摘要
目的构建乳腺癌患者抗Her-2/neu全人源性噬菌体单链抗体可变区(ScFv)文库,筛选抗Her-2/neu单链抗体。方法收集40例乳腺癌患者前哨淋巴结(SLN),提取总RNA,反转录为cDNA作模板,在目前常用的引物基础上重新设计可变区的引物,用PCR扩增全套抗体可变区,构建T载体库。并改造pCANTAB-5E构建了pCANTAB-Linker。将T载体库酶切连接入pCANTAB-Linker构建出ScFv文库。最后构建噬菌体单链抗体库并进行初级筛选。结果成功改良ScFv文库的构建方法。初步得到12株对人乳腺癌组织有高亲和力的Her-2/neu单链抗体。结论改良了ScFv文库的构建方法,可以获得较大库容量的单链抗体库,并从中筛选到人源性抗Her-2/neu单链抗体。
Objective To construct a phage single-chain fragment variable region（ScFv） antibody library of human-derived antibreast cancer Her-2/neu,and screen out anti-Her-2/neu specific single-chain antibody from the library.Methods Total RNA was extracted from 40 sentinel lymph nodes（SLN） of breast cancer patients and reversely transcribed into cDNA.After a group of the primers of variable regions were redesigned based on the current commonly used ones,the PCR was used in amplified a full set of antibody variable regions and constructed T vector library.The pCANTAB-5E was constructed with pCANTAB-Linker.T vector library was digested with endonuclease and connected into a ScFv library,the phage display library of ScFv antibody against Her-2/ neu of breast cancer was set up and used to screen out the single-chain antibody.Results The 12 clones of E.Coli TG1 were obtained,which produced high affinity antibodies against human breast cancer.Conclusion It is demonstrated that a large capacity phase displayed ScFv library of anti-Her-2/neu of human breast cancer can be obtained and the human anti-Her-2/neu antibodies are screened out by the modified method for constructing ScFv library.
出处
《生物医学工程与临床》
CAS
2010年第2期161-166,F0002,共7页
Biomedical Engineering and Clinical Medicine
