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目的:为构建高质量乳腺癌噬菌体展示文库,用pKE-2质粒载体经卡那抗性筛选富集开放阅读框架(open-reading frame ORF)内阳性克隆。方法:用Trizol试剂提取乳腺癌组织总RNA,分离纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上定向EcoR/Hind接头,用EcoR和Hind消化接头,使形成两端分别带有EcoR和Hind粘性末端的双链cDNA,将其连接于带有EcoR和Hind末端的pKE-2质粒载体,转化大肠杆菌DH5α,经卡那抗性筛选,富集ORF克隆。结果:经过ORF筛选,文库中ORF克隆比例达到80%,较筛选前的5%提高75%。
OBJECTIVE: To construct a high quality breast cancer phage display library, positive clones enriched in the open-reading frame ORF were screened by kanamycin resistance using pKE-2 plasmid vector. Methods: Trizol reagent was used to extract total RNA from breast cancer tissues. The mRNA was isolated and purified, and double-stranded cDNA was synthesized by reverse transcription. A directional EcoR / Hind linker was added to the ends of the double-stranded cDNA and the linker was digested with EcoR and Hind to form a double-stranded cDNA with cohesive ends of EcoR and Hind at both ends, respectively, which were ligated to pKE- 2 plasmid vector, transformed into E. coli DH5α, screening by kanamycin, enriched ORF clones. Results: After ORF screening, the proportion of ORF clones in the library reached 80%, which was 75% higher than that of 5% before screening.