摘要
目的:为提取高纯度hnRNPA2/B1蛋白以满足制备特异性hnRNPA2/B1单克隆抗体的需要,特进行hnRNPA2/B1cDNA制备。方法:采用PCR法从人平滑肌细胞克隆并扩增hnRNPA2/B1全长基因,新扩增的PCR反应物加入含有TOPOTA载体,TOPO克隆反应物2μl加入感受态大肠杆菌SOC介质中混匀培养、筛选、M13引物测序。结果:实验从20个白色菌落中经PCR扩增出基因片段,对其中6个克隆的质粒进行DNA序列测定,发现其中1个质粒含有特定的人hnRNPA2/B1基因序列。结论:采用PCR法可从人平滑肌细胞克隆、制备出hnRNPA2/B1全长基因cDNA,这将为提取高纯度hnRNPA2/B1蛋白、制备用于早期诊断肺癌的抗hnRNPA2/B1单克隆抗体具有重要意义。
Objective:To extract highly purified hnRNP A2/B1 protein,the preparation of hnRNP A2/B1 cDNA is necessary. Methods:Full- length hnRNP A2/B1 cDNA of human smooth muscle cell had been cloned and amplified by PCR, the new amplified reactant 2 μl added to TOPO TA vector and medium of competent bacillus coli were cultured, screened ,at last sequenced by M13 primer. Results :The essay had amplified fragment of gene from 20 white colons, and sequenced the DNA. among 6 plasmids ,eventually one plasmid has been found the sequence of hnRNP A2/B1 . Conclusion:The full - length hnRNP A2/B1 cDNA of human smooth muscle cell could be cloned by PCR. It would be very significant for extraction of highly purified hnRNP A2/B1 protein and the preparation of its monoclonal antibody using in early diagnosis of lung cancer.
出处
《临床肿瘤学杂志》
CAS
2006年第1期7-10,共4页
Chinese Clinical Oncology
基金
安徽省科技厅重点科研基金项目(01023024
05023086)
安徽省教育厅自然科学基金项目(2004KJ238ZC)