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轻链库
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  light chain library
     About 680bp PCR products were obtained and then were digested with restriction enzymes for ligation into the pComb3 vector. For the construction of the light chain library, the light chain repertoires were cloned into the phagemid pComb3. The ligated DNA was electroporated into competent E. coli XL 1-Blue cells.
     用SacⅠ和XbaⅠ酶切轻链PCR产物和噬粒载体pComb3,凝胶回收酶切产物,通过T4 DNA连接酶将轻链基因片段克隆到pComb3载体上,电穿孔转染大肠杆菌感受态细胞XL1-Blue,构建轻链库
短句来源
     First cloning variable region gene of light and heavy chain into Psp73-T respectively to get the light chain library and heavy chain library.
     首先用前面阐述的T载体方法,先将轻链、重链可变区基因片段分别克隆入PSp73*中,构建出轻链库和重链库。
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  “轻链库”译为未确定词的双语例句
     The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.
     轻链PCR产物和噬粒载体pComb3经Sac Ⅰ和Xba Ⅰ酶切,由T4 DNA连接酶连接,电穿孔转化感受态大肠杆菌XL1-Blue,扩增后提取噬粒,构建轻链库
短句来源
     coli XL 1-Blue by Electroporation. The transformed cells were infected with VCSM13 helper phage toyield recombinant phage antibody Fabs. The phagemids abstracted from amplified E.
     轻链库和重链Fd段PCR产物经Xho Ⅰ和Spe Ⅰ酶切,以同样方法连接后转化XL1-Blue,加入辅助噬菌体VCSM13,产生噬菌体抗体全库。
短句来源
     Half-nested PCR was employed to amplify the heavy chain Fd and light chain k. Firstly, the k genes were cloned into phagemid pComb3 and electrotransformed into E. Coli to construct a K-chain library which contained 2.88×10~6 separate clones,the k gene positive ratio is 60%.
     先将轻链基因克隆到pComb3中,电转化TG1菌构建成κ轻链文库,经30次电转后将轻链库混合,库容量达到2.88×10~6cfu,轻链插入率为60%。
短句来源
     Methods Vκ and the Fd fragment of the anti keratin Fab were replaced sequentially with diverse Vκ genes and Fd fragments derived from lymphocytes of healthy donors.
     方法分离、扩增多样性人免疫球蛋白轻链基因和IgG IgM重链Fd段基因 ,分别构建轻链库和重链库。
短句来源
     The shuffled libraries were panned using off rate selection process to isolate antibodies with increased affinity.
     先以原始克隆的重链与轻链库随机搭配进行轻链替换 ,对形成的换链库采用解离速率筛选法 (off ratese lection)选取亲和力提高的克隆 ;
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  相似匹配句对
     Use of library;
     的利用;
短句来源
     A ffinity mature of hum an anti-TNF-αFabthr-ough constructing a light chain second library
     构建轻链次级提高人抗TNF-α抗体的亲和力
短句来源
     Repository's Development History
     仓储的发展
短句来源
     Conclusion:According to the sequenceing and ELISA assay,we suggest that κ chain is a human anti-HBs-κ immunoglobuin.
     结论 :利用抗原抗体特异性反应从噬菌体抗体中筛选到抗HBs轻链基因。
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  light chain library
The subsequent ligation of 1 Fd genes into the light chain library resulted in a combinatorial library with 2i10' members.
      


おurrently, the antiTNFα monoclonal antibody (Mab) is considered to be one of the most promising agents against septicemia and endotoxin shock. It is believed that the Mabs made from human are superior than that from animals for the purpose of patient treatment. In order to produce human antiTNFα Mab, a pooled human immunoglobulin combined phage libraries (from HAV, HBV and HCV patients) were used to select antibody fragment with pure guided antigenTNFα. The results demonstrated that a specific phage antibody...

おurrently, the antiTNFα monoclonal antibody (Mab) is considered to be one of the most promising agents against septicemia and endotoxin shock. It is believed that the Mabs made from human are superior than that from animals for the purpose of patient treatment. In order to produce human antiTNFα Mab, a pooled human immunoglobulin combined phage libraries (from HAV, HBV and HCV patients) were used to select antibody fragment with pure guided antigenTNFα. The results demonstrated that a specific phage antibody against TNFα was obtained, it only reacted with TNFα and did not react with other cytokines such as IFNγ, TPO etc and antigens of HAV, HBV and HCV in ELISA test.Further the authors constructed a light chain library to obtain an another light chain which was able to substitute the original light chain, and formed a new phage antibody with higher TNFα binding activity. Finally, the new phage antibody was expressed in E.coli as a soluble antibody fragment and the product of expressing was demonstrated to have a higher TNFα bind activities. The sequence analysis of the antibody gene showed that it contained a heavy chain belonging to human IgG Ⅲ group family and a κⅢ light chain.

应用抗TNF单抗治疗菌血症和内毒素休克已证明是一种有效的策略,人源抗TNF抗体较鼠单抗或其它动物来源的单抗具有更大的应用前景。我们用一个混合的人抗体组合噬菌体抗体库,针对人重组TNFα进行筛选获得了对人重组TNFα具有亲和性和特异性的噬菌体抗体。然后我们又建立一个人抗体轻链库,用以对筛选到的抗体进行轻链置换。结果获得了与人重组TNFα具有更高亲和力的噬菌体抗体。对该抗体在大肠杆菌中所做的可溶性表达表明,其Fab片段能与人重组TNFα结合。DNA测序表明该抗体属IgGⅢ亚类并含有一条κⅢ亚类的轻链

Objective To improve the affinity of a human anti keratin Fab. Methods Vκ and the Fd fragment of the anti keratin Fab were replaced sequentially with diverse Vκ genes and Fd fragments derived from lymphocytes of healthy donors. The shuffled libraries were panned using off rate selection process to isolate antibodies with increased affinity. Clones thus obtained were analyzed by ELISAs and sequence analysis. Results By shuffling the Vκ, antibodies with a 10 fold improved affinity were isolated. And a further...

Objective To improve the affinity of a human anti keratin Fab. Methods Vκ and the Fd fragment of the anti keratin Fab were replaced sequentially with diverse Vκ genes and Fd fragments derived from lymphocytes of healthy donors. The shuffled libraries were panned using off rate selection process to isolate antibodies with increased affinity. Clones thus obtained were analyzed by ELISAs and sequence analysis. Results By shuffling the Vκ, antibodies with a 10 fold improved affinity were isolated. And a further improvement was obtained by Fd fragment shuffling, resulted in an affinity of 7.9×10 10 /mol/L, nearly 23 fold higher than that of the original Fab. Conclusion Chain shuffling is an effective method for affinity maturation of antibodies isolated from phage antibody library.

目的 通过链替换技术对一株人源性抗角蛋白Fab单抗进行体外亲和力成熟。方法分离、扩增多样性人免疫球蛋白轻链基因和IgG IgM重链Fd段基因 ,分别构建轻链库和重链库。先以原始克隆的重链与轻链库随机搭配进行轻链替换 ,对形成的换链库采用解离速率筛选法 (off ratese lection)选取亲和力提高的克隆 ;然后再将获得的轻链基因与重链库基因重组进行重链替换 ,筛选亲和力进一步提高的克隆并进行亲和力测定、可变区基因序列分析等一系列鉴定。结果 替换轻链后所获抗体的亲和力提高到原来的 10倍 ;替换重链后亲和力进一步提高达到 7.9× 10 1 0 mol L ,是原始克隆的近 2 3倍。结论 链替换技术是提高抗体库中所获Fab抗体亲和力的有效手段。

Objective:To construct a naive human Fab fragment phage display library.Methods:Peripheral blood lymphocytes were isolated from 800 ml blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were PCR amplified and the amplification products were ligated into the phagemid vector pComb3,then the ligated sample was transformed into competent E.coli XL1-Blue.The transformed cells were infected with VCSM13 helper phage to yield...

Objective:To construct a naive human Fab fragment phage display library.Methods:Peripheral blood lymphocytes were isolated from 800 ml blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were PCR amplified and the amplification products were ligated into the phagemid vector pComb3,then the ligated sample was transformed into competent E.coli XL1-Blue.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as SacⅠ,XbaⅠ,SpeⅠand XhoⅠto monitor the insertion of the light chain or heavy chain Fd genes.Results:By combination of light chain and heavy chain genes,an antibody library containing 5.4×10~6 clones was obtained,and the cutting of enzymes showed that there were light chain or heavy chain Fd genes of the phagemids.Conclusion:This study can construct a human naive Fab phage display library which is useful to immunotheropy.

目的通过构建人天然F ab段噬菌体抗体库为恶性肿瘤的免疫治疗提供新方法。方法取4个健康献血员新鲜血液各200m l,分离淋巴细胞,提取总RNA,经逆转录合成cDNA,设计多对引物,以PCR扩增抗体轻链和重链Fd段cDNA。轻链PCR产物和质粒载体pCom b3经SacⅠ和X baⅠ酶切,由T 4 DNA连接酶连接,电穿孔转化感受态大肠杆菌XL 1-B lue,扩增后提取质粒,构建轻链库轻链库和重链Fd段PCR产物经X hoⅠ和SpeⅠ酶切,同样方法连接转化XL 1-B lue,加入辅助噬菌体VCSM 13产生噬菌体抗体全库。酶切鉴定轻链库和全库有无插入片段。结果提取的淋巴细胞总RNA质量好,部分重链Fd片段、部分κ轻链和λ轻链cDNA得到扩增。F ab全库库容达5.4×106,酶切证实有插入片段。结论本研究构建的人天然F ab段噬菌体抗体库可为进一步筛选特异性抗体及从肿瘤组织中提取肿瘤抗原用于免疫治疗奠定基础。

 
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