【摘要】 目的　扩增人乳腺癌抗体全套可变区基因 ,将其克隆到T载体来构建T载体文库。方法　收集乳腺癌患者外周转移淋巴结 30例 ,TRIzol法提取总RNA、纯化mRNA ,行RT PCR扩增 ,制备T载体 ,用于PCR产物的克隆。结果　总RNA纯度高 ,完整性好 ,mRNA获得率为 1%。经RT PCR ,VH、Vλ、Vκ的每一条 5 ’端引物均与其相应的 3’端引物成功地扩增出了可变区基因片段 ,轻、重链T载体库分别为 1.7× 10 8和 3.5× 10 8。结论　所采用的引物能够10 0 %扩增目的片段 ,T载体过渡能显著提高克隆效率 ,为噬菌体抗体库的构建和筛选奠定了基础。更多还原

【Abstract】 Objective To amplify all human V genes of breast carcinoma by RT-PCR. The products of PCR were cloned into T-ve ctor to construct clone library. Methods Peripheral lympha dens of 30 patients with breast carcinoma were collected, from which total RNA w as extracted by TRIzol Reagent, and mRNA was purified with Oligotex mRNA kit. Th en cDNA was derived through reverse transcription. PCR products were amplified a nd cloned into T-vector to construct clone libraries. Results Total RNA was extracted purely and intactly, so was mRNA which accounts for 1% of the former. By RT-PCR, every pair of primers amplified the V gene efficie ntly. Repertoires of VH and VL were 3.5×10 8and 1.7×10 8 respectivel y. Conclusion The set of primers adopted are able to recognize 100% of functional V genes with high efficiency. T-vector will facilitate the digestion, which lays a foundation for construction and selection of phage libra ry.更多还原