弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4的构建
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摘要
目的构建弓形虫抗原基因真核表达质粒pVAX1-SAG1和pVAX1-SAG4。方法根据弓形虫RH标准株SAG1和SAG4基因序列分别设计一对特异引物(分别含有HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ内切酶位点),PCR扩增目的基因,并将这两段基因分别克隆至PEGM-T Easy载体,经菌落PCR和双酶切鉴定;用HindⅢ/BamHⅠ和BamHⅠ/XbaⅠ分别双酶切PEGM-T Easy-SAG1、PEGM-T Easy-SAG4和pVAX1,得到含内切酶位点的SAG1和SAG4基因片段,分别与pVAX1连接,构建pVAX1-SAG1和pVAX1-SAG4真核表达质粒,经菌落PCR和双酶切鉴定。结果PCR扩增得到目的基因SAG1和SAG4,测序结果分别与弓形虫RH标准株SAG1和SAG4基因比较,符合率均为100%。构建PEGM-T Easy-SAG1、PEGM-T Easy-SAG4克隆质粒和pVAX1-SAG1、pVAX1-SAG4真核表达质粒,分别经菌落PCR和双酶切鉴定,显示722bp的SAG1基因片段和511bp的SAG4基因片段均插入载体PEGM-T Easy和pVAX1中。结论成功克隆了pVAX1-SAG1、pVAX1-SAG4真核表达质粒,为弓形虫基因疫苗应用研究奠定了基础。
Objective To construct pVAX1-SAG1and pVAX1-SAG4eukaryotic expression plasmids of an antigen gene of Toxoplasma gondii. Methods A pair of specific primers was designed and synthesized in accordance with the SAG1and SAG4gene sequences of T.gondii(RH strain).These sequences contain the HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ endonuclease cleavage sites.The genes SAG1and SAG4were amplified with PCR using genomic DNA fromT.gondii as a template.The PCR products were cloned into a PEGM-T Easy vector and were identified by digestion with restriction enzymes and colony PCR;The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4and pVAX1vectors were digested with HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ,and then the target fragments SAG1and SAG4with endonuclease cleavage sites were subcloned into a pVAX1vector and identified by digestion with restriction enzymes and colony PCR. Results The target DNA fragments SAG1and SAG4yielded by PCR were sequenced and the results were compared with the reported sequences of the SAG1and SAG4genes of T.gondii(RH strain).The rate of concordance was 100%.The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4,pVAX1-SAG1and pVAX1-SAG4were found to contain the target gene fragments SAG1(722bp)and SAG4(511bp)according to digestion with restriction enzymes and colony PCR. Conclusion The eukaryotic expression plasmids pVAX1-SAG1and pVAX1-SAG4encoding the SAG1and SAG4proteins of T.gondii were constructed,laying the foundation for studies using vaccines from T.gondii genes.
Objective To construct pVAX1-SAG1and pVAX1-SAG4eukaryotic expression plasmids of an antigen gene of Toxoplasma gondii. Methods A pair of specific primers was designed and synthesized in accordance with the SAG1and SAG4gene sequences of T.gondii(RH strain).These sequences contain the HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ endonuclease cleavage sites.The genes SAG1and SAG4were amplified with PCR using genomic DNA fromT.gondii as a template.The PCR products were cloned into a PEGM-T Easy vector and were identified by digestion with restriction enzymes and colony PCR;The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4and pVAX1vectors were digested with HindⅢ/BamH Ⅰand BamH Ⅰ/XbaⅠ,and then the target fragments SAG1and SAG4with endonuclease cleavage sites were subcloned into a pVAX1vector and identified by digestion with restriction enzymes and colony PCR. Results The target DNA fragments SAG1and SAG4yielded by PCR were sequenced and the results were compared with the reported sequences of the SAG1and SAG4genes of T.gondii(RH strain).The rate of concordance was 100%.The recombinant plasmid PEGM-T Easy-SAG1and PEGM-T Easy-SAG4,pVAX1-SAG1and pVAX1-SAG4were found to contain the target gene fragments SAG1(722bp)and SAG4(511bp)according to digestion with restriction enzymes and colony PCR. Conclusion The eukaryotic expression plasmids pVAX1-SAG1and pVAX1-SAG4encoding the SAG1and SAG4proteins of T.gondii were constructed,laying the foundation for studies using vaccines from T.gondii genes.
引文
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[12]杨雯,万孝玲,田春林,等.弓形虫表面抗原SAG4基因的克隆、表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2009,27(4):340-3.
[2]Oka Y,Akbar SM,Horiik N,et al.Mechanism and therapeutic potential of DNA-based immunization against the envelope proteins of hepatitis B virus in normal and transgenic mice[J].Immunology,2001,103(1):90-7.
[3]赵焕阁,韦祎,黄用豪,等.弓形虫ROP18基因原核表达载体的构建及表达[J].中国病原生物学杂志,2013,8(6):527-9,534.
[4]张晓磊,张进顺,朱晓波,等.刚地弓形虫RH株ROP11基因的克隆与真核表达载体的构建[J].中国病原生物学杂志,2013,8(11):1002-4,1016.
[5]周光前,Hazrimarstmm S.DNA疫苗接种的免疫学机理、优越性及安全性[J].国外医学:生物制品分册,1997,20(2):53-5.
[6]高世同,吴少庭,龙彩虹,等.弓形虫pVAX1-SAG2真核表达质粒的构建及其诱导的小鼠免疫应答[J].中国人兽共患病杂志,2004,20(8):658-61.
[7]Kasper LH,Bradley M S,Pfefferkorn ER,et al.Idem ification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies[J].J Immunol,1984,132(1):443-9.
[8]Alexandre J,Jebbari H,Biuethmann H,et a1.Immunological control of Toxoplasma gondii and appropriate vaccine design[J].Curr Top Microbiol Immunol,1996,219:188-90.
[9]杨雯,田春林,朱穗京,等.弓形虫表面抗原SAG1基因的克隆表达、纯化及鉴定[J].广西医科大学学报,2010,27(2):205-8.
[10]龚娅,陈晓光,冯明钊.弓形虫P30基因重组质粒的构建及其免疫效果[J].中国寄生虫学与寄生虫病杂志,2001,19(5):282-6.
[11]易艳军,许丽芳,周支香,等.弓形虫P30基因-乳酸乳球菌重组质粒的构建及其表达[J].中国病原生物学杂志,2012,7(6):440-2.
[12]杨雯,万孝玲,田春林,等.弓形虫表面抗原SAG4基因的克隆、表达和鉴定[J].中国寄生虫学与寄生虫病杂志,2009,27(4):340-3.