| 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). |
|
| 摘要点击次数: 1021 |
| 全文下载次数: 879 |
| 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. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|