鸡源志贺菌IpaC基因表达载体的构建及其在毕赤酵母中的表达
摘要
为使IpaC基因表达产物保持天然的生物学活性,实现在酵母表达系统中高效表达,本研究在不改变IpaC蛋白氨基酸序列的情况下,根据酵母密码子偏爱性优化合成IpaC#基因,分别构建真核表达载体pGAPZαA-IpaC和pGAPZαA-IpaC#,然后将线性化重组质粒电转化至毕赤酵母X-33中,对阳性转化子用SDS-PAGE及Westernblot检测其表达产物。结果表明,经SDS-PAGE及Westernblot检测,仅在pGAPZαA-IpaC#/X-33重组菌表达产物菌体沉淀中检测到大小为70ku的目的蛋白,实现了鸡源志贺菌IpaC#基因在毕赤酵母中的胞内表达,从而验证毕赤酵母表达系统与外源基因序列的相关性,为进一步提高鸡源志贺菌IpaC基因在毕赤酵母中的高效表达提供科学依据,对研究该病的发病机制具有重要意义。
引文
[1]许兰菊,王川庆,胡攻政,等.鸡志贺氏菌在我国的发现及其病原特性研究[J].中国预防兽医学报,2004,26(4):281-286.
[2]Terry C M,Picking W L,Birket S E,et al.The C-terminus of Ipa C is required for effector activities related to Shigella invasion of host cells[J].Microbial Pathogenesis,2008,45(4):282-289.
[3]Shaikh N,Terajima J,Watanabe H.Ipa C of Shigella binds to the Cterminal domain ofβ-catenin[J].Microbial Pathogenesis,2003,35(3):107-117.
[4]Brzu S,Benjelloun-Touimi Z,Phalipon A,et al.Functional analysis of the Shigella flexneri Ipa C invasin by insertional mutagenesis[J].Infection and immunity,1997,65(5):1599-1605.
[5]Kim S,Kim J,Seo G.Iron oxide nanoparticle-impregnated powder-activated carbon(Ipa C)for NOM removal in MF membrane water treatment system[J].Desalination and Water Treatment,2013,51(31/32/33):6392-6400.
[6]刘红英.人志贺菌对雏鸡的致病性及人、鸡志贺菌某些重要基因的比较研究[D].郑州:河南农业大学,2008.
[7]杨霞,陈陆,许兰菊,等.鸡源鲍氏志贺菌hn03株的分子鉴定与演化分析[J].畜牧兽医学报,2009,40(3):402-408.
[8]杨霞.利用分子遗传标记技术对鸡鲍氏志贺菌的鉴定和菌株起源研究[D].郑州:河南农业大学,2007.
[9]苗银萍,蒋大伟,许兰菊,等.鸡源志贺菌Ipa C基因的克隆及原核表达[J].中国兽医学报,2013,33(9):1352-1357.
[10]陈志宝,宋博翠.弗氏志贺菌侵袭性基因Ipa C诱饵质粒的构建及其自激活作用[J].中国生物制品学杂志,2010,23(6):590-593.
[11]孙素霞,王红,王勇,等.福氏志贺氏菌毒力基因Ipa C在大肠杆菌中表达的初步研究[J].中国人兽共患病杂志,2003,19(6):29-31.
[12]Ahmad M,Hirz M,Pichler H,et al.Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production[J].Applied Microbiology and Biotechnology,2014,98(12):5301-5317.
[13]Borgheresi R A M B,Palma M S,Ducancel F,et al.Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris[J].Toxicon,2001,39(8):1211-1218.
[14]Li J W,Liu Y,Li K,et al.Cloning of a fusion gene containing h GM-CSF gene and gene encoding L protein of HBV and expression in Pichia pastoris[J].Hepato-Gastroenterology,2009,57(99/100):578-582.
[15]Scorer C A,Buckholz R G,Clare J J,et al.The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris[J].Gene,1993,136(1/2):111-119.
[16]姚斌,张春义,王建华.高效表达具有生物活性植酸酶的毕赤酵母[J].中国科学C辑,1998,28(3):237-243.
[17]陈惠,赵海霞,王红宁,等.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量[J].中国生物化学与分子生物学报,2005,21(2):171-175.
[18]Chang S W,Shieh C J,Lee G C,et al.Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris[J].Applied Microbiology and Biotechnology,2005,67(2):215-224.
[19]陈熙,陈毓,齐小雨,等.人溶菌酶定点突变基因在毕赤酵母中的表达及活性分析[J].江苏农业学报,2014,30(6):1396-1401.
[20]董亚青,朱文斗,王琳琳,等.鸡α-干扰素在毕赤酵母中的表达及其表达条件优化[J].江苏农业科学,2015,43(11):275-277.
[2]Terry C M,Picking W L,Birket S E,et al.The C-terminus of Ipa C is required for effector activities related to Shigella invasion of host cells[J].Microbial Pathogenesis,2008,45(4):282-289.
[3]Shaikh N,Terajima J,Watanabe H.Ipa C of Shigella binds to the Cterminal domain ofβ-catenin[J].Microbial Pathogenesis,2003,35(3):107-117.
[4]Brzu S,Benjelloun-Touimi Z,Phalipon A,et al.Functional analysis of the Shigella flexneri Ipa C invasin by insertional mutagenesis[J].Infection and immunity,1997,65(5):1599-1605.
[5]Kim S,Kim J,Seo G.Iron oxide nanoparticle-impregnated powder-activated carbon(Ipa C)for NOM removal in MF membrane water treatment system[J].Desalination and Water Treatment,2013,51(31/32/33):6392-6400.
[6]刘红英.人志贺菌对雏鸡的致病性及人、鸡志贺菌某些重要基因的比较研究[D].郑州:河南农业大学,2008.
[7]杨霞,陈陆,许兰菊,等.鸡源鲍氏志贺菌hn03株的分子鉴定与演化分析[J].畜牧兽医学报,2009,40(3):402-408.
[8]杨霞.利用分子遗传标记技术对鸡鲍氏志贺菌的鉴定和菌株起源研究[D].郑州:河南农业大学,2007.
[9]苗银萍,蒋大伟,许兰菊,等.鸡源志贺菌Ipa C基因的克隆及原核表达[J].中国兽医学报,2013,33(9):1352-1357.
[10]陈志宝,宋博翠.弗氏志贺菌侵袭性基因Ipa C诱饵质粒的构建及其自激活作用[J].中国生物制品学杂志,2010,23(6):590-593.
[11]孙素霞,王红,王勇,等.福氏志贺氏菌毒力基因Ipa C在大肠杆菌中表达的初步研究[J].中国人兽共患病杂志,2003,19(6):29-31.
[12]Ahmad M,Hirz M,Pichler H,et al.Protein expression in Pichia pastoris:recent achievements and perspectives for heterologous protein production[J].Applied Microbiology and Biotechnology,2014,98(12):5301-5317.
[13]Borgheresi R A M B,Palma M S,Ducancel F,et al.Expression and processing of recombinant sarafotoxins precursor in Pichia pastoris[J].Toxicon,2001,39(8):1211-1218.
[14]Li J W,Liu Y,Li K,et al.Cloning of a fusion gene containing h GM-CSF gene and gene encoding L protein of HBV and expression in Pichia pastoris[J].Hepato-Gastroenterology,2009,57(99/100):578-582.
[15]Scorer C A,Buckholz R G,Clare J J,et al.The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris[J].Gene,1993,136(1/2):111-119.
[16]姚斌,张春义,王建华.高效表达具有生物活性植酸酶的毕赤酵母[J].中国科学C辑,1998,28(3):237-243.
[17]陈惠,赵海霞,王红宁,等.植酸酶基因中稀有密码子的改造提高其在毕赤酵母中的表达量[J].中国生物化学与分子生物学报,2005,21(2):171-175.
[18]Chang S W,Shieh C J,Lee G C,et al.Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris[J].Applied Microbiology and Biotechnology,2005,67(2):215-224.
[19]陈熙,陈毓,齐小雨,等.人溶菌酶定点突变基因在毕赤酵母中的表达及活性分析[J].江苏农业学报,2014,30(6):1396-1401.
[20]董亚青,朱文斗,王琳琳,等.鸡α-干扰素在毕赤酵母中的表达及其表达条件优化[J].江苏农业科学,2015,43(11):275-277.