巨型艾美耳球虫配子体蛋白EmGAM56的原核表达及其抗原性鉴定
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摘要
本研究将巨型艾美耳球虫Emgam56基因去除信号肽后的ORF序列连接至pET-28a(+)载体,构建表达载体pET-28-Emgam56,转化表达宿主菌大肠杆菌BL21(DE3),经IPTG诱导表达;分析表达产物的可溶性和重组蛋白的分子相对质量,并进一步鉴定重组配子体蛋白rEmGAM56的抗原性。SDS-PAGE分析表明,表达载体能表达56000左右蛋白质,主要以可溶性蛋白的形式存在;Western blot检测表明,rEmGAM56蛋白能被鼠抗rEmGam56多克隆抗体和巨型艾美耳球虫感染鸡的康复血清特异性识别,显示重组蛋白具有良好的抗原性。本研究结果为研制重组配子体抗原亚单位疫苗奠定了基础。
The Emgam56 gene of Eimeria maxima deleted signal peptide encoding region was cloned into pET-28a(+)of prokaryotic expression vector,then pET-28a(+)-Emgam56 was transformed into E.coli BL21(DE3)to analyze whether the constructed vector could express inferred recombinant EmGAM56 and its solubility,and characterizing the antigenicity of the recombinant EmGAM56.SDS-PAGE showed the constructed vector expressed a protein around 56 000,as soluble form.And immunoblot assays demonstrated that the rEmGAM56 could be recognized by both the rehabilitation serum of chicken infected with E.maximaand mouse anti-rEmGAM56 polyclonal antibody,and showed good antigenicity and specificity.The results showed that rEmGAM56 could be used to develop recombinant subunit vaccine against coccidiosis.
The Emgam56 gene of Eimeria maxima deleted signal peptide encoding region was cloned into pET-28a(+)of prokaryotic expression vector,then pET-28a(+)-Emgam56 was transformed into E.coli BL21(DE3)to analyze whether the constructed vector could express inferred recombinant EmGAM56 and its solubility,and characterizing the antigenicity of the recombinant EmGAM56.SDS-PAGE showed the constructed vector expressed a protein around 56 000,as soluble form.And immunoblot assays demonstrated that the rEmGAM56 could be recognized by both the rehabilitation serum of chicken infected with E.maximaand mouse anti-rEmGAM56 polyclonal antibody,and showed good antigenicity and specificity.The results showed that rEmGAM56 could be used to develop recombinant subunit vaccine against coccidiosis.
引文
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[16]孙卫国,张灵霞,熊志红,等.人角质细胞生长因子2在不同原核表达系统中的表达差异[J].生物技术通讯,2014,25(5):669-675.
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[18]DENG T.Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization[J].FEBS Lett,1997,409(2):269-72.
[19]WALLACH M G,MENCHER D,YARUS S,et al.Eimeria maxima:identification of gametocyte protein antigen[J].Exp Parasitol,1989,68(1):49-56.
[2]SHARMAN P A,SMITH N C,WALLACH M G,et al.Chasing the golden egg:vaccination against poultry coccidiosis[J].Parasite Immunol,2010,32(8):590-598.
[3]SHIRLEY M W,SMITH A L,BLAKE D P.Challenges in the successful control of the avian coccidia[J].Vaccine,2007,25(30):5540-5547.
[4]HAO L,LIU X,ZHOU X,et al.Transient transfection of Eimeria tenella using yellow or red fluorescent protein as a marker[J].Mol Biochem Parasitol,2007,153(2):213-215.
[5]PEEK H W,LANDMAN W J.Coccidiosis in poultry:anticoccidial products,vaccines and other prevention strategies[J].Vet Q,2011,31(3):143-161.
[6]TEWARI A K,MAHARANA B R.Control of poultry coccidiosis:changing trends[J].J Parasitol Dis,2011,35(1):10-17.
[7]WALLACH M G.The development of CoxAbic?a novel vaccine against coccidiosis[J].World Poult,2002,18(1):2-4.
[8]彭昊,陶建平,许金俊,等.巨型艾美尔球虫配子体gam8 2基因的克隆表达与免疫原性分析[J].中国兽医学报,2010,30(9):1203-1207.
[9]彭昊,许金俊,刘丹丹,等.巨型艾美尔球虫重组蛋白Gam82-Y和重组质粒pcDNA-gam82的免疫保护效果研究[J].中国预防兽医学报,2011,33(9):728-732.
[10]许金俊,姜慧捷,陶建平,等.巨型艾美球虫gam56基因的截短原核表达及其产物的免疫保护效果[J].中国预防兽医学报,2009,31(11):892-898.
[11]姜慧婕.巨型艾美球虫配子体gam56基因的克隆与表达及其免疫保护性试验[D].江苏扬州:扬州大学,2008.
[12]BELLI S I,WITCOMBE D,WALLACH M G,et al.Functional genomics of gam56:characterization of the role of a 56kilodalton sexual stage antigen in oocyst wall formation in Eimeria maxima[J].Int J Parasitol,2002,32(14):1727-1737.
[13]BELLI S I,LEE M,THEBO P,et al.Biochemical characterisation of the 56and 82 kilodalton immunodominant gametocyte antigens fromEimeria maxima[J].Int J Parasitol,2002,32(1):21-32.
[14]BELLI S I,MAI K,SKENE C D,et al.Characterisation of the antigenic and immunogenic properties of bacterially expressed,sexual stage antigens of the coccidian parasite,Eimeria maxima[J].Vaccine,2004,22:4316-4325.
[15]张晓燕,丁隽,刘群.巨型艾美耳球虫配子体gam56基因的克隆与表达[J].中国兽医杂志,2007,43(11):12-14.
[16]孙卫国,张灵霞,熊志红,等.人角质细胞生长因子2在不同原核表达系统中的表达差异[J].生物技术通讯,2014,25(5):669-675.
[17]CAMPOS J A,ALEDO J C,SEGURA J A,et al.Expression of recombinant human L-glutaminase in Escherichia coli:polyclonal antibodies production and immunological analysis of mouse tissues[J].Biochim Biophys Acta,2003,1648(1/2):17-23.
[18]DENG T.Bacterial expression and purification of biologically active mouse c-Fos proteins by selective codon optimization[J].FEBS Lett,1997,409(2):269-72.
[19]WALLACH M G,MENCHER D,YARUS S,et al.Eimeria maxima:identification of gametocyte protein antigen[J].Exp Parasitol,1989,68(1):49-56.