高勇强,阳青兰,胡丽,梁瑜,刘彦,张愉快,王可耕,肖建华.pcDNA3.1/ SjDLC 和pcDNA3.1/mIFN-γ重组质粒构建及表达.[J].中南医学科学杂志.,2017,(6):558-561, 579.
pcDNA3.1/ SjDLC 和pcDNA3.1/mIFN-γ重组质粒构建及表达
Construction and expression of the pcDNA3.1/SjDLCand pcDNA 3.1/mIFN-γ
投稿时间:2016-07-20  修订日期:2017-08-28
DOI:10.15972/j.cnki.43-1509/r.2017.06.006
中文关键词:  DNA疫苗  动力蛋白轻链  干扰素-γ
英文关键词:DNA vaccine  SjDLC  IFN-γ
基金项目:湖南省教育厅科学研究项目(编号:13C824);湖南省研究生科研创新项目(CX2015B415);湖南省分子靶标新药研究协同创新中心资助项目(NO.2015-351);特殊病原体防控湖南省重点实验室资助项目(NO.2014-5). 
作者单位
高勇强 南华大学医学院病原生物学研究所 湖南 衡阳 421001 
阳青兰 南华大学医学院病原生物学研究所 湖南 衡阳 421001 
胡丽 南华大学医学院病原生物学研究所 湖南 衡阳 421001
南华大学护理学院内护教研室 
梁瑜 南华大学医学院病原生物学研究所 湖南 衡阳 421001
南华大学医学院寄生虫学教研室 
刘彦 南华大学医学院病原生物学研究所 湖南 衡阳 421001
南华大学医学院寄生虫学教研室 
张愉快 南华大学医学院病原生物学研究所 湖南 衡阳 421001 
王可耕 南华大学医学院病原生物学研究所 湖南 衡阳 421001 
肖建华 南华大学医学院病原生物学研究所 湖南 衡阳 421001 
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中文摘要:
      目的 构建日本血吸虫动力蛋白轻链DNA疫苗和小鼠干扰素真核表达质粒,研究其在HeLa细胞及动物体内的表达。方法设计特异性引物,PCR扩增获取动力蛋白轻链(SjDLC)与干扰素-γ(IFN-γ)基因,克隆于pcDNA3.1真核质粒,经双酶切及测序鉴定。将两种重组质粒分别转染至HeLa细胞,Western blot鉴定其表达。真核质粒经左腿股四头肌免疫小鼠,在末次免疫2周后,PCR法检测小鼠肌组织内SjDLC和IFN-γ基因,免疫组织化学法检测基因的表达,MTT法检测T细胞增殖水平。结果PCR和双酶切能检测到280 bp的SjDLC基因及480 bp的IFN-γ基因;Western blot显示在12 kDa和19 kDa处有阳性反应条带,与SjDLC和IFN-γ分子量大小一致,两种基因能在HeLa细胞中表达。PCR及免疫组化显示两种基因能在小鼠肌肉组织中存在和表达。MTT法显示两种质粒均能刺激T细胞增殖。结论成功构建pcDNA3.1/SjDLC和pcDNA 3.1/ mIFN-γ真核质粒,两种质粒能在HeLa细胞和动物体内表达。
英文摘要:
      Objective To construct the dynein light chain of Schistosoma japonicum DNA vaccine and IFN-γ recombinant plasmid of mice,so as to investigate their expression in HeLa cells and mouse.MethodsA pair of primers designed by PRIMER5.0 software was synthesized;the gene fragments of SjDLC and mIFN-γ were amplified by PCR,and inserted into eukaryotic expression plasmid pcDNA3.1,where the recombinant plasmids were identified with restrictive enzymes and sequenced.The recombinant vectors pcDNA3.1/SjDLC and pcDNA3.1 /mIFN-γ were transfected into HeLa cells;the expressed proteins were identified by Western blot.These plasmids were injected into BALB /c mice too,the gene of SjDLC and IFN-γ in quadriceps femoris were identified by PCR.The expression of SjDLC,IFN-γ were observed with immunohistochemistry.The proliferation of splenic lymphocytes in mice was detected by MTT.ResultsThe 280 bp of SjDLC gene and 480 bp of IFN-γ gene were observed through PCR and double enzyme digestion;Western blot displayed that positive response bands in 12 kDa and 19 kDa which is consistent with the molecular weight of SjDLC and IFN-γ were successfully expressed in HeLa cells.PCR and immunohistochemistry showed that two genes expressed in mouse muscle tissue.MTT showed that two plasmids could stimulate the proliferation of T cells.ConclusionThe pcDNA /SjDLC DNA vaccine and recombinant plasmid pcDNA /mIFN-γ were successfully constructed and then expressed in HeLa cells and mouse.
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