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     Human anti TNF α antibody Fabs with higher affinity was obtained through replacing the light chain fragment facilitated by constructing a light chain second library.
     通过构建轻链二级库的方法 ,对人源抗 TNF-α单抗 Fab进行轻链置换 ,并筛选出具有更高亲和力的人源抗 TNF- α单抗的 Fab.
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     AimToobtainhumananti TNF αantibodyFabswithhigheraffinitythroughreplacingthelightchainofthehumananti TNF αantibodyFabfragmentbyconstructingalightchainsecondlibrary.
     目的使用构建轻链次级库的方法,对已经获得的人源抗TNFα单抗mAb的Fab进行轻链置换,并筛选具有更高亲和力的人源抗TNFαmAb的Fab。
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     PERMUTATION OF SEQUENCES
     序列的置换
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     Quick Trickle Permutation
     全距置换
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     Diagnosis of light chain deposition disease in early stage
     早期轻链沉积病的诊断
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     kD and 28? kD respectively.
     轻链分子量在28 kD左右。
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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

Human anti TNF α antibody Fabs with higher affinity was obtained through replacing the light chain fragment facilitated by constructing a light chain second library. Firstly, amplify the repertoire of the human light chains genes from human peripheral blood lymphocytes using RT PCR technique and assemble them randomly with the heavy chain gene of the anti TNF α antibody so as to construct a second light chain library which comprises single heavy chain gene of the antibody to TNF α and all of the human...

Human anti TNF α antibody Fabs with higher affinity was obtained through replacing the light chain fragment facilitated by constructing a light chain second library. Firstly, amplify the repertoire of the human light chains genes from human peripheral blood lymphocytes using RT PCR technique and assemble them randomly with the heavy chain gene of the anti TNF α antibody so as to construct a second light chain library which comprises single heavy chain gene of the antibody to TNF α and all of the human light chain genes.Τhen select the antibody clones with higher binding affinity. Some new phage displayed antibodies, which had higher affinity for TNF α, were obtained from the new library through three rounds of biopanning. It is conclued that constructing light chain second library could effectively improve the affinity of the antibody, and could be a valid method to overcome the disadvantage of the lower affinity of the human antibody from phage display.

通过构建轻链二级库的方法 ,对人源抗 TNF-α单抗 Fab进行轻链置换 ,并筛选出具有更高亲和力的人源抗 TNF- α单抗的 Fab.首先用 RT- PCR技术扩增正常人全套抗体轻链基因 ,并与已获得的人源抗 TNF- α单抗的重链基因配对 ,构建人源抗 TNF- α噬菌体抗体轻链二级库 ,然后筛选与 TNF- α具有更高亲和力的克隆 .经过三轮的生物淘筛 ( biopanning) ,获得了比原来的人源抗 TNF-α单抗 Fab具有更高亲和力的人源抗体 Fab.轻链二级库筛选法能够有效使抗体的亲和力提高 ,为解决噬菌体抗体库法制备的人源抗体的亲和力较低这一难题提供了一种有效的方法 .

AimToobtainhumananti TNF αantibodyFabswithhigheraffinitythroughreplacingthelightchainofthehumananti TNF αantibodyFabfragmentbyconstructingalightchainsecondlibrary.MethodsFirstlyamplifytherepertoireofthehumanlightchainsgenesfromhumanperipheralbloodlympho-cytesusingRT PCRandassemblethemrandomlywiththeheavychaingeneoftheanti TNFαantibodysoastoconstructasecondlightchainlibrarywhichcomprisesasingleheavychaingeneoftheantibodytoTNFαandallofthehumanlightchaingenes.Thenselecttheantibodycloneswithhigherbindingaffinity.ResultsSomenewphagedisplayedantibodieswhichhadhigheraffinityforTNFαwereobtainedfromthenewlibrarythroughthreeroundsofbiopanning.Conclusionconstructinglightchainsecondlibrarycouldeffectivelyimprovetheaffinityoftheantibodyandcouldbeavalidmethodtoovercomethedis-advantageoftheloweraffinityofthehumanantibodyfromphagedisplay....

AimToobtainhumananti TNF αantibodyFabswithhigheraffinitythroughreplacingthelightchainofthehumananti TNF αantibodyFabfragmentbyconstructingalightchainsecondlibrary.MethodsFirstlyamplifytherepertoireofthehumanlightchainsgenesfromhumanperipheralbloodlympho-cytesusingRT PCRandassemblethemrandomlywiththeheavychaingeneoftheanti TNFαantibodysoastoconstructasecondlightchainlibrarywhichcomprisesasingleheavychaingeneoftheantibodytoTNFαandallofthehumanlightchaingenes.Thenselecttheantibodycloneswithhigherbindingaffinity.ResultsSomenewphagedisplayedantibodieswhichhadhigheraffinityforTNFαwereobtainedfromthenewlibrarythroughthreeroundsofbiopanning.Conclusionconstructinglightchainsecondlibrarycouldeffectivelyimprovetheaffinityoftheantibodyandcouldbeavalidmethodtoovercomethedis-advantageoftheloweraffinityofthehumanantibodyfromphagedisplay.

目的使用构建轻链次级库的方法,对已经获得的人源抗TNFα单抗mAb的Fab进行轻链置换,并筛选具有更高亲和力的人源抗TNFαmAb的Fab。方法首先,采用RTPCR扩增正常人全套抗体轻链基因,并与已获得的人源抗TNFαmAb的重链基因配对,构建人源抗TNFα噬菌体抗体的轻链次级库。然后筛选与TNFα具有更高亲和力的克隆。结果经过3轮的生物淘筛biopanning,获得了较原来的人源抗TNFαmAbFab具有更高亲和力的人源抗体Fab。结论轻链次级库筛选法,能有效地提高抗体的亲和力,为解决噬菌体抗体库法制备的人源抗体亲和力较低这一难题,提供了一种有效的方法。

 
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