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人乳腺癌全套抗体可变区基因的扩增与克隆

Amplification of all human V genes of breast carcinoma from peripheral lymphad ens

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【作者】 吕勇刚窦科峰吴昱彤赵爱志刘杰陈刚陶开山药立波

【Author】 Lü Yonggang, Dou Kefeng, Wu Yutong, Zhao Aizhi, Liu Jie, Chen Gang, Tao Kaisha n, Yao Libo (Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710033, China)

【机构】 第四军医大学西京医院肝胆外科唐都医院信息科第四军医大学西京医院基础部药理学教研室西安交通大学第一医院血液科第四军医大学西京医院基础部生物化学教研室 陕西西安710033陕西西安710033陕西西安710038陕西西安710032陕西西安710061陕西西安710033

【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。

【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.

【基金】 国家自然科学基金资助 (No .30 371 399)
  • 【文献出处】 西安交通大学学报(医学版) ,Journal of Xi’an Jiaotong University(Medical Sciences) , 编辑部邮箱 ,2004年06期
  • 【分类号】R737.9
  • 【被引频次】3
  • 【下载频次】64
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