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弓形虫GRA7基因的克隆表达及免疫分析

CLONING AND EXPRESSION OF DENSE GRANULE ANTIGEN 7 OF TOXOPLASMA GONDH
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摘要 本文构建了刚地弓形虫RH株致密颗粒蛋白7(GRA7)基因原核表达重组质粒,进行蛋白表达并分析重组蛋白的免疫反应性。设计合成引物,从弓形虫RH株基因组DNA中特异扩增出GRA7基因片段并进行序列分析。目的片段用EcoRI和BamHI双酶切后分别构建重组质粒pET32a-GRA7和pET28a-GRA7,而后转人大肠埃希菌BL21(DE3)中诱导表达,Westernblotting分析表达蛋白。结果显示,从弓形虫RH株中成功扩增出大小约710bp的GRA7基因片段,构建的重组质粒经双酶切和PCR鉴定及测序,目的基因片段与预期相符。重组质粒pET32a-GRA7经IPTG诱导后,可高效表达重组蛋白,Westernblotting分析显示重组GRA7蛋白能被弓形虫慢性感染血清识别,而重组质粒pET28a-GRA7未能诱导表达出目的蛋白。原核表达的重组GRA7抗原具有特异的免疫反应性,为弓形虫的诊断应用和疫苗研究奠定了基础。 To construct prokaryotic expression plasmid pET28a-GRA7 and pET32a-GRA7 containing the dense granulesantigen 7 (GRAT) gene of T. gondii RH strain. The gene fragment GRA7 was amplified by PCR from genomic DNA of T. gondii RH strain. The GRA7 gene was digested by the restriction endonuclease EcoR I and BamH I , and then was further snbcloned into plasmid pET28a ( + ) and pET32a ( + ) , respectively. The plasmid pET28a-GRA7 and pET32a-GRA7 were then transformed into BL21 (DE3) to express the recombinant protein with IPTG induction. The expression product was identified by SDS-PAGE and Western blotting. The plasmid pET28a-GRA7 and pET32a-GRA7 were confirmed with Enzyme digestion and PCR analysis, respectively. With IPTG induction, the recombinant protein GRA7 was expressed in a fusional form in pET32a-GRA7/BL21 (DE3) , while pET28a-GRA7/BL21 (DE3) was fail to express the recombinant protein. Western blotting showed that the recombinant protein could be specifically recognized by sera from mice chronically infected by T. gondii, which shows the recombinant GRA7 expresses a specific immunoreactivity, those built a foundation for the further studies on T. gondii diagnosis and vaccine.
出处 《寄生虫与医学昆虫学报》 CAS 2013年第1期7-11,共5页 Acta Parasitologica et Medica Entomologica Sinica
基金 基金项目:国家自然科学基金项目(No.30972578 31030066) 教育部博士点项目(20094433120013,20094433120016) 广东省自然科学基金项目(9151051501000075) 广东省“大学生创新实验计划”项目(1212110025,1212110027) 南方医科大学大学生课外科研项目(GWXS20110101,GWXS20110205)
关键词 刚地弓形虫 致密颗粒蛋白7(GRA7) 基因克隆 原核表达 免疫反应性 Toxoplasma gondii Dense granule antigen 7 (GRA7) Gene clone Prokaryotic expression Immunoreactivity
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参考文献9

  • 1郑斌,郑焕钦,何蔼,李卓雅,曹爱莲,张瑞琳,詹希美.弓形虫不同分离株致密颗粒蛋白基因的序列分析及其在大肠埃希菌中的表达[J].中国寄生虫学与寄生虫病杂志,2005,23(2):100-105. 被引量:3
  • 2赵焕阁,李煜,谭光宏,黄凤迎,王华,郭峻莉,周松林,黄用豪.弓形虫MIC8基因的克隆及真核表达质粒的构建[J].中国热带医学,2011,11(7):805-807. 被引量:1
  • 3Caffaro, C. E. and J. C. Boothroyd 2011 Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells. Eukaryot. Cell, 10 (8): 1 095-1 099.
  • 4Cesbron-Delauw, M. F. 1994 Dense-granule organelles of Toxoplasma gondii: their role in the host-parasite relationship. Parasitol. Today, 10 (8) : 293-296.
  • 5Cong, H. , E. J. Mui, W. H. Witola et al. 2012 Toxoplasma gondii HLA-B* 0702-restricted GRA7 (20-28) peptide with adjuvants and a universal helper T cell epitope elicits CD8 ( + ) T cells producing interferon-Y and reduces parasite burden in HLA-B* 0702 mice. Hum. lmmunol., 73 (1): 1-10.
  • 6Koethe, M. , S. Port, M. Ludewig et al. 2011 Prevalence of specific IgG-antibodies against Toxoplasma gondii in domestic turkeys determined by kinetic ELISA based on recombinant GRA7 and GRA8. Vet. Parasitol. , 180 (3--4): 179-190.
  • 7Sadeghiani, G., M. Zare, J. Babaie et al. 2009 Heterologous production of dense granule GRA7 antigen of Toxoplasma gondii in Escherichia coli. Southeast Asian J. Trop. Med. Public Health, 40 (4) : 692-700.
  • 8Tilley, M., M. E. Fichera, M. E. Jerome et al. 1997 Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins. Infect. lmmun. , 65 (11) : 4 598-4 605.
  • 9Vercammen, M., T. Scorza, K. Huygen et al. 2000 DNA vaccination with genes encoding Toxoplasma gondii antigens GRAI, GRA7, and ROP2 induces partially protective immunity against lethal challenge in mice. Infect. lmmun. , 68 ( 1 ) : 38-45.

二级参考文献27

  • 1李学瑞,赵象忠,王艳华,张德林.弓形虫微线体蛋白研究进展[J].中国人兽共患病学报,2006,22(4):368-370. 被引量:8
  • 2Henning KesseLer, AngeLika Henn-Gotz et al. Microneme protein8--a new essentiaL invasion factor in ToxopLasma gondii [J] . CeLL Science, 2008,121 (7) : 947-956.
  • 3Loutit JS . BartoneLLa infections [J] . Current CLinicaL Topics in Infectious Diseases, 1997,17: 269-290.
  • 4ALaa Bassuny IsmaeL, Sekkai D, CoLLin C, et al . The MIC3 gene of ToxopLasma gondii is a noveL potent vaccine candidate against toxopLasmosis[J ] . Infect Immun, 2003,71 : 6222-6228.
  • 5Beghetto E,NieLsen HV,DeL Porto P,et ak Combination of antigenic regions of ToxopLasma gondii microneme proteins induces protective immunity against oraL infection with parasite cyst s [J] . Infect Dis, 2005,191 : 637-645.
  • 6Lourenco EV,Bernardes ES,SiLva NM,et al. Immunization wit h MIC1 and MIC4 induces protective immunity against ToxopLasma gondi i [ J ]. Microbes Infect, 2006,8 ( 5 ) : 1244-1251.
  • 7BhopaLe GM . I)eveLopment of a vaccine for toxopLasmosis:current status[J] . Microbes Infect,2003,5 (5):457-4621.
  • 8J萨姆布鲁克 金冬雁等(译).分子克隆实验指南(第二版)[M].北京:科学出版社,1999.267-269.
  • 9Tilley M, Fichera ME, Jerome ME, et al. Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins[J]. Infect Immun, 1997, 65:4598-4605.
  • 10Charf H, Darcy F, Torpier G, et al. Toxoplasma gondii: Characterization and localization of antigens secreted from tachyzoites [J].Exp Parasital, 1990, 71:114-124.

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