猪β-defensin-2基因的定点突变及其初步抑菌活性分析
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摘要
猪β-defensin-2以其广谱高效的杀菌活性、不易使微生物对其产生耐药性及细胞毒性小等特点倍受人们的关注。随着对抗菌肽结构、活性、抗菌肽作用机制及其基因表达调控机制的了解,设计一种高效的、有利于人类健康的抗菌肽作抗生素替代品是完全可行的。所以,本实验利用生物信息学工具预测猪β-defensin-2蛋白结构,结合猪β-defensin-2防御素的结构特征,有选择的对氨基酸进行定点突变,构建获得七个猪β-defensin-2的突变体mpBD2-1~7,借此在蛋白中造成一点、多点突变,从而开发新型的生物活性更高的猪β-defensin-2基因。主要研究内容如下:
(1)以实验室已经构建的pBD2基因为模板,通过设计不同的PCR引物引入七个不同位点的突变,并将其分别连接至PMD19-T载体上,经过PCR、双酶切及其最后的测序,证明不同的突变基因分别插入到表达载体PET30a中,成功构建出七个不同原核表达载体PET30a-mpBD2-1~7 ,最后转化至大肠杆菌BL21(DE3)PlysE中,得到七个表达菌株BL21-PET30a -mpBD2-1~7。
(2)对重组的突变工程菌株BL21-PET30 -mpBD2-1~7分别进行诱导表达,表达产物经镍离子亲和层析柱纯化,初步获得纯化目的蛋白。
(3)检测七种重组蛋白的抑菌活性。抑菌结果显示,取样品最小抑菌浓度20μg/mL与三种有害菌株共培养,观察其动态生长趋势,结果显示24h时,mpBD2-4、5、7的重组蛋白对三种菌的抑菌活性均高于未突变的PBD2;mpBD2-1、2、3、6对不同菌株出现了相应的低于对照PBD2的抑菌结果。
(4)检测七种重组蛋白的溶血活性。结果显示1、实验组的mpBD2的溶血率在各个终浓度条件下均低于对照组pBD2。2、重组mpBD2蛋白的溶血率最大为10%左右。3、mpBD2-3溶血率在整个对比数据中,溶血率最低,只有5%。
综上所述,本研究成功克隆了七个突变的重组猪β-defensin-2基因mpBD2-1~7,并和未突变的pBD2进行抑菌活性对比,初步获得抑菌活性高于pBD2的重组蛋白mpBD2-4、5、7,为进一步研究生物活性和抑菌作用机理奠定了基础。
Porcineβ-defensin-2 has attracted increasing attention for its characteristics such as efficient broad-spectrum antimicrobial activity,difficult to make microbes resistant to it and weak cytotoxicity. With the understanding of antimicrobial peptides’structure,activity,antibacterial mechanism and regulation of gene expression,it's entirely feasible to design a kind of antimicrobial peptide which is efficient and good for human health to replace antibiotics. In our study,we forecast the protein structure of porcineβ-defensin-2 by bioinformatics tools and make site-directed mutagenesis selectively in the amino acid sequence ,only to acquire the construction of porcineβ-defensin-2 mutant called mpBD2.We make one or more point mutant in the protein to exploit new-type porcineβ-defensin-2 with more biological activity. The main research contents are as follows:
1. Tthe Laboratory has been constructed pBD2 gene as template, by designing PCR primers to introduce the different sites of seven different mutations, and their respective connected to the PMD19-T vector, after PCR, restriction enzyme digestion and end sequencing Demonstrate the different mutations were inserted into the expression vector PET30a in the successful construction of the seven different prokaryotic expression vector PET30a-mpBD2-1 ~ 7, the final transformed into E. coli BL21 (DE3) PlysE, the obtained expression strain BL21-PET30a seven -mpBD2-1 ~ 7.
2. Recombinant strains with mutations in BL21-PET30-mpBD2-1~7 were induced for expression, the expressed product by nickel ion affinity chromatography purification, the initial purpose of the purified protein.
3. Detection of antibacterial activity of seven recombinant proteins. Antibacterial results show that the minimum inhibitory concentration 20μg/mL take samples of harmful strains were cultured with three to observe the dynamic growth trend, the results show 24h time, mpBD2-4, 5,7 of the recombinant protein on the inhibition of three bacteria Activity was higher than mutation pBD2; mpBD2-1,2,3,6 of different strains were lower than the corresponding control pBD2 antibacterial results.
4. Detection of hemolytic activity of seven of the recombinant protein. The results showed that 1. the experimental group mpBD2 hemolytic rate in all conditions, the final concentration was lower than the control group pBD2. 2.recombinant protein mpBD2 maximum hemolysis rate of 10%. 3. mpBD2-3 hemolytic rate in the comparison data, hemolysis was the lowest,only 5%.
In summary, we successfully cloned seven mutant recombinant porcineβ-defensin-2 gene mpBD2-1 ~ 7, and with and without mutations pBD2 antibacterial activity compared to the initial access to the reorganization of antibacterial activity than pBD2 Protein mpBD2-4, 5,7, in order to further study the biological activity and inhibition mechanism of the foundation.
(1)以实验室已经构建的pBD2基因为模板,通过设计不同的PCR引物引入七个不同位点的突变,并将其分别连接至PMD19-T载体上,经过PCR、双酶切及其最后的测序,证明不同的突变基因分别插入到表达载体PET30a中,成功构建出七个不同原核表达载体PET30a-mpBD2-1~7 ,最后转化至大肠杆菌BL21(DE3)PlysE中,得到七个表达菌株BL21-PET30a -mpBD2-1~7。
(2)对重组的突变工程菌株BL21-PET30 -mpBD2-1~7分别进行诱导表达,表达产物经镍离子亲和层析柱纯化,初步获得纯化目的蛋白。
(3)检测七种重组蛋白的抑菌活性。抑菌结果显示,取样品最小抑菌浓度20μg/mL与三种有害菌株共培养,观察其动态生长趋势,结果显示24h时,mpBD2-4、5、7的重组蛋白对三种菌的抑菌活性均高于未突变的PBD2;mpBD2-1、2、3、6对不同菌株出现了相应的低于对照PBD2的抑菌结果。
(4)检测七种重组蛋白的溶血活性。结果显示1、实验组的mpBD2的溶血率在各个终浓度条件下均低于对照组pBD2。2、重组mpBD2蛋白的溶血率最大为10%左右。3、mpBD2-3溶血率在整个对比数据中,溶血率最低,只有5%。
综上所述,本研究成功克隆了七个突变的重组猪β-defensin-2基因mpBD2-1~7,并和未突变的pBD2进行抑菌活性对比,初步获得抑菌活性高于pBD2的重组蛋白mpBD2-4、5、7,为进一步研究生物活性和抑菌作用机理奠定了基础。
Porcineβ-defensin-2 has attracted increasing attention for its characteristics such as efficient broad-spectrum antimicrobial activity,difficult to make microbes resistant to it and weak cytotoxicity. With the understanding of antimicrobial peptides’structure,activity,antibacterial mechanism and regulation of gene expression,it's entirely feasible to design a kind of antimicrobial peptide which is efficient and good for human health to replace antibiotics. In our study,we forecast the protein structure of porcineβ-defensin-2 by bioinformatics tools and make site-directed mutagenesis selectively in the amino acid sequence ,only to acquire the construction of porcineβ-defensin-2 mutant called mpBD2.We make one or more point mutant in the protein to exploit new-type porcineβ-defensin-2 with more biological activity. The main research contents are as follows:
1. Tthe Laboratory has been constructed pBD2 gene as template, by designing PCR primers to introduce the different sites of seven different mutations, and their respective connected to the PMD19-T vector, after PCR, restriction enzyme digestion and end sequencing Demonstrate the different mutations were inserted into the expression vector PET30a in the successful construction of the seven different prokaryotic expression vector PET30a-mpBD2-1 ~ 7, the final transformed into E. coli BL21 (DE3) PlysE, the obtained expression strain BL21-PET30a seven -mpBD2-1 ~ 7.
2. Recombinant strains with mutations in BL21-PET30-mpBD2-1~7 were induced for expression, the expressed product by nickel ion affinity chromatography purification, the initial purpose of the purified protein.
3. Detection of antibacterial activity of seven recombinant proteins. Antibacterial results show that the minimum inhibitory concentration 20μg/mL take samples of harmful strains were cultured with three to observe the dynamic growth trend, the results show 24h time, mpBD2-4, 5,7 of the recombinant protein on the inhibition of three bacteria Activity was higher than mutation pBD2; mpBD2-1,2,3,6 of different strains were lower than the corresponding control pBD2 antibacterial results.
4. Detection of hemolytic activity of seven of the recombinant protein. The results showed that 1. the experimental group mpBD2 hemolytic rate in all conditions, the final concentration was lower than the control group pBD2. 2.recombinant protein mpBD2 maximum hemolysis rate of 10%. 3. mpBD2-3 hemolytic rate in the comparison data, hemolysis was the lowest,only 5%.
In summary, we successfully cloned seven mutant recombinant porcineβ-defensin-2 gene mpBD2-1 ~ 7, and with and without mutations pBD2 antibacterial activity compared to the initial access to the reorganization of antibacterial activity than pBD2 Protein mpBD2-4, 5,7, in order to further study the biological activity and inhibition mechanism of the foundation.
引文
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