摘要
目的构建小鼠M IP-1α(mM IP-1α)基因和白喉杆菌外毒素DT 390基因的真核融合表达载体,并对其功能进行初步研究。方法通过RT-PCR获得mM IP-1α基因,插入含DT 390基因的真核质粒SRα,获得真核表达载体SR-αmM IP-1-αDT 390。重组载体经PCR、限制性内切酶酶切及DNA序列测定鉴定,用Po lyF ect脂质体转染N IH 3T 3细胞,然后用免疫荧光技术检测目的蛋白的表达,并利用转染N IH 3T 3细胞后的培养上清体外测定mM IP-1-αDT 390融合蛋白的选择性细胞毒活性。结果成功构建真核表达质粒SR-αmM IP-1-αDT 390,mM IP-1-αDT 390融合蛋白可在N IH 3T 3细胞株中正确表达,细胞转染上清对小鼠活化T淋巴细胞具有较强的细胞抑制效应。结论mM IP-1-αDT 390融合基因真核表达载体的获得及功能的部分研究为进一步研究其抗炎效果和临床应用奠定基础。
Objective To construct a novel recombinant immunotoxin （IT） expression vector by fusing mouse macrophage inflammatory protein-1α gene （rnMIP-1α） into a truncated diphtheria toxin gene （DT390）, and examine the expression of mMIP-1α-DT390 fusion protein in NIH3T3 cells. Methods rnMIP-1α cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction （RT-PCR）, and inserted in the expression plasmid SRα, that includes the DT390 gene, to form the recombinant vector SRa-rnMIP-1α- DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT. Results We have successfully constructed a recombinant immunotoxin expression vector named as SRce-mMIP-1α-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte. Conclusion The SRα-raMIP-1α-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2007年第1期1-5,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家863高科技计划(项目编号2002AA214101)
四川省应用基础项目(04JY029-002)资助
关键词
巨噬细胞炎症蛋白-1Α
白喉杆菌外毒素
重组免疫毒素
真核表达
Macrophage inflammatory protein-1α （MIP-1α） Diphtheria Toxin （DT） Recombinant immunotoxin Eukaryotic expression
