摘要
目的本研究拟构建人乳腺癌T7噬菌体展示文库,为进一步筛选特异性高的乳腺癌分子标志物原奠定基础。方法确诊乳腺癌患者手术标本20例,Trizol法提取总RNA,逆转录合成双链cDNA后,与T7Select 10-3载体连接,体外包装并扩增得到人乳腺癌T7噬菌体展示cDNA文库。结果构建了乳腺癌T7噬菌体展示cDNA文库,文库滴度2.1×107pfu/ml。随机从文库抽取50个克隆PCR扩增插入片段,电泳结果显示乳腺癌文库插入率98%(49/50)。结论成功构建了乳腺癌T7噬菌体展示cDNA文库,为筛选和鉴定乳腺癌相关抗原奠定基础。
Objective To construct and evaluate T7 phage display cDNA library from human breast carcinoma patients. Methods Total RNA was extracted from 20 breast carcinoma tissues using Trizol,and reversed cDNA was inserted into T7 Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro. Plaque assay and PCR assay were used to evaluate the library. Results The human breast carcinoma T7 phage display cDNA library was established. The titer of the library was 2.1 × 10^7 pfu/ml using Plaque Assay. PCR Amplification of random plaques showed an insert ratio of 98% （49/50） ,the insert range is 300 bp to 1 500 bp. Conclusion The human breast carcinoma T7 phage display cDNA library was constructed successfully, and it will lay solid foundation for selecting and identifying breast carcinoma related antigens.
出处
《中国实验诊断学》
2015年第11期1823-1825,共3页
Chinese Journal of Laboratory Diagnosis
基金
嘉兴市科技计划(2012AY1075-7)
作者简介
韩冬，38岁，男，博士，副教授，主要从事肿瘤免疫研究。
