泰勒虫重组抗原Tams1-spag基因原核表达与纯化
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摘要
为了在低成本的情况下,得到高浓度、高纯度的环形泰勒虫表面重组抗原的融合蛋白,设定表达过程中不同的OD600nm、IPTG浓度与诱导时间,探索最佳表达条件。通过KCl染色法切胶后分别经透析袋电洗脱法与快速离心法回收目的蛋白。对上述试验的蛋白样品进行SDS-PAGE和Western blot检测。结果表明,在OD600nm=1时加入浓度为1mmol/L IPTG,过夜诱导时,重组蛋白的表达量最大。在KCl染色法切胶回收目的条带的基础上,电洗脱法与快速离心法均能得到同等浓度的高纯度目的蛋白,经Western blot检测重组蛋白的生物活性没有改变,仍具有良好的反应原性。
To study the purification method of the of fusion protein of Theileria annulata surface recombinant antigens and to obtain high purity products at low cost,different parameters were set in order to obtain the optimum of expression.The experimental parameters included OD600 nm,IPTG concentration induction time.Firstly,the target proteins were isolated by cutting the gel slices that contained the right bands which were stained by KCl solution.Secondly,the recycle protein were recovered by electrical elution and high centrifugation.Through SDS-PAGE and Western-blot,the experimental results indicated that the maximal quantity of expression of recombinant proteins was confirmed when IPTG concentration was 1mmol/Land OD600nm=1,the induced time was stay overnight.The two methods can obtain the same purified products and high purity.Western-blot assay indicated that the recombinant protein were not denaturated,and had good antigencity.
To study the purification method of the of fusion protein of Theileria annulata surface recombinant antigens and to obtain high purity products at low cost,different parameters were set in order to obtain the optimum of expression.The experimental parameters included OD600 nm,IPTG concentration induction time.Firstly,the target proteins were isolated by cutting the gel slices that contained the right bands which were stained by KCl solution.Secondly,the recycle protein were recovered by electrical elution and high centrifugation.Through SDS-PAGE and Western-blot,the experimental results indicated that the maximal quantity of expression of recombinant proteins was confirmed when IPTG concentration was 1mmol/Land OD600nm=1,the induced time was stay overnight.The two methods can obtain the same purified products and high purity.Western-blot assay indicated that the recombinant protein were not denaturated,and had good antigencity.
引文
[1]汪明.兽医寄生虫学[M].北京:中国农业大学出版社,2008:332.
[2]农业部畜牧兽医局.一、二、三类动物疫病释义[M].北京:中国农业出版社,2004:97.
[3]蔺红玲,张继瑜,魏小娟,等.环形泰勒虫主要膜蛋白的研究进展[J].黑龙江畜牧兽医,2011,(05):22-24.
[4]Raiendran C,RavD D.Diagnosis of tropical bovine theileriosis by ELISA with recombinant merozoite surface protein of Theileria annulata(Tams1)[J].J Parasit Dis,2014,38(1):41-45.
[5]Darghouth M A,Boulter N R,Gharbi M,et al.Vaccination of calves with an attenuated cell line of Theileria annulataand the sporozoite antigen SPAG-1produces a synergistic effect[J].Vet Parasitol,2006,142(1-2):54-62.
[6]Ghoneim A M,EI-Fayomy A O.Targeting tams-1gene results in underestimation of Theileria annulata infection in diseased cattle in Egypt[J].Acta Parasitol,2014,59(1):85-90.
[7]郑进峰.环形泰勒虫TaSP基因的克隆、原核表达及PCR诊断方法的建立[D].河南郑州:河南农业大学,2013.
[8]魏玉荣,易忠,马文戈,等.泰勒焦虫表面抗原Tams1、Spag1和Tasp基因的克隆与表达[J].新疆农业科学.2013,(11):2136-2142
[9]魏玉荣,易忠,马文戈,等.泰勒焦虫重组蛋白Tasp-Tams1-Spag1间接ELISA检测方法的建立[J].新疆农业大学学报.2013,36(5):360-365.
[10]冯延叶.变性蛋白复性装置研制及包涵体蛋白变性和复性技术研究[D].上海:华东理工大学,2013.
[11]刘镕,钟沁萍,蒋明森,等.一种获得高纯度包涵体蛋白的简便方法[J].中国寄生虫学与寄生虫病杂志.2011,29(5):399-401.
[12]Lee G H,Cooney D.The economics of inclusion body processing[J].Bioprocess Biosyst Eng,2006,29(2):73-90.
[13]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
[14]潘辉.新疆卡拉库尔羊Fas基因的克隆、原核表达及抗原性分析[D].新疆塔里木:塔里木大学,2012.
[15]Bio-rad laboratories.Model 422 electro-eluter instruction manual[M].US:Bio-rad Laboratories,2005:1-11.
[16]李友娟,郭凤达,葛均青,等.鳗鲡疱疹病毒ORF51基因的原核表达及多克隆抗体的制备[J].福建农林大学学报,2014(5):490-494.
[2]农业部畜牧兽医局.一、二、三类动物疫病释义[M].北京:中国农业出版社,2004:97.
[3]蔺红玲,张继瑜,魏小娟,等.环形泰勒虫主要膜蛋白的研究进展[J].黑龙江畜牧兽医,2011,(05):22-24.
[4]Raiendran C,RavD D.Diagnosis of tropical bovine theileriosis by ELISA with recombinant merozoite surface protein of Theileria annulata(Tams1)[J].J Parasit Dis,2014,38(1):41-45.
[5]Darghouth M A,Boulter N R,Gharbi M,et al.Vaccination of calves with an attenuated cell line of Theileria annulataand the sporozoite antigen SPAG-1produces a synergistic effect[J].Vet Parasitol,2006,142(1-2):54-62.
[6]Ghoneim A M,EI-Fayomy A O.Targeting tams-1gene results in underestimation of Theileria annulata infection in diseased cattle in Egypt[J].Acta Parasitol,2014,59(1):85-90.
[7]郑进峰.环形泰勒虫TaSP基因的克隆、原核表达及PCR诊断方法的建立[D].河南郑州:河南农业大学,2013.
[8]魏玉荣,易忠,马文戈,等.泰勒焦虫表面抗原Tams1、Spag1和Tasp基因的克隆与表达[J].新疆农业科学.2013,(11):2136-2142
[9]魏玉荣,易忠,马文戈,等.泰勒焦虫重组蛋白Tasp-Tams1-Spag1间接ELISA检测方法的建立[J].新疆农业大学学报.2013,36(5):360-365.
[10]冯延叶.变性蛋白复性装置研制及包涵体蛋白变性和复性技术研究[D].上海:华东理工大学,2013.
[11]刘镕,钟沁萍,蒋明森,等.一种获得高纯度包涵体蛋白的简便方法[J].中国寄生虫学与寄生虫病杂志.2011,29(5):399-401.
[12]Lee G H,Cooney D.The economics of inclusion body processing[J].Bioprocess Biosyst Eng,2006,29(2):73-90.
[13]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.
[14]潘辉.新疆卡拉库尔羊Fas基因的克隆、原核表达及抗原性分析[D].新疆塔里木:塔里木大学,2012.
[15]Bio-rad laboratories.Model 422 electro-eluter instruction manual[M].US:Bio-rad Laboratories,2005:1-11.
[16]李友娟,郭凤达,葛均青,等.鳗鲡疱疹病毒ORF51基因的原核表达及多克隆抗体的制备[J].福建农林大学学报,2014(5):490-494.