摘要
用Trizol提取6周龄白莱航鸡胸腺组织的总RNA,再用Oligo-dT纤维素富集mRNA后,用RT-PCR分别扩增出鸡CD4和CD8α基因cDNA。PCR产物切胶回收后连入T载体,测序正确后再连入pcDNA3.1(+)。将重组质粒pcDNA3.1-CD4和pcDNA3.1-CD8分别转染COS-7细胞,用已知的抗鸡CD4和CD8的单克隆抗体检测到转染细胞中CD4和CD8α分子的表达,这说明构建的真核表达质粒正确,为下一步用DNA免疫方法研制抗鸡CD4和CD8的单克隆抗体以及研究鸡CD4和CD8分子的结构和功能打下基础。
Total RNAs of thymocytes were extracted from 6-wk-old Leghorn chicken, mRNAs were isolated from total RNAs using oligo-dT cellulose, then CD4 and CD8α cDNAs were amplified with RT-PCR using the purified mRNAs as the templates. The sequences were verified through sequencing after CD4 and CD8α cDNAs were ligated into pGEM T easy vector. The eukaryotic expression plasmids were constructed by cloning the CD4 and CD8α cDNA into (pcDNA3.1(+)) respectively, and then the COS-7 cells were transfected with recombinant plasmid pcDNA3.1-CD4 or (pcDNA3.1-CD8.) 40h after transfection the expression of CD4 or CD8 gene on COS-7 cells could be detected using indirect immunofluorescent assay (IFA) with murine monoclonal antibody against chicken CD4 or CD8, indicating that the correct constructs of eukaryotic expression plasmids were obtained and it will provide useful materials for the development of mAb-CD4, mAb-CD8 and for the study of the structure and functions of chicken CD4 and CD8 molecules.
出处
《扬州大学学报（农业与生命科学版）》
CAS
CSCD
2004年第4期56-60,共5页
Journal of Yangzhou University：Agricultural and Life Science Edition
基金
全国优秀博士学位论文作者专项资金资助项目(FANEDD:200358)
江苏省六大人才高峰项目(G2002-026)
