猪硒蛋白W的原核表达及IgY多克隆抗体制备
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摘要
试验旨在优化硒蛋白W(selenoprotein W,SelW)的原核表达系统,并评估SelW的免疫原性和基于IgY抗体检测猪体内SelW的可行性。将获得的猪SelW基因序列密码子进行优化、合成并连接至pET-32a(+)表达载体中,构建原核表达重组质粒pET32a(+)-SelW,转化E.coli BL21(DE3)宿主菌中,进行IPTG诱导表达,SDS-PAGE鉴定重组猪SelW的表达情况,并免疫产蛋鸡制备抗SelW的IgY多克隆抗体,通过间接ELISA检测IgY抗体滴度,采用Western blotting检测IgY识别抗原的特异性。SDS-PAGE结果显示,SelW基因在原核细胞中成功表达,得到了大小约为33 ku的重组蛋白,IPTG最佳诱导浓度及时间分别为0.5 mmol/L和6 h;可溶性分析结果显示,重组蛋白SelW主要以包涵体形式存在。间接ELISA检测结果显示,免疫后45 d的IgY多克隆抗体的效价可达1∶51 200。Western blotting检测结果显示,重组蛋白具有良好的免疫原性,所制备的IgY抗体与SelW的亲和力较高。本试验成功构建并优化了SelW蛋白的原核表达系统,提高了SelW蛋白的表达量,获得了具有良好免疫原性的SelW蛋白,制备的IgY抗体能特异性识别猪肌肉组织中的SelW,可用于检测猪组织中SelW的表达、预防硒中毒和缺硒性疾病的监测等,为进一步探究SelW的生物学功能奠定基础。
The aim of the experiment was to optimize the prokaryotic expression system of selenoprotein W(SelW),and evaluate the immunogenicity of SelW and the feasibility of detecting SelW in swine based on IgY antibodies.The obtained gene sequence codon of porcine SelW was optimized,synthesized and ligated into pET-32 a(+) expression vector,and the prokaryotic expression recombinant plasmid pET32 a(+)-SelW was constructed and transformed into E.coli BL21 host strain,which was induced by IPTG.The expression of SelW in recombinant swine was identified by SDS-PAGE,and the IgY polyclonal antibody against SelW was prepared by immunizing laying hens,the IgY antibody titer was detected by indirect ELISA,the specificity of the IgY recognition antigen was examined using Western blotting.The results of SDS-PAGE showed that SelW gene was successfully expressed in prokaryotic cells,and a recombinant protein with a size of about 33 ku was obtained.The optimal expression concentration and time of IPTG were 0.5 mmol/L and 6 h,respectively.Soluble analysis results showed that the recombinant protein existed mainly in the form of inclusion bodies.The results of indirect ELISA showed that the titer of IgY polyclonal antibody at 45 days after immunization could reach 1∶51 200.Western blotting results showed that the recombinant protein had good immunogenicity,and the prepared IgY antibody had higher affinity with SelW.This experiment successfully constructed and optimized the prokaryotic expression system of SelW,increased the expression of SelW,and obtained SelW with good immunogenicity,and the prepared IgY antibody specifically recognized SelW in swine muscle tissue,which could be used to detect the expression of SelW in swine tissues.By measuring the expression of SelW to reflect the level of selenium in swine,it could be used to prevent selenium poisoning and selenium-deficient disease monitoring,and laid a foundation for further exploration of SelW biological function.
The aim of the experiment was to optimize the prokaryotic expression system of selenoprotein W(SelW),and evaluate the immunogenicity of SelW and the feasibility of detecting SelW in swine based on IgY antibodies.The obtained gene sequence codon of porcine SelW was optimized,synthesized and ligated into pET-32 a(+) expression vector,and the prokaryotic expression recombinant plasmid pET32 a(+)-SelW was constructed and transformed into E.coli BL21 host strain,which was induced by IPTG.The expression of SelW in recombinant swine was identified by SDS-PAGE,and the IgY polyclonal antibody against SelW was prepared by immunizing laying hens,the IgY antibody titer was detected by indirect ELISA,the specificity of the IgY recognition antigen was examined using Western blotting.The results of SDS-PAGE showed that SelW gene was successfully expressed in prokaryotic cells,and a recombinant protein with a size of about 33 ku was obtained.The optimal expression concentration and time of IPTG were 0.5 mmol/L and 6 h,respectively.Soluble analysis results showed that the recombinant protein existed mainly in the form of inclusion bodies.The results of indirect ELISA showed that the titer of IgY polyclonal antibody at 45 days after immunization could reach 1∶51 200.Western blotting results showed that the recombinant protein had good immunogenicity,and the prepared IgY antibody had higher affinity with SelW.This experiment successfully constructed and optimized the prokaryotic expression system of SelW,increased the expression of SelW,and obtained SelW with good immunogenicity,and the prepared IgY antibody specifically recognized SelW in swine muscle tissue,which could be used to detect the expression of SelW in swine tissues.By measuring the expression of SelW to reflect the level of selenium in swine,it could be used to prevent selenium poisoning and selenium-deficient disease monitoring,and laid a foundation for further exploration of SelW biological function.
引文
[1] 郑良焰,张琴,刘碧涛,等.不同硒浓度日粮对小鼠肝脏和睾丸组织中部分硒蛋白mRNA水平的影响[J].中国畜牧兽医,2013,40(3):38-42.ZHENG L Y,ZHANG Q,LIU B T,et al.Effect of different dietary selenium concentration on mRNA levels of some selenoprotein in mice liver and testis[J].China Animal Husbandry & Veterinary Medicine,2013,40(3):38-42.(in Chinese)
[2] LIU Q,YANG J,CAI J,et al.Analysis of the interactions between thioredoxin and 20 selenoproteins in chicken[J].Biological Trace Element Research,2017,179(2):304-317.
[3] 王斌,王盼.硒的功能与心血管疾病的研究进展[J].国外医学(医学地理分册),2013,34(1):20-22.WANG B,WANG P.Progress on selenium function and cardiovascular disease[J].Foreign Medical Sciences (Section of Medgeography),2013,34(1):20-22.(in Chinese)
[4] DONG H,CHEN W,SUN C,et al.Identification,characterization of selenoprotein W and its mRNA expression patterns in response to somatostatin 14,cysteamine hydrochloride,17β-estradiol and a binary mixture of 17β-estradiol and cysteamine hydrochloride in topmouth culter (Erythroculter ilishaeformis)[J].Fish Physiology and Biochemistry,2017,43(1):115-126.
[5] LIU W,YAO H,ZHAO W,et al.Selenoprotein W was correlated with the protective effect of selenium on chicken myocardial cells from oxidative damage[J].Biological Trace Element Research,2016,171(2):419-426.
[6] ALKAN Z,DUONG F L,HAWKES W C.Selenoprotein W controls epidermal growth factor receptor surface expression,activation and degradation via receptor ubiquitination[J].Biochimica et Biophysica Acta,2015,1853(5):1087-1095.
[7] JEON Y H,KO K Y,LEE J H,et al.Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein[J].Biochimica et Biophysica Acta (BBA)-Molecular Cell Research,2016,1863(1):10-18.
[8] 高爱琴,朱玉霞,雷振华,等.有机硒与维生素E对肉仔鸡肉质性状、血液生化指标及抗氧化指标的影响[J].中国畜牧兽医,2017,44(6):1694-1700.GAO A Q,ZHU Y X,LEI Z H,et al.Effect of organic selenium and vitamin E on meat quality quality,blood biochemical indexes and antioxidant indexes in broiler[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1694-1700.(in Chinese)
[9] XU M,SUN D,LI J,et al.Monoclonal antibodies against avian selenoprotein W[J].Hybridoma,2011,30(6):563-566.
[10] BOULLION R D,MOKELKE E A,WAMHOFF B R,et al.Porcine model of diabetic dyslipidemia:Insulin and feed algorithms for mimicking diabetes mellitus in humans[J].Comparative Medicine,2003,53(1):42-52.
[11] HLINAK A,SCHADE R.Egg yolk antibodies,state of the art and future prospects[J].Altex,1996,13(5):5-9.
[12] SCHADE R.Chicken egg yolk antibodies (IgY-technology):A review of progress in production and use in research and human and veterinary medicine[J].Atla-Alternatives to Laboratory Animals,2005,33(2):129-154.
[13] ZHANG X Y,CHEN H X,TIAN Z H,et al.Chicken monoclonal IgY antibody:A novel antibody development strategy[J].Avian Biology Research,2010,3(3):97-106.
[14] POLSON A,WECHMAR M B V,REGENMORTEL M H.Isolation of viral IgY antibodies from yolks of immunized hens[J].Immunological Communications,1980,9(5):475-493.
[15] HE J X,HU J J,THIRUMALAI D,et al.Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of gentamicin residues[J].Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes,2016,51(1):8-13.
[16] HE J X,WANG Y,ZHANG X Y.Preparation of artificial antigen and development of indirect competitive ELISA based on chicken IgY for the detection of acid orangeⅡin food samples[J].Food Analytical Methods,2016,9(2):378-384.
[17] YEH J Y,BEILSTEIN M A,ANDREWS J S,et al.Tissue distribution and influence of selenium status on levels of selenoprotein W[J].The FASEB Journal,1995,9(5):392-396.
[18] CAO L,ZHANG L,ZENG H,et al.Analyses of selenotranscriptomes and selenium concentrations in response to dietary selenium deficiency and age reveal common and distinct patterns by tissue and sex in telomere-dysfunctional mice[J].Journal of Nutrition,2017,147(10):1858-1866.
[19] 吴东,夏伦志,陈丽园,等.富硒日粮对波杂肉山羊体组织硒储留和肉品质的影响[J].中国草食动物,2008,28(6):40-41.WU D,XIA L Z,CHEN L Y,et al.Effects of selenium-enriched diets on selenium storage and meat quality of meat goats[J].China Herbivores,2008,28(6):40-41.(in Chinese)
[20] WHANGER P D.Selenoprotein expression and function—Selenoprotein W[J].Biochimica et Biophysica Acta General Subjects,2009,1790(11):1448-1452.
[21] KOVACS-NOLAN J,MINE Y.Improving the Safety and Quality of Eggs and Egg Products[M].Woodhead publishing limited:Filip Van Immerseel,Yves Nys,Maureen Bain,2011.
[22] REN H,YANG W,THIRUMALAI D,et al.A comparative evaluation of six principal IgY antibody extraction methods[J].Atla-Alternatives to Laboratory Animals,2016,44(1):11-20.
[23] WHANGER P D.Selenoprotein W:A review[J].Cellular & Molecular Life Sciences,2000,57(13-14):1846-1852.
[24] 李文锋,梅敏敏,黄兴国,等.新型鸭呼肠孤病毒σB蛋白基因密码子的优化及原核表达[J].中国畜牧兽医,2018,45(10):2700-2706.LI W F,MEI M M,HUANG X G,et al.Optimization and prokaryotic expression of the σB protein gene codons of the new-type duck reovirus[J].China Animal Husbandry & Veterinary Medicine,2018,45(10):2700-2706.(in Chinese)
[25] 张永宁,张舟,诸明欣,等.A型塞内卡病毒VP2蛋白原核表达及其多克隆抗体的制备[J].中国畜牧兽医,2019,46(1):256-263.ZHANG Y N,ZHANG Z,ZHU M X,et al.Prokaryotic expression of the VP2 protein of Senecavirus A and preparation of its polyclonal antibodies[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):256-263.(in Chinese)
[2] LIU Q,YANG J,CAI J,et al.Analysis of the interactions between thioredoxin and 20 selenoproteins in chicken[J].Biological Trace Element Research,2017,179(2):304-317.
[3] 王斌,王盼.硒的功能与心血管疾病的研究进展[J].国外医学(医学地理分册),2013,34(1):20-22.WANG B,WANG P.Progress on selenium function and cardiovascular disease[J].Foreign Medical Sciences (Section of Medgeography),2013,34(1):20-22.(in Chinese)
[4] DONG H,CHEN W,SUN C,et al.Identification,characterization of selenoprotein W and its mRNA expression patterns in response to somatostatin 14,cysteamine hydrochloride,17β-estradiol and a binary mixture of 17β-estradiol and cysteamine hydrochloride in topmouth culter (Erythroculter ilishaeformis)[J].Fish Physiology and Biochemistry,2017,43(1):115-126.
[5] LIU W,YAO H,ZHAO W,et al.Selenoprotein W was correlated with the protective effect of selenium on chicken myocardial cells from oxidative damage[J].Biological Trace Element Research,2016,171(2):419-426.
[6] ALKAN Z,DUONG F L,HAWKES W C.Selenoprotein W controls epidermal growth factor receptor surface expression,activation and degradation via receptor ubiquitination[J].Biochimica et Biophysica Acta,2015,1853(5):1087-1095.
[7] JEON Y H,KO K Y,LEE J H,et al.Identification of a redox-modulatory interaction between selenoprotein W and 14-3-3 protein[J].Biochimica et Biophysica Acta (BBA)-Molecular Cell Research,2016,1863(1):10-18.
[8] 高爱琴,朱玉霞,雷振华,等.有机硒与维生素E对肉仔鸡肉质性状、血液生化指标及抗氧化指标的影响[J].中国畜牧兽医,2017,44(6):1694-1700.GAO A Q,ZHU Y X,LEI Z H,et al.Effect of organic selenium and vitamin E on meat quality quality,blood biochemical indexes and antioxidant indexes in broiler[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1694-1700.(in Chinese)
[9] XU M,SUN D,LI J,et al.Monoclonal antibodies against avian selenoprotein W[J].Hybridoma,2011,30(6):563-566.
[10] BOULLION R D,MOKELKE E A,WAMHOFF B R,et al.Porcine model of diabetic dyslipidemia:Insulin and feed algorithms for mimicking diabetes mellitus in humans[J].Comparative Medicine,2003,53(1):42-52.
[11] HLINAK A,SCHADE R.Egg yolk antibodies,state of the art and future prospects[J].Altex,1996,13(5):5-9.
[12] SCHADE R.Chicken egg yolk antibodies (IgY-technology):A review of progress in production and use in research and human and veterinary medicine[J].Atla-Alternatives to Laboratory Animals,2005,33(2):129-154.
[13] ZHANG X Y,CHEN H X,TIAN Z H,et al.Chicken monoclonal IgY antibody:A novel antibody development strategy[J].Avian Biology Research,2010,3(3):97-106.
[14] POLSON A,WECHMAR M B V,REGENMORTEL M H.Isolation of viral IgY antibodies from yolks of immunized hens[J].Immunological Communications,1980,9(5):475-493.
[15] HE J X,HU J J,THIRUMALAI D,et al.Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of gentamicin residues[J].Journal of Environmental Science and Health Part B-Pesticides Food Contaminants and Agricultural Wastes,2016,51(1):8-13.
[16] HE J X,WANG Y,ZHANG X Y.Preparation of artificial antigen and development of indirect competitive ELISA based on chicken IgY for the detection of acid orangeⅡin food samples[J].Food Analytical Methods,2016,9(2):378-384.
[17] YEH J Y,BEILSTEIN M A,ANDREWS J S,et al.Tissue distribution and influence of selenium status on levels of selenoprotein W[J].The FASEB Journal,1995,9(5):392-396.
[18] CAO L,ZHANG L,ZENG H,et al.Analyses of selenotranscriptomes and selenium concentrations in response to dietary selenium deficiency and age reveal common and distinct patterns by tissue and sex in telomere-dysfunctional mice[J].Journal of Nutrition,2017,147(10):1858-1866.
[19] 吴东,夏伦志,陈丽园,等.富硒日粮对波杂肉山羊体组织硒储留和肉品质的影响[J].中国草食动物,2008,28(6):40-41.WU D,XIA L Z,CHEN L Y,et al.Effects of selenium-enriched diets on selenium storage and meat quality of meat goats[J].China Herbivores,2008,28(6):40-41.(in Chinese)
[20] WHANGER P D.Selenoprotein expression and function—Selenoprotein W[J].Biochimica et Biophysica Acta General Subjects,2009,1790(11):1448-1452.
[21] KOVACS-NOLAN J,MINE Y.Improving the Safety and Quality of Eggs and Egg Products[M].Woodhead publishing limited:Filip Van Immerseel,Yves Nys,Maureen Bain,2011.
[22] REN H,YANG W,THIRUMALAI D,et al.A comparative evaluation of six principal IgY antibody extraction methods[J].Atla-Alternatives to Laboratory Animals,2016,44(1):11-20.
[23] WHANGER P D.Selenoprotein W:A review[J].Cellular & Molecular Life Sciences,2000,57(13-14):1846-1852.
[24] 李文锋,梅敏敏,黄兴国,等.新型鸭呼肠孤病毒σB蛋白基因密码子的优化及原核表达[J].中国畜牧兽医,2018,45(10):2700-2706.LI W F,MEI M M,HUANG X G,et al.Optimization and prokaryotic expression of the σB protein gene codons of the new-type duck reovirus[J].China Animal Husbandry & Veterinary Medicine,2018,45(10):2700-2706.(in Chinese)
[25] 张永宁,张舟,诸明欣,等.A型塞内卡病毒VP2蛋白原核表达及其多克隆抗体的制备[J].中国畜牧兽医,2019,46(1):256-263.ZHANG Y N,ZHANG Z,ZHU M X,et al.Prokaryotic expression of the VP2 protein of Senecavirus A and preparation of its polyclonal antibodies[J].China Animal Husbandry & Veterinary Medicine,2019,46(1):256-263.(in Chinese)