日本血吸虫谷胱甘肽-S-转移酶抗原表位与霍乱毒素B亚基融合蛋白在Bac-to-Bac杆状病毒/昆虫细胞的表达
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摘要
PCR扩增日本血吸虫28 000谷胱甘肽-S-转移酶(Sj28GST)的主要抗原表位与霍乱毒素B亚基(CTB)的融合基因, CTB-Sj28GST经SalⅠ和SphⅠ双酶切后定向克隆至转移质粒pFastBac,构建重组转移质粒pFastBac-CTB-Sj28GST,转化DH10Bac感受态细胞进行基因的同源重组,提取重组病毒,用M13通用引物以及CTB-Sj28GST引物PCR鉴定阳性后转染草地贪夜蛾细胞(Sf9),收集亲代重组病毒反复感染Sf9细胞进行病毒扩增, PCR鉴定重组病毒。重组病毒感染细胞出现病变时,用抗Sj28GST多克隆抗体间接免疫荧光(IFA)鉴定重组蛋白的表达情况,蛋白质印迹(Western blotting)鉴定病毒感染细胞裂解液,分析重组蛋白的抗原性。研究结果表明, Sj28GST含有4个抗原表位,长189 bp,与CTB融合后长519 bp。重组转移质粒pFastBac-CTB-Sj28GST的PCR及序列鉴定与预期一致。Sf9转染细胞获得的重组病毒经PCR鉴定,获得与预期一致的519 bp片段。IFA鉴定表明,重组病毒感染的Sf9细胞呈现绿色荧光,未感染的对照细胞无绿色荧光。Western blotting鉴定在约Mr22 000处有特异性的蛋白条带,与预期目的蛋白大小一致。该重组蛋白能被抗Sj28GST多克隆抗体识别。
To induce mucosal protective immunity for schistosomiasis, 4 major epitopes from a leading vaccine antigen of Schistosoma japonicum 28 000 glutathione-S-transferase(Sj28 GST) was fused with cholera toxin B subunit(CTB) as a mucosal adjuvant and the fusion protein was expressed as recombinant protein in insect cell Sf9 using Bac-to-Bac baculovirus expression system. The coding DNA for fusion CTB-Sj28 GST was PCR amplified from previous constructed plasmid and then subcloned into transposed plasmid pFastBac using Sal Ⅰ and Sph Ⅰ sites. The recombinant pFastBac-CTB-Sj28 GST plasmid DNA was transformed into DH10 Bac for homologous recombination. The successfully obtained recombinant viruses were identified by PCR using M13 universal primers and CTB-Sj28 GST specific primer and then transfected into Sf9 insect cells. The recombinant viruses were amplified by repetitively infecting Sf9 cells. The expressed recombinant fusion CTB-Sj28 GST in infected insect cells was identified by indirect immunofluorescent assay(IFA), and the expressed fusion protein in the cell lysates identified by Western blotting with polyclonal antibody against Sj28 GST. Results demonstrated that a 519 bp DNA fragment containing 4 epitopes of S j28 GST(189 bp) fused with CTB coding DNA was successfully amplified and cloned into pFastBac plasmid to construct p FastBac-CTB-Sj28 GST. The obtained recombinant viruses from infected Sf9 cells contained the same size of fusion DNA identified by PCR. The transfected Sf9 insect cells enabled to express fusion CTB-Sj28 GST identified by IFA with anti-Sj28 GST specific antibody. Western blotting showed a specific band at the Mr22 000, the same size as the expected molecular weight of fusion CTB-Sj28 GST, which could be recognized by the polyclonal antibody against-Sj28 GST.
To induce mucosal protective immunity for schistosomiasis, 4 major epitopes from a leading vaccine antigen of Schistosoma japonicum 28 000 glutathione-S-transferase(Sj28 GST) was fused with cholera toxin B subunit(CTB) as a mucosal adjuvant and the fusion protein was expressed as recombinant protein in insect cell Sf9 using Bac-to-Bac baculovirus expression system. The coding DNA for fusion CTB-Sj28 GST was PCR amplified from previous constructed plasmid and then subcloned into transposed plasmid pFastBac using Sal Ⅰ and Sph Ⅰ sites. The recombinant pFastBac-CTB-Sj28 GST plasmid DNA was transformed into DH10 Bac for homologous recombination. The successfully obtained recombinant viruses were identified by PCR using M13 universal primers and CTB-Sj28 GST specific primer and then transfected into Sf9 insect cells. The recombinant viruses were amplified by repetitively infecting Sf9 cells. The expressed recombinant fusion CTB-Sj28 GST in infected insect cells was identified by indirect immunofluorescent assay(IFA), and the expressed fusion protein in the cell lysates identified by Western blotting with polyclonal antibody against Sj28 GST. Results demonstrated that a 519 bp DNA fragment containing 4 epitopes of S j28 GST(189 bp) fused with CTB coding DNA was successfully amplified and cloned into pFastBac plasmid to construct p FastBac-CTB-Sj28 GST. The obtained recombinant viruses from infected Sf9 cells contained the same size of fusion DNA identified by PCR. The transfected Sf9 insect cells enabled to express fusion CTB-Sj28 GST identified by IFA with anti-Sj28 GST specific antibody. Western blotting showed a specific band at the Mr22 000, the same size as the expected molecular weight of fusion CTB-Sj28 GST, which could be recognized by the polyclonal antibody against-Sj28 GST.
引文
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[6] Mudd PA,Martins MA,Ericsen AJ,et al. Vaccine-induced CD8+T cells control AIDS virus replication[J]. Nature,2012,491(7422):129-133.
[7] Takala-Harrison S,Clark TG,Jacob CG,et al. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia[J]. Proc Natl Acad Sci USA,2013,110(1):240-245.
[8]蒋玉文,杨京岚,许崇波,等.霍乱毒素B亚单位基因的克隆及其核苷酸序列分析[J].中国兽医学报,2000,20(4):343-346.
[9] Philipps B,Forstner M,Mayr LM. A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells[J]. Biotechnol Prog,2005,21(3):708-711.
[10]唐莉,王胜军. GITRL的分子结构[J].生命的化学,2009,29(3):386-390.
[11]马洁,王胜军,毛朝明,等.小鼠GITRL融合蛋白的表达、纯化及免疫原性的研究[J].中国免疫学杂志,2009,25(8):675-677.
[2] Capron A,Dessaint JP,Capron M,et al. Vaccine strategies against schistosomiasis[J]. Immunobiology,1992,184(2/3):282-294.
[3]李传明,石佑恩.日本血吸虫大陆株26kDa谷胱甘肽S-转移酶编码区基因的克隆及序列测定[J].寄生虫与医学昆虫学报,1997,4(1):6-11.
[4] Smith DB,Rubira MR,Simpson RJ,et al. Expression of an enzymatically active parasite molecule in Escherichia coli:Schistosoma japonicum glutathione S-transferase[J]. Mol Biochem Parasitol,1988,27(2/3):249-256.
[5]李焱,余新炳,吴忠道,等.日本血吸虫26kDa谷胱甘肽S-转移酶基因真核表达载体的构建及序列测定[J].中国人兽共患病杂志,2000,16(6):28-30.
[6] Mudd PA,Martins MA,Ericsen AJ,et al. Vaccine-induced CD8+T cells control AIDS virus replication[J]. Nature,2012,491(7422):129-133.
[7] Takala-Harrison S,Clark TG,Jacob CG,et al. Genetic loci associated with delayed clearance of Plasmodium falciparum following artemisinin treatment in Southeast Asia[J]. Proc Natl Acad Sci USA,2013,110(1):240-245.
[8]蒋玉文,杨京岚,许崇波,等.霍乱毒素B亚单位基因的克隆及其核苷酸序列分析[J].中国兽医学报,2000,20(4):343-346.
[9] Philipps B,Forstner M,Mayr LM. A baculovirus expression vector system for simultaneous protein expression in insect and mammalian cells[J]. Biotechnol Prog,2005,21(3):708-711.
[10]唐莉,王胜军. GITRL的分子结构[J].生命的化学,2009,29(3):386-390.
[11]马洁,王胜军,毛朝明,等.小鼠GITRL融合蛋白的表达、纯化及免疫原性的研究[J].中国免疫学杂志,2009,25(8):675-677.
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