重组刚地弓形虫微线料体蛋白MIC10的制备与特性分析
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摘要
目的制备重组刚地弓形虫微线体蛋白10(MIC10),并对其免疫学特性进行分析。方法利用逆转录PCR(RT-PCR)技术从弓形虫总RNA中反转录制备第一链cDNA,再利用特异性引物从cDNA中扩增出编码MIC10的基因片段,构建重组表达质粒pET28a-MIC10,转化入大肠埃希菌BL21(DE3)后用异丙基硫代半乳糖苷(IPTG)进行诱导表达,通过镍螯合亲和层析法纯化重组MIC10蛋白。用纯化的重组蛋白免疫家兔,制备抗MIC10多克隆抗体,采用Western blot分析重组MIC10蛋白的免疫学特性。结果采用RT-PCR法从弓形虫cDNA中反转录扩增到长度约为416bp的特异性DNA,基因序列分析证实为编码MIC10的基因片段;制备的重组表达质粒pET28a-MIC10转化大肠埃希菌BL21(DE3)后经IPTG诱导,能表达重组MIC10蛋白;用纯化的重组MIC10蛋白免疫家兔,血清抗体效价达到1∶200 000以上。Western blot分析显示该抗体能识别弓形虫排泄分泌抗原中的天然MIC10分子而不识别弓形虫成虫抗原,证明MIC10为外分泌蛋白。结论制备了具有天然免疫原性的重组弓形虫MIC10蛋白,并证明该蛋白为外分泌蛋白。
Objectives To prepare a recombinant microneme protein 10(MIC10)fromToxoplasma gondii and to analyze its immunological characteristics. Methods Reverse transcription PCR(RT-PCR)was first used to prepare firststrand cDNA from Toxoplasma total RNA,and then the first-strand cDNA was used as a template to amplify the DNA fragment encoding the MIC10 mature peptide with gene-specific primers.This gene fragment was cloned into the plasmid pET28 a(+)to construct the recombinant expression plasmid pET28 a-MIC10.The recombinant expression plasmid pET28 a-MIC10 was transfected into E.coli BL21(DE3)competent cells to develop an engineered bacterium.Isopropylβ-D-1-thiogalactopyranoside(IPTG)was used to induce positive clones of the engineered bacterium to express the recombinant MIC10 protein.The purified recombinant MIC10 protein was prepared using nickel chelating affinity chromatography.A rabbit was immunized with the purified recombinant MIC10 protein to prepare IgG polyclonal antibodies against the MIC10 protein.The immunological characteristics of the recombinant MIC10 protein were analyzed using Western blotting. Results A specific DNA band of 416 bp was amplified from the first-strand cDNA of T.gondii,and the results of DNA sequencing verified that this DNA fragment is the gene encoding the MIC10 protein of T.gondii.IPTG induced the engineered bacterium containing the recombinant expression plasmid pET28 a-MIC10 to express the recombinant MIC10 protein.The titer of serum IgG antibodies against the recombinant protein MIC10 in a rabbit immunized with the pure recombinant MIC10 protein was higher than 1:200 000.The natural MIC10 protein of the excreted and secretory antigen(ESA)of T.gondii was recognized by the purified antibody IgG against the recombinant MIC10 protein,which further confirmed that MIC10 is an exocrine protein. Conclusion Recombinant MIC10 protein from T.gondii was successfully prepared with natural immunogenicity,and findings have indicated that this protein is an exocrine protein.
Objectives To prepare a recombinant microneme protein 10(MIC10)fromToxoplasma gondii and to analyze its immunological characteristics. Methods Reverse transcription PCR(RT-PCR)was first used to prepare firststrand cDNA from Toxoplasma total RNA,and then the first-strand cDNA was used as a template to amplify the DNA fragment encoding the MIC10 mature peptide with gene-specific primers.This gene fragment was cloned into the plasmid pET28 a(+)to construct the recombinant expression plasmid pET28 a-MIC10.The recombinant expression plasmid pET28 a-MIC10 was transfected into E.coli BL21(DE3)competent cells to develop an engineered bacterium.Isopropylβ-D-1-thiogalactopyranoside(IPTG)was used to induce positive clones of the engineered bacterium to express the recombinant MIC10 protein.The purified recombinant MIC10 protein was prepared using nickel chelating affinity chromatography.A rabbit was immunized with the purified recombinant MIC10 protein to prepare IgG polyclonal antibodies against the MIC10 protein.The immunological characteristics of the recombinant MIC10 protein were analyzed using Western blotting. Results A specific DNA band of 416 bp was amplified from the first-strand cDNA of T.gondii,and the results of DNA sequencing verified that this DNA fragment is the gene encoding the MIC10 protein of T.gondii.IPTG induced the engineered bacterium containing the recombinant expression plasmid pET28 a-MIC10 to express the recombinant MIC10 protein.The titer of serum IgG antibodies against the recombinant protein MIC10 in a rabbit immunized with the pure recombinant MIC10 protein was higher than 1:200 000.The natural MIC10 protein of the excreted and secretory antigen(ESA)of T.gondii was recognized by the purified antibody IgG against the recombinant MIC10 protein,which further confirmed that MIC10 is an exocrine protein. Conclusion Recombinant MIC10 protein from T.gondii was successfully prepared with natural immunogenicity,and findings have indicated that this protein is an exocrine protein.
引文
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[14]Carruthers VB,Sibley LD.Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts[J].Eur J Cell Biol,1997,73(2):114-23.
[15]Dubremetz JF,Achbarou A,Bermudes D,et al.Kinetics and pattern of organelle exocytosis during Toxoplasma gondii hostcell interaction[J].Parasitol Res,1993,79(5):402-8.
[16]曲道峰.弓形虫微线体蛋白MIC3生物学特性及免疫原性研究[D].杭州:浙江大学,2011.
[17]Fourmaux MN,Achbarou A,Mercereau-Puijalon O,et al.The MIC1microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface[J].Mol Biochem Parasitol,1996,83(2):201-10.
[18]Brecht S,Carruthers VB,Ferguson DJ,et al.The toxoplasma micronemal protein MIC4is an adhesin composed of six conserved apple domains[J].J Biol Chem,2001,276(6):4119-27.
[2]He JJ,Ma J,Wang JL,et al.Analysis of miRNA expression profiling in mouse spleen affected by acute Toxoplasma gondii infection[J].Infect Genet Evol,2016(37):137-42.
[3]Dubey JP,Jones JL.Toxoplasma gondii infection in humans and animals in the United States[J].Int J Parasitol,2008,38(11):1257-78.
[4]华海涌,唐凤,刘一新,等.江苏省孕妇弓形虫感染情况调查[J].中国血吸虫病防治杂志2013,25(1):56-8,79.
[5]刘转转,杨燕萍,殷国荣,等.刚地弓形虫苹果酸脱氢酶基因的克隆表达及免疫原性分析[J].中国寄生虫学与寄生虫病杂志,2013,8(1):12-6.
[6]Torrey EF,Yolken RH.Toxoplasma oocysts as a public health problem[J].Trends Parasitol,2013,29(8):380-4.
[7]Wang H,Wang T,Luo Q,et al.Prevalence and genotypes of Toxoplasma gondii in pork from retail meat stores in Eastern China[J].Int J Food Microbiol,2012,157(3):393-7.
[8]王艳华,李学瑞,张德林,等.弓形虫病诊断方法研究进展[J].中国兽医寄生虫病,2008,21(1):28-33.
[9]刘楠,张晓磊,郭玲玲,等.刚地弓形虫ROP16-ROP18复合基因真核表达载体的构建[J].中国病原生物学杂志,2017,12(1):57-61.
[10]刘荣荣,张进顺,张晓磊,等.刚地弓形虫棒状体蛋白10的生物信息学分析[J].中国病原生物学杂志,2017,12(9):855-9.
[11]郑斌,尹志奎,史明珠,等.刚地弓形虫MIC8CTD作用蛋白的筛选及鉴定[J].中国病原生物学杂志,2014,9(3):255-7.
[12]Hoff EF,Cook SH,Sherman GD,et al.Toxoplasma gondii:molecular cloning and characterization of a novel 18-kDa secretory antigen,TgMIC10[J].Exp Parasitol,2001,97(2):77-88.
[13]张居作,陈汉忠,徐君飞.我国弓形虫的感染现状[J].动物医学进展,2008,10(7):101-4.
[14]Carruthers VB,Sibley LD.Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts[J].Eur J Cell Biol,1997,73(2):114-23.
[15]Dubremetz JF,Achbarou A,Bermudes D,et al.Kinetics and pattern of organelle exocytosis during Toxoplasma gondii hostcell interaction[J].Parasitol Res,1993,79(5):402-8.
[16]曲道峰.弓形虫微线体蛋白MIC3生物学特性及免疫原性研究[D].杭州:浙江大学,2011.
[17]Fourmaux MN,Achbarou A,Mercereau-Puijalon O,et al.The MIC1microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface[J].Mol Biochem Parasitol,1996,83(2):201-10.
[18]Brecht S,Carruthers VB,Ferguson DJ,et al.The toxoplasma micronemal protein MIC4is an adhesin composed of six conserved apple domains[J].J Biol Chem,2001,276(6):4119-27.