抗人CD44单克隆抗体HI313生物学功能研究及其单链抗体的构建
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摘要
实验目的:探讨小鼠抗人CD44单克隆抗体(HI313)对髓系白血病细胞增殖、分化的影响。构建HI313单链抗体(ScFv),为进一步治疗性抗体开发打下基础。
实验方法:通过流式细胞仪检测,比较小鼠抗人CD44单克隆抗体HI313与HI44a的亲合力及竞争抑制作用;以胎盘兰计数和MTS法检测HI313对白血病细胞系增殖抑制的影响;Annexin V/PI和The Cycle TESE~(TM) PLUSDNA Reagent Kit检测HI313对NB4细胞凋亡及细胞周期影响;以及HI313对AML病人血细胞诱导分化作用;RT-PCR的方法克隆小鼠杂交瘤HI313的抗体轻、重链可变区基因,利用overlap-PCR方法构建HI313-ScFv融合基因,定向克隆入原核表达载体pET22b,构建重组质粒pET22b/HI313-ScFv。将重组质粒转染入BL21感受态菌株,在IPTG存在条件下,诱导表达HI313单链抗体。所得融合蛋白经复性、镍柱纯化后,通过流式细胞术检测其与天然细胞膜CD44蛋白的结合活性和特异性。
实验结果:通过THP-1和KG1a细胞的检测,HI313克隆的相对亲合力分别为1.2nM和1.34nM;HI44a克隆的相对亲和力分别为3.5nM和5.89nM;免疫竞争实验表明HI313与HI44a识别不同抗原表位;HI313单克降抗体对NB4白血病细胞系具有强烈的抑制增殖作用,并使NB4细胞周期停留在G0/G1期;对AML病人血细胞诱导分化作用结果显示HI313可使髓系表面的成熟标志表达量增加。扩增HI1313重链可变区基因357bp,HI313轻链可变区基因339bp;成功构建HI313-ScFv单链抗体,能与KG1a细胞表面抗原结合。
实验结论:HI313单克隆抗体具有高亲和力;且与HI44a克隆结合的不是一个抗原表位;HI313对NB4细胞具有增殖抑制作用,作用机制可能与阻滞细胞周期于G0/G1期相关;HI313能有效诱导AML病人血细胞的分化,并对AML病人的各亚型有一定的促分化作用,提示此抗体具有针对AML进行靶向治疗的潜力,为HI313人抗体的进一步基因工程改造及临床应用奠定了基础。
Objective:
To study the biological functions of mouse anti human monoclonal antibody-HI313 , desire to construct anti-CD44 single chain fragment variable based on HI313 monoclonal antibody (HI1313-ScFv). These results provide a potential target and basis for developing CD44-targeted therapy in AML.
Methods:
Through observations on HI313 and HI44a monoclonal antibody affinity and competitive inhibition by flow cytometry; to analyze the affection on some CD44+ leukemia cell lines caused by HI313, we use MTS and trypan-blue dye . Annexin V/PI and The Cycle TESE~(TM) PLUS DNA Reagent Kit detect the growing inhibition and cycle of the cells; Detect HI313 effect in reverse the differentiation blockage of AML cell. Amplified both heavy chain and light chain variable region gene of HI313 McAb by RT-PCR, than splice them into ScFv in form of VH-linker-VL.That recombinant gene fragment fragment was cloned into plasmid pET22b and induced to express the recombinant protein, HI313-ScFv, by IPTG in E.coli BL21.We purified the output by Ni Sepharose~(TM), denatured/re-natured and detected it binding activity with natural cell through Flow cytometry.
Result:
The relative affinities of HI313 tested by THP-1 and KG1a are 1.2nM and 1.34nM, while the ones of HI44a are 3.5nM and 5.89nM. The affinities of HI313 and HI44a are both less than the normal 10nM.The FACS shows that there is no competitive inhibition between HI313 and HI44a although they are both monoclonal antibodies of CD44, which indicates that the two antibodies do not share the same epitope.The HI313 monoclonal antibody can significantly inhibit the proliferation of the leukemia cell line NB4 by blocking the cell cycle on the G_0/G_1 point, while has little effects on other cell lines.Inducing differentiation of AML hemocytes shows that HI313 can increase the expression of myelogenous mature antigens.
The variable region gene of both HI313 heavy chain (339bp) and light chain (357bp) was cloned.To construct The spliced ScFv fusion gene with linker by overlap-PCR. All long and successfully cloned into PET22b plasmid to construct pET22b/HI313-ScFv expression vector. That can high express recombinant protein in E.coli induced by IPTG. The recombinant ScFv protein can bind nature CD44 molecular.
Conclusion:
Both HI313 and HI44a possess high affinity, HI313 monoclonal antibody can inhibit growth of NB4 cell and induced those cell cycle retained G0/G1 stage. After construction of HI313-ScFv. we should keep moving to study its potential usage and reform it for future clinical application.
实验方法:通过流式细胞仪检测,比较小鼠抗人CD44单克隆抗体HI313与HI44a的亲合力及竞争抑制作用;以胎盘兰计数和MTS法检测HI313对白血病细胞系增殖抑制的影响;Annexin V/PI和The Cycle TESE~(TM) PLUSDNA Reagent Kit检测HI313对NB4细胞凋亡及细胞周期影响;以及HI313对AML病人血细胞诱导分化作用;RT-PCR的方法克隆小鼠杂交瘤HI313的抗体轻、重链可变区基因,利用overlap-PCR方法构建HI313-ScFv融合基因,定向克隆入原核表达载体pET22b,构建重组质粒pET22b/HI313-ScFv。将重组质粒转染入BL21感受态菌株,在IPTG存在条件下,诱导表达HI313单链抗体。所得融合蛋白经复性、镍柱纯化后,通过流式细胞术检测其与天然细胞膜CD44蛋白的结合活性和特异性。
实验结果:通过THP-1和KG1a细胞的检测,HI313克隆的相对亲合力分别为1.2nM和1.34nM;HI44a克隆的相对亲和力分别为3.5nM和5.89nM;免疫竞争实验表明HI313与HI44a识别不同抗原表位;HI313单克降抗体对NB4白血病细胞系具有强烈的抑制增殖作用,并使NB4细胞周期停留在G0/G1期;对AML病人血细胞诱导分化作用结果显示HI313可使髓系表面的成熟标志表达量增加。扩增HI1313重链可变区基因357bp,HI313轻链可变区基因339bp;成功构建HI313-ScFv单链抗体,能与KG1a细胞表面抗原结合。
实验结论:HI313单克隆抗体具有高亲和力;且与HI44a克隆结合的不是一个抗原表位;HI313对NB4细胞具有增殖抑制作用,作用机制可能与阻滞细胞周期于G0/G1期相关;HI313能有效诱导AML病人血细胞的分化,并对AML病人的各亚型有一定的促分化作用,提示此抗体具有针对AML进行靶向治疗的潜力,为HI313人抗体的进一步基因工程改造及临床应用奠定了基础。
Objective:
To study the biological functions of mouse anti human monoclonal antibody-HI313 , desire to construct anti-CD44 single chain fragment variable based on HI313 monoclonal antibody (HI1313-ScFv). These results provide a potential target and basis for developing CD44-targeted therapy in AML.
Methods:
Through observations on HI313 and HI44a monoclonal antibody affinity and competitive inhibition by flow cytometry; to analyze the affection on some CD44+ leukemia cell lines caused by HI313, we use MTS and trypan-blue dye . Annexin V/PI and The Cycle TESE~(TM) PLUS DNA Reagent Kit detect the growing inhibition and cycle of the cells; Detect HI313 effect in reverse the differentiation blockage of AML cell. Amplified both heavy chain and light chain variable region gene of HI313 McAb by RT-PCR, than splice them into ScFv in form of VH-linker-VL.That recombinant gene fragment fragment was cloned into plasmid pET22b and induced to express the recombinant protein, HI313-ScFv, by IPTG in E.coli BL21.We purified the output by Ni Sepharose~(TM), denatured/re-natured and detected it binding activity with natural cell through Flow cytometry.
Result:
The relative affinities of HI313 tested by THP-1 and KG1a are 1.2nM and 1.34nM, while the ones of HI44a are 3.5nM and 5.89nM. The affinities of HI313 and HI44a are both less than the normal 10nM.The FACS shows that there is no competitive inhibition between HI313 and HI44a although they are both monoclonal antibodies of CD44, which indicates that the two antibodies do not share the same epitope.The HI313 monoclonal antibody can significantly inhibit the proliferation of the leukemia cell line NB4 by blocking the cell cycle on the G_0/G_1 point, while has little effects on other cell lines.Inducing differentiation of AML hemocytes shows that HI313 can increase the expression of myelogenous mature antigens.
The variable region gene of both HI313 heavy chain (339bp) and light chain (357bp) was cloned.To construct The spliced ScFv fusion gene with linker by overlap-PCR. All long and successfully cloned into PET22b plasmid to construct pET22b/HI313-ScFv expression vector. That can high express recombinant protein in E.coli induced by IPTG. The recombinant ScFv protein can bind nature CD44 molecular.
Conclusion:
Both HI313 and HI44a possess high affinity, HI313 monoclonal antibody can inhibit growth of NB4 cell and induced those cell cycle retained G0/G1 stage. After construction of HI313-ScFv. we should keep moving to study its potential usage and reform it for future clinical application.
引文
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