论文部分内容阅读
背景:CTLA-4lg的制备过程比较复杂,单克隆抗体的价格比较昂贵,而且应用CTLA-4lg单克隆抗体注射本身存在着血浆药物浓度不稳定的问题。目的:探讨Ctla4lg表达质粒构建的可行性。设计、时间及地点:基因水平观察实验,于2005/2006在中国医科大学中心实验室完成。材料:雄性Wistar大鼠10只,近交系昆明小鼠10只。方法:提取Wistar大鼠总RNA,反转录成cDNA第1链,PCR扩增CTLA4基因,将其和真核表达载体pcDNA3酶切、连接。转化感受态菌DH5α,培养后挑取阳性克隆、提取质粒,酶切鉴定重组子、测序,最后转染近交系昆明小鼠,应用Westen Blot法检测血清CTLA4lg水平。主要观察指标:构建的Ctla4lg表达质粒基因序是否正确以及能否在小鼠体内进行蛋白表达。结果:对含有pcDNA3-CTLA4质粒的LB菌液进行鉴定测序,目的基因片断大小为288bp,与Genebank上公布的CTLA4序列完全一致,质粒构建正确。利用阳性脂质体载体包被pcDNA3-CTLA4lg转染小鼠,有3只在第7天血清检测CTLA4Ig阳性,显示pcDNA3-CTLA4lg能够在鼠肌细胞内表达。结论:利用基因合成和重组技术成功构建了真核表达载体pcDNA3-CTLA4Ig,利用脂质体法成功的将pcDNA3-CTLA4lg转染到鼠肌细胞内并进行表达。
BACKGROUND: The preparation of CTLA-4lg is complicated. Monoclonal antibodies are expensive, and the injection of CTLA-4lg monoclonal antibody itself poses the problem of unstable plasma drug concentration. Objective: To investigate the feasibility of constructing Ctla4lg expression plasmid. DESIGN, TIME AND SETTING: The gene level observation experiment was performed at the Central Laboratory of China Medical University in 2005/2006. MATERIALS: Ten male Wistar rats and ten Kunming mice were inbred. Methods: The total RNA of Wistar rats was extracted and reverse transcribed into the first strand of cDNA. The CTLA4 gene was amplified by PCR and ligated with the eukaryotic expression vector pcDNA3. The competent cells were transformed into DH5α. Positive clones were picked out after cultivation. Plasmids were extracted and identified by restriction enzyme digestion. The recombinant plasmids were sequenced. Finally, Kunming mice were inoculated with inbred strain. Westen Blot method was used to detect serum CTLA4lg levels. MAIN OUTCOME MEASURES: Ctla4lg expression plasmid constructed correctly and whether the protein expression in mice. Results: The LB strain containing pcDNA3-CTLA4 plasmid was identified and sequenced. The size of the target gene fragment was 288bp, which was identical with the CTLA4 sequence published on Genebank. The plasmid was constructed correctly. The positive liposome vector coated pcDNA3-CTLA4lg transfected mice, 3 were detected on the 7th day serum CTLA4Ig positive, indicating that pcDNA3-CTLA4lg can be expressed in rat muscle cells. Conclusion: The eukaryotic expression vector pcDNA3-CTLA4Ig was successfully constructed by gene synthesis and recombination technology. The pcDNA3-CTLA4lg was successfully transfected into rat myocytes by liposome and expressed.