【作者】 陈小军；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2009， 博士

【摘要】 背景:CD33是髓系细胞分化抗原,分子量为67KD,主要分布在髓系血细胞,特别是在分化的早期阶段。其胞内区含有免疫酪氨酸抑制基序（ITIM）,所以它可能具有通过募集信号分子来调节细胞的生长与分化等功能。CD33在90%以上的急性髓系白血病患者都有表达。在造血干细胞表面没有表达,在成熟的粒细胞和其他组织也没有表达,因此CD33成为髓系白血病治疗的良好靶点。有文献证实AML细胞表面免疫共刺激分子CD80表达低下或缺乏,是导致白血病细胞不能被机体细胞毒性T细胞（CTL）有效地识别杀伤,从而逃逸宿主免疫监视的重要原因之一,而T细胞也因缺乏第二信号而无能。因此可以利用CD33表面抗原作为AML白血病细胞的表面标记,构建CD80胞外区-抗人CD33单链抗体（ExCD80-CD33scFv）融合基因、表达融合蛋白,通过融合蛋白中抗人CD33scFv将CD80胞外区结合到AML白血病细胞表面,刺激肿瘤特异性CTL细胞活化,从而增强抗肿瘤免疫。目的:构建及表达抗人CD33单链抗体（CD33scFv）基因,并检测其生物活性;扩增CD80胞外区,构建ExCD80-CD33scFv融合基因,并在大肠杆菌中表达融合蛋白,进一步检测融合蛋白的生物活性。方法:HIM3-4是血研所研制的分泌鼠源性抗人CD33单克隆抗体的杂交瘤细胞,并已获得国际人类白细胞分化抗原协作组会议正式命名。我们利用RT-PCR技术分别克隆了抗人CD33单克隆抗体HIM3-4的轻、重链可变区基因,并利用短肽（Gly4Ser）₃作为linker,通过overlap PCR方法组装成抗人CD33scFv。将获得的抗体基因克隆到具有强启动子的PET28a（+）载体上,应用IPTG诱导表达,纯化的表达产物经证明具有与靶抗原人类CD33特异结合的能力。CD80 DNA序列由王敏教授惠赠,我们通过另行设计带所需酶切位点的引物来扩增CD80胞外区,在此基础上,利用人血清白蛋白（HAS）第三结构域亲水性的403-427位氨基酸作为链间连接肽将抗人CD33单链抗体及人类细胞表面抗原CD80胞外区基因分别经酶切、纯化后克隆至原核表达载体PET22b（+）中,经IPTG诱导,在大肠杆菌内获得了融合蛋白的表达。融合蛋白的成功构建与表达,为下一步探讨血液肿瘤的免疫治疗机制奠定了基础。结果:1.通过RT-PCR方法,利用通用引物从HIM3-4杂交瘤细胞中克隆了抗体轻、重链可变区基因,分别为321bp和366bp,我们利用（GGGG）₃为连接肽构建了抗人CD33基因工程单链抗体,并在PET28a（+）中得到表达,蛋白大小约为31KD,经复性后具有与人CD33结合活性。2克隆出CD80胞外区序列,大小为633bp。3利用大小为25Aa的链间链接肽,将CD80胞外区与CD33ScFv连为融合表达基因,并在PET22b（+）中得到表达,蛋白大小约为55KD,经复性后具有与人CD33抗原结合活性。结论:1.从HIM3-4杂交瘤细胞中克隆了抗体轻、重链可变区基因,经测序为功能性重排的抗体可变区基因。2用（Gly₄Ser）₃连接肽基因将VH、VL连接成scFv基因,并用表达载体PET28a（+）表达了scFv蛋白。3.扩增了人类ExCD80 cDNA序列,测序显示序列正确。4.构建了PET22b（+）-ExCD80-CD33scFv融合基因表达质粒,并在含有稀有密码子的宿主菌Rosetta（DE3）中获得了表达。并初步探讨了其生物学功能。更多还原

【Abstract】 Background:CD33,a normal self-antigen that is a Mr 67,000 cell surface glycoprotein largely restricted to the myeloid/monocytic lineage.It contains a conserved immunoreceptor tyrosine based inhibitory motif(ITIM) in the cytoplasmic tails,so it may have roles in modulating cellular functions via recruitment of signaling molecules.CD33 was selected as the target antigen because of its over expression on 90%of acute myeloid leukemia(AML) blasts and its lack of expression on pluripotent stem cells (12-14),and it is not expressed on mature granulocytes and other tissues either.Many reports confirmed that lack or low expression of costimulatory molecule CD80 on AML cells surfaces is one of most important reasons that CTL can not recognize and kill cancer cells efficiently.It is a recognized phenomenon that T cells are rendered anergic due to the lack of costimulatory molecules expression by tumor cells.Based on the above information,we constructed CD80-anti human CD33 single chain fragment variable(scFv) fusion gene and express it in prokaryocytic expression vector.By this mean, we can modulate the AML cells with fusion protein to enhance the tumor-specific immunity.Objectives:To construct and express the single chain variable fragments (scFv) gene against human CD33 antigen,and characterize its bioactivity.Ampligy the extracellular region of CD80.Construct CD80-anti human CD33 single chain fragment variable(scFv) fusion gene and express it in prokaryocytic expression vector,and then detect the biological activity of the fusion protein.Methods:The genes encoding the light and heavy chain variable regions were cloned by RT—PCR from a murine hybridoma cell line HIM3-4,which could produce monoclonal antibody(mAb) against human CD33 antigen and is named by International workshop on human leukocyte differentiation antigens.Then the light and heavy chain variable regions were fused together by a short peptide linker containing 15 amino acid(Gly4Ser)3 using splice-overlap extensive PCR.The recombinant anti-CD33 scFv was subcloned into the expression vector pET28a(+)and expressed in E.coli Rosetta after induction by IPTG.Flow cytometry analysis showed that the scFv could react with human CD33 antigen.CD80 sequence was kindly presented by professor Wang Min.The extracellular region of CD80 was amplified by PCR with primers containing designed restriction enzymes.The hydrophilic fragments of 403-427 from domainⅢof HSA was choosed as an interlinker for ExCD80-CD33ScFv construction.The fusion gene was cloned into PET22b(+),after the fusion protein was expressed with IPTG in E.coli.Rosetta(DE3),its biological activity was detected with indirect immunological fluorescence experiment.Results:1 SDS-PAGE and Western blot analysis showed that the recombinant anti-CD33 scFv gene was expressed in the form of inclusion body in E.coli Rosetta(DE3),and the purified fusion protein was obtained after a series of purification steps including cell lysis,inclusion body solubilization, Ni2+ metal affinity chromatography and protein refolding.Flow cytometry(FCM) analysis showed that the scFv could react with human CD33 antigen,the VL was 321bp,the VH was 366bp.2 The extracellular region of CD80 was cloned by PCR,it is about 633bp.3 structing a ExCD80-CD33ScFv sequence in PET22b(+) vector by genetic engineering,then expressing it in the E.Coli Rosetta(DE3). The fusion protein was 55KSD fragment with human CD33-binding specificity after renaturation.Conclusions:(1) The VL and VH gene of CD33ScFv were cloned from hybridoma cell line HIN3-4.(2) Combinant anti-CD33 scFv gene has been successfully constructed and expressed in E.coli Rosetta(DE3).(3) the ExCD80 gene was successfully amplified.4 PET22b(+)-ExCD80-CD33ScFv was successfully expressed in Rosetta(DE3),its biological activity was detected initialy.更多还原