【摘要】 目的:构建针对B细胞淋巴瘤Daudi细胞系噬菌体抗体库,从中筛选特异性抗体,并对所筛选出的阳性克隆进行鉴定。方法:以Daudi细胞免疫BALB/c小鼠,ELISA检测抗血清滴度后,分离抗体阳性的小鼠脾淋巴细胞,提取RNA。利用RT-PCR扩增抗体к轻链和重链Fd片段,经SacI/XbaI和XhoI/SpeI双酶切,依次克隆入噬菌体载体pComb3H-SS的相应酶切位点,并电转化大肠杆菌XL1-Blue,以辅助噬菌体VCSM13进行超感染,构建B细胞淋巴瘤Daudi细胞系特异性Fab噬菌体抗体库。以Daudi细胞为抗原对抗体库进行6轮"吸附-洗脱-扩增"筛选,并通过ELISA法对随机挑选的克隆进行抗原结合活性测定,获得的阳性克隆进一步做DNA序列测定、在大肠杆菌XL1-Blue中进行可溶性表达,并用Western blot对表达产物的特异性作了鉴定。结果:构建了容量为3.13×107的抗人B细胞淋巴瘤Daudi细胞系的Fab噬菌体抗体库,并筛选获得了与Daudi细胞系特异性识别结合的4株阳性克隆。氨基酸序列分析结果显示:阳性克隆的重链、轻链可变区序列分别与基因库中已注册的鼠源性免疫球蛋白重链、轻链可变区序列有80%～94%和88%～95%同源性。阳性克隆成功在大肠杆菌细胞中可溶性表达,Western blot结果表明所获得的可溶性表达产物可与Daudi细胞膜抗原特异性结合。结论:成功地构建Fab噬菌体抗体库并筛选出针对Daudi细胞系膜抗原的抗体,为进一步研究B细胞淋巴瘤的免疫治疗提供了实验基础。更多还原

【Abstract】 AIM:To generate and screen the specific Fab phage antibody library against human Daudi cell strain in B-lymphoma and identify the positive clones.METHODS:BALB/c mice were immunized with Daudi cells,and antisera were titrated by ELISA.Following the demonstration of sufficient antibody titer,total RNA was extracted from splenic lymphocytes of the immunized mice and RT-PCR was used to amplify κ light chain and Fd fragments of heavy chain.After restrictive digestion with Sac I/Xba I and Xho I/Spe I,the κ light chain and the Fd fragments were successively inserted into the phagemid vector pComb3H-SS and then electroporated into E.coli XL1-Blue.The specific Fab phage antibody library against Daudi cell strain in human B-lymphoma was constructed by infection of helper phage VCSM13.Following six rounds of biopanning with Daudi cells,the antigen binding activities of random clones were tested by ELISA to select the positive clones,which were further DNA sequenced,expressed in E.coli XL1-Blue and identified by Western blot.RESULTS:The Fab phage antibody library with 3.13×107 size was constructed and four positive clones which specifically recognized Daudi cell strain were isolated.In amino acid sequences,the variable heavy domains(VH)were found to be 80%-94% and variable light domains(VL)88%-95% homologous with respective murine germline genes in GenBank.Furthermore,soluble Fab antibodies of the positive clones were successfully expressed in E.coli XL1-Blue and the reactivity with the membrane proteins of Daudi cells was demonstrated by Western blot.CONCLUSION:Fab phage antibody library is successfully constructed and specific antibodies against membrane antigens in Daudi cells are obtained,which provides an experimental foundation for the further investigation of B-lymphoma immunotherapy.更多还原