枯草芽孢杆菌表达载体的构建及猪源表皮生长因子的表达
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摘要
本文首先以枯草芽孢杆菌(B.subtilis)穿梭载体pDG150为基础,构建了含有5个克隆位点的表达载体pTD160,并在此基础上构建了带有淀粉酶信号肽的分泌表达载体pTD161和可用于外源蛋白与胞衣蛋白cotG融合表达的表面展示载体pTD162。将脂肪酶基因lipa分别插入pTD161和pTD162,在B.subtilis 168中表达,其LipA酶活性分别为23.4U/mg和15.3U/mg,表明pTD载体具有良好的表达性能。通过无抗生素压力传代培养10代后,发现其稳定性仍为96.5%和95.0%,证明pTD载体具备较高的稳定性。将密码子优化后的猪源表皮生长因子基因pegf分别导入pTD161和pTD162,转化B.subtilis 168获得工程菌Bs168-pTD161-pegf和Bs168-pTD162-pegf,ELISA法证明pEGF在工程菌中表达量分别达到365ng/mL和285ng/mL,证明pEGF获得高效表达和展示。
Firstly,based on the shuttle vector pDG150 of Bacillus subtilis,the expression vector pTD160 containing five cloning sites was constructed.Secondly,the expression vector pTD161 harboring a signal peptide of amylase Q and the surface display vector pTD162 which allows fusion expression of foreign proteins with cotG protein were constructed.The LipA gene was inserted into pTD161 and pTD162 respectively and expressed in B.subtilis 168.The activity of LipA was 23.4 U/mg and 15.3 U/mg respectively,indicating that the pTD vectors had good expression performance.The stability of pTD vectors was 96.5%and 95.0%respectively after 10 generations of non-antibiotic pressure subculture.The porcine epidermal growth factor gene(pegf)optimized by codon of B.subtilis was introduced into pTD161 and pTD162 respectively,and then transformed into B.subtilis 168,generating the engineering strains Bs168-pTD161-pegf and Bs168-pTD162-pegf.ELISA assay showed that the expression of pEGF in these engineering strains reached 365 ng/mL and 285 ng/mL respectively,which indicated the pEGF had been well expressed in B.subtilis 168.
Firstly,based on the shuttle vector pDG150 of Bacillus subtilis,the expression vector pTD160 containing five cloning sites was constructed.Secondly,the expression vector pTD161 harboring a signal peptide of amylase Q and the surface display vector pTD162 which allows fusion expression of foreign proteins with cotG protein were constructed.The LipA gene was inserted into pTD161 and pTD162 respectively and expressed in B.subtilis 168.The activity of LipA was 23.4 U/mg and 15.3 U/mg respectively,indicating that the pTD vectors had good expression performance.The stability of pTD vectors was 96.5%and 95.0%respectively after 10 generations of non-antibiotic pressure subculture.The porcine epidermal growth factor gene(pegf)optimized by codon of B.subtilis was introduced into pTD161 and pTD162 respectively,and then transformed into B.subtilis 168,generating the engineering strains Bs168-pTD161-pegf and Bs168-pTD162-pegf.ELISA assay showed that the expression of pEGF in these engineering strains reached 365 ng/mL and 285 ng/mL respectively,which indicated the pEGF had been well expressed in B.subtilis 168.
引文
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[15]蔡恒,陈忠军,万红贵,等.地衣芽孢杆菌α-淀粉酶信号肽的序列分析及其在大肠杆菌中的分泌特性[J].华北农学报,2008,23(2):106-109.
[16]朱婧斐.L-阿拉伯糖异构酶在枯草芽孢杆菌中的芽孢表面展示及酶学性质研究[D].镇江:江苏大学,2016.
[17]杜萍萍,张莹莹,申培立,等.一株产耐高温碱性脂肪酶菌株的筛选及产酶条件优化[J].食品工业科技,2015,36(9):188-193.
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[19]周珍文,胡旭初,邓秋连,等.枯草杆菌芽孢抵抗胃肠道环境的耐性评估[J].热带医学杂志,2008,8(3):235-237.
[20]邱永乾.微生物脂肪酶库构建及磷脂酶D的筛选与应用[D].青岛:中国海洋大学,2015.
[21]邱权,詹志春,周樱,等.饲料中微生态制剂应用研究[J].饲料工业,2016,37(2):55-58.
[2]Lee D N,Chuang Y S,Chiou H Y,et al.Oral administration recombinant porcine epidermal growth factor enhances the jejunal digestive enzyme genes expression and activity of early-weaned piglets[J].JAnim Physiol Anim Nutr,2010,92(4):463-470.
[3]王定越.重组猪EGF乳酸乳球菌的构建及其对早期断奶仔猪肠道健康的影响[D].成都:四川农业大学,2013.
[4]Carpenter G,Cohen S.Human epidermal growth factor and the proliferation of human fibroblasts[J].Journal of Cellular Physiology,2010,88(2):227.
[5]李超,郭春华,刘棋,等.猪表皮生长因子及其应用前景[J].畜牧与兽医,2009,41(12):93-96.
[6]王勇飞,段叶辉,夏连喜,等.重组猪表皮生长因子对早期断奶仔猪生长性能的影响[J].动物营养学报,2014,26(12):3787-3792.
[7]钟泽民,赖强,余希尧,等.猪表皮生长因子在植物乳杆菌中的表达及其活性检测[J].生物工程学报,2015,31(9):1325-1334.
[8]汪洋,杨桂香,吴波浪,等.猪表皮生长因子在毕赤酵母中的表达及鉴定[J].中国农业科学,2007,40(11):2593-2599.
[9]李情敏,何名芳,张凤英,等.枯草芽孢杆菌液态发酵的研究[J].中国酿造,2016,35(2):43-47.
[10]Hilgren J,Swanson K M,Diez-Gonzalez F,et al.Susceptibilities of Bacillus subtilis,Bacillus cereus,and a virulent Bacillus anthracis spores to liquid biocides[J].Journal of Food Protection,2009,72(2):360-360.
[11]Han X,Shiwa Y,Itoh M,et al.Molecular cloning and sequence analysis of an extracellular protease from four Bacillus subtilis strains[J].Journal of the Agricultural Chemical Society of Japan,2013,77(4):870-873.
[12]Wurm D J,Veiter L,Ulonska S,et al.The E.coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake[J].Applied Microbiology&Biotechnology,2016,100(20):8721-8729.
[13]Johnsen P J,Dubnau D,Levin B R.Episodic selection and the maintenance of competence and natural transformation in Bacillus subtilis[J].Genetics,2009,181(4):1521-1533.
[14]Ringquist S,Shinedling S,Barrick D,et al.Translation initiation in Escherichia coli:sequences within the ribosome-binding site[J].Molecular Microbiology,2010,6(9):1219-1229.
[15]蔡恒,陈忠军,万红贵,等.地衣芽孢杆菌α-淀粉酶信号肽的序列分析及其在大肠杆菌中的分泌特性[J].华北农学报,2008,23(2):106-109.
[16]朱婧斐.L-阿拉伯糖异构酶在枯草芽孢杆菌中的芽孢表面展示及酶学性质研究[D].镇江:江苏大学,2016.
[17]杜萍萍,张莹莹,申培立,等.一株产耐高温碱性脂肪酶菌株的筛选及产酶条件优化[J].食品工业科技,2015,36(9):188-193.
[18]李志辉.枯草芽孢杆菌穿梭表达载体和表面展示载体的构建[D].保定:河北农业大学,2014.
[19]周珍文,胡旭初,邓秋连,等.枯草杆菌芽孢抵抗胃肠道环境的耐性评估[J].热带医学杂志,2008,8(3):235-237.
[20]邱永乾.微生物脂肪酶库构建及磷脂酶D的筛选与应用[D].青岛:中国海洋大学,2015.
[21]邱权,詹志春,周樱,等.饲料中微生态制剂应用研究[J].饲料工业,2016,37(2):55-58.