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)
