本文關(guān)鍵詞：抗人CD44單克隆抗體HI313生物學(xué)功能研究及其單鏈抗體的構(gòu)建　出處：《中國協(xié)和醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CD44單克隆抗體 HI313克隆 增殖 分化 單鏈抗體

【摘要】： 實(shí)驗(yàn)?zāi)康?探討小鼠抗人CD44單克隆抗體(HI313)對髓系白血病細(xì)胞增殖、分化的影響。構(gòu)建HI313單鏈抗體(ScFv),為進(jìn)一步治療性抗體開發(fā)打下基礎(chǔ)。 實(shí)驗(yàn)方法:通過流式細(xì)胞儀檢測,比較小鼠抗人CD44單克隆抗體HI313與HI44a的親合力及競爭抑制作用;以胎盤蘭計(jì)數(shù)和MTS法檢測HI313對白血病細(xì)胞系增殖抑制的影響;Annexin V/PI和The Cycle TESE~(TM) PLUSDNA Reagent Kit檢測HI313對NB4細(xì)胞凋亡及細(xì)胞周期影響;以及HI313對AML病人血細(xì)胞誘導(dǎo)分化作用;RT-PCR的方法克隆小鼠雜交瘤HI313的抗體輕、重鏈可變區(qū)基因,利用overlap-PCR方法構(gòu)建HI313-ScFv融合基因,定向克隆入原核表達(dá)載體pET22b,構(gòu)建重組質(zhì)粒pET22b/HI313-ScFv。將重組質(zhì)粒轉(zhuǎn)染入BL21感受態(tài)菌株,在IPTG存在條件下,誘導(dǎo)表達(dá)HI313單鏈抗體。所得融合蛋白經(jīng)復(fù)性、鎳柱純化后,通過流式細(xì)胞術(shù)檢測其與天然細(xì)胞膜CD44蛋白的結(jié)合活性和特異性。 實(shí)驗(yàn)結(jié)果:通過THP-1和KG1a細(xì)胞的檢測,HI313克隆的相對親合力分別為1.2nM和1.34nM;HI44a克隆的相對親和力分別為3.5nM和5.89nM;免疫競爭實(shí)驗(yàn)表明HI313與HI44a識別不同抗原表位;HI313單克降抗體對NB4白血病細(xì)胞系具有強(qiáng)烈的抑制增殖作用,并使NB4細(xì)胞周期停留在G0/G1期;對AML病人血細(xì)胞誘導(dǎo)分化作用結(jié)果顯示HI313可使髓系表面的成熟標(biāo)志表達(dá)量增加。擴(kuò)增HI1313重鏈可變區(qū)基因357bp,HI313輕鏈可變區(qū)基因339bp;成功構(gòu)建HI313-ScFv單鏈抗體,能與KG1a細(xì)胞表面抗原結(jié)合。 實(shí)驗(yàn)結(jié)論:HI313單克隆抗體具有高親和力;且與HI44a克隆結(jié)合的不是一個抗原表位;HI313對NB4細(xì)胞具有增殖抑制作用,作用機(jī)制可能與阻滯細(xì)胞周期于G0/G1期相關(guān);HI313能有效誘導(dǎo)AML病人血細(xì)胞的分化,并對AML病人的各亞型有一定的促分化作用,提示此抗體具有針對AML進(jìn)行靶向治療的潛力,為HI313人抗體的進(jìn)一步基因工程改造及臨床應(yīng)用奠定了基礎(chǔ)。
[Abstract]:Objective: To explore the effect of murine anti human CD44 monoclonal antibody (HI313) on proliferation and differentiation of myeloid leukemia cells, and construct HI313 single chain antibody (ScFv), so as to lay a foundation for further development of therapeutic antibodies.
Methods: by flow cytometry, affinity and competition comparison of mouse monoclonal antibody HI313 against human CD44 and HI44a inhibition; HI313 detected by trypan blue counting and MTS method in inhibiting proliferation of leukemia cell lines; Annexin V/PI and The Cycle TESE~ PLUSDNA Reagent Kit (TM) effect on the cell cycle and the detection of HI313 the apoptosis of NB4 cells; and HI313 of AML patients blood cell differentiation induced by RT-PCR antibody; cloned mouse hybridoma HI313 light and heavy chain variable gene, construct HI313-ScFv fusion gene by overlap-PCR method and cloned into the prokaryotic expression vector pET22b to construct recombinant plasmid pET22b/HI313-ScFv. plasmid was transfected into BL21 competent strains and in the condition of IPTG, induced expression of HI313 scFv. The fusion protein after refolding, nickel column purification, by detecting the natural fine flow cytometry The binding activity and specificity of CD44 protein in the cell membrane.
Results: the detection of THP-1 and KG1a cells, the relative affinity of HI313 clones were 1.2nM and 1.34nM; the relative affinity of HI44a clones were 3.5nM and 5.89nM; immune competition experiments show that HI313 and HI44a recognize different antigen epitope; monoclonal antibody HI313 to inhibit the proliferation of NB4 strong leukemia cell line NB4, and the cell cycle arrest in the G0/G1 phase of AML patients; blood cell differentiation results show that HI313 can make the mature myeloid surface marker expression increased. Amplification of HI1313 gene of heavy chain variable region of 357bp gene, 339bp HI313 light chain variable region; the successful construction of HI313-ScFv single chain antibody binds to KG1a cell surface antigen.
Conclusion: HI313 monoclonal antibody with high affinity; and combined with HI44a cloning is not an epitope; HI313 can inhibit the proliferation of NB4 cells. The mechanism may be related to cell cycle arrest in G0/G1 phase; HI313 can effectively induce the differentiation of blood cells in AML patients, and AML patients have different subtypes differentiation of the antibody against AML with the targeted therapeutic potential, laid the foundation for further improvement of genetic engineering HI313 antibody and its clinical application.

【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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1 張濤;抗人CD44單克隆抗體HI313生物學(xué)功能研究及其單鏈抗體的構(gòu)建[D];中國協(xié)和醫(yī)科大學(xué);2008年

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本文編號：1369269