750μmol/L. Evaluation of thermal stability indicated that Cec Md possessed well thermal stability and its structure was not destroyed and, accordingly its activity was maintained in a high temperature condition. Results demonstrated that lymphocyte transformation rates in peripheral blood in SD rats and Sanhuang chickens were increased by Cec Md, which demonstrated that this peptide played an active part in regulatory function in cellular immunity.
引文
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[48] Powersand J P, Hancock R E W. The relationship between peptide structureand antibacterial activity, Peptides, 2003, 24: 1681-1691.
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[50] Matsuzaki K. Why and how are peptide-lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes, Biochim Biophys Acta, 1999, 1462: 1-10.
[51] Hancock R E W, Chapple D S. Peptide antibiotics, Antimicrob Agents Chemother, 1999, 43: 1317-1323.
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[54] Holak T A, Engstr?m A, Kraulis P J, et al. The solution conformation of the antibacterial peptide cecropin A: a nuclear magnetic resonance and dynamical simulated annealing study. Biochemistry, 1988, 27: 7620-7629.
[55] Boman H G, Faye I, Gudmundsso G. H, et al. Cell-free immunity in Cecropia. A model system for antibacterial proteins. Eur J Biochem, 1991, 201: 23-31.
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[64]王来城,王金星,王来元.家蝇防御素基因cDNA的克隆及序列分析.动物学报,2003, 49(3):408-413.
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