摘要
目的构建一种新型重组免疫毒素DT390-Rantes的真核表达质粒,并对这种免疫毒素的功能进行初步研究。方法通过RT-PCR的方法,从小鼠肝脏中获得Rantes基因;将Rantes基因插入到含有DT390片段的真核质粒SRα中,构建成重组质粒;重组质粒转入大肠杆菌JM109,筛选出含有正确插入片段的克隆;限制性内切酶酶切图谱分析重组质粒,并进行DNA序列分析;用脂质体介导法转染NIH3T3细胞,免疫荧光检测重组质粒的表达情况;MTT法测定DT390-Rantes的表达活性。结果经过酶切分析及DNA测序证实,Rantes基因正确插入到真核表达质粒SRα中,并在NIH3T3细胞中表达;MTT法证实,重组免疫毒素DT390-Rantes能在体外杀伤活化的T细胞。结论成功地构建了一种新型重组免疫毒素真核表达质粒DT390-Rantes-SRα,该质粒可在真核细胞中瞬时表达,其转染上清对活化的T细胞具有杀伤作用。
Objective To construct a novel eukaryotic expression plasmid for recombinant immunotoxin DT390-Rantes and perform preliminary analysis of its function. Methods The gene fragment coding for Rantes was obtained from the liver tissues of C57BL/6 mice using RT-PCR, and inserted into the eukaryotic expression plasmid SRa containing DT390 gene to construct the recombinant plasmid DT390-Rantes-SRa, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonuclease digestion and DNA sequencing. The recombinance plasmid DT390-Rantes-SRa was transfected into NIH3T3 cells and its expression was observed by immunofluorescence detection. The activity of the expressed DT390-Rantes in vitro was evaluated by MTT assay. Results The gene fragment of Rantes was correctly inserted into the eukaryotic expression plasmid SRa as verified by restriction endonuclease digestion and DNA sequencing, and could be expressed in NIH3T3 cells. MTT assay confirmed that the expression product DT390-Rantes could kill activated T cells in vitro. Conclusions The recombinant eu- karyotic expression plasmid DT390-Rantes-SRa is successfully constructed and expressed in eukaryotic cells. The expressed product can specifically kill activated T cells in vitro.
出处
《第一军医大学学报》
CAS
CSCD
北大核心
2005年第11期1344-1347,共4页
Journal of First Military Medical University
基金
国家863高科技计划项目(2002AA214101)
国家自然基金委创新研究群体基金(30221001)
四川省应用基础项目(04JY029-002)~~
