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抗人ppGalNAc-T2单克隆抗体制备以及慢病毒介导ppGalNAc-T2基因表达对白血病细胞恶性表型的影响
Generation of Anti-human ppGalNAc-T2 Monoclonal Antibody and the Effect of ppGalNAc-T2 Gene Expression Mediated by Lentivirus on the Malignant Phenotype of Leukemia Cell Line
【作者】 刘春亮;
【导师】 吴士良;
【作者基本信息】 苏州大学 , 生物化学与分子生物学, 2011, 硕士
【摘要】 目的:O-糖基化异常与多种疾病密切相关,ppGalNAc-Ts酶家族作为催化黏蛋白型O-聚糖合成的起始酶,在肿瘤中出现的ppGalNAc-Ts异常表达已经被认为是肿瘤特异聚糖结构产生的重要机制之一。有研究发现,与正常组织或细胞相比,ppGalNAc-T2在结肠癌、乳腺癌等肿瘤中存在异常表达。为了研究ppGalNAc-T2基因在表达及其功能,本实验制备特异性抗人ppGalNAc-T2单克隆抗体,并对该抗体的特性及应用进行分析。方法:(1)抗原制备:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建pET-32a(+)-ppGalNAc-T2原核表达重组载体,载体经鉴定后转化至大肠杆菌BL21宿主菌,采用自动诱导方案表达ppGalNAc-T2融合蛋白;融合蛋白经凝胶纯化并鉴定后获得免疫用抗原;(2)杂交瘤及单克隆抗体制备:免疫BALB/c小鼠,利用细胞融合技术构建杂交瘤细胞,经间接ELISA和流式细胞术双筛选方法来获得稳定、高效分泌抗ppGalNAc-T2抗体的杂交瘤细胞株;然后采用小鼠体内腹水诱生法大量制备腹水型单抗;(3)单克隆抗体特性分析及其应用:测定单克隆抗体的亚类,然后将单克隆抗体用于间接ELISA,原核蛋白及真核蛋白的Western Blot分析,流式细胞术以及细胞免疫荧光分析。结果:筛选出一株稳定分泌抗人ppGalNAc-T2单克隆抗体的杂交瘤细胞株,命名为LW-5F3,保藏号NO.C2010106;该单抗亚类为IgMκ;Western blot分析该单克隆抗体不仅识别原核表达的ppGalNAc-T2融合蛋白,还特异识别L929-ppGalNAc-T2转基因细胞表达的ppGalNAc-T2真核蛋白;流式细胞术分析检测到白血病Jurkat细胞和肝癌HepG2细胞均表达ppGalNAc-T2蛋白,并对肝癌HepG2细胞中ppGalNAc-T2蛋白表达情况进行了免疫荧光分析。第二部分:慢病毒介导ppGalNAc-T2基因表达对白血病细胞恶性表型的影响目的:白血病细胞株在经全反式维甲酸诱导分化过程中ppGalNAc-T2基因出现表达异常,而且有研究发现抑制O-糖基化可影响结肠癌和淋巴瘤等肿瘤的生长恶化。我们推测ppGalNAc-T2的表达及其控制合成的O-糖链在白血病中可能有着重要的作用。为此我们利用慢病毒转染手段,来研究ppGalNAc-T2基因表达对白血病Jurkat细胞恶性表型的影响,来探讨O-糖基化在白血病中的作用。方法:(1)慢病毒过表达载体构建:利用PCR的方法从人293T细胞中克隆ppGalNAc-T2的编码区cDNA,构建慢病毒重组质粒venus-ppGalNAc-T2,将重组质粒及包装质粒共转染至293T细胞产生病毒颗粒,毒液经纯化并进行滴度测定;(2)慢病毒RNAi载体构建:设计并合成靶向沉默ppGalNAc-T2基因表达的shRNA及negative control,经退火后连接至慢病毒载体YH1上,病毒产生及滴度测定等操作同上;(3)白血病Jurkat细胞转染:将(1)和(2)所得慢病毒感染Jurkat细胞,然后进行经流式分选仪分选YFP阳性细胞,每个单细胞单独培养为单克隆细胞株,经流式细胞术,半定量RT-PCR和Western blot鉴定后,获得Jurkat细胞ppGalNAc-T2过表达及RNAi模型;(4)实验分析ppGalNAc-T2表达改变后,Jurkat细胞增殖、粘附以及TransWell迁移能力的改变。结果:成功构建了重组慢病毒ppGalNAc-T2过表达载体和慢病毒RNAi载体,感染Jurkat细胞经流式细胞仪分选,半定量RT-PCR和Western blot实验鉴定获得了稳定转染Jurkat细胞株;MTT实验,细胞粘附实验和Transwell细胞迁移实验分析表明ppGalNAc-T2过表达可促进Jurkat细胞的增殖,粘附及细胞迁移能力;当ppGalNAc-T2表达被RNAi沉默后,Jurkat细胞的增殖,粘附和迁移能力降低。
【Abstract】 Objective: UDP-N-acetyl-D-galactosamine polypeptide N-acetylgalactosaminy- ltransferases (ppGalNAc-Ts) regulate the initial key step of mucin O-glycosylation. ppGalNAc-T2 as a key member of ppGalNAc-T family, was recently described abbrant expression in oral squamous cell carcinoma, colorectal and breast carcinoma. In order to gain further insight into the role of ppGalNAc-T2, we decide to produce the anti-human ppGalNAc-T2 monoclonal antibody.Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into prokaryotic expression vector pET32a(+) and transformed into E.coli BL21(DE3). After induced by auto-induction, the recombinant protein was expressed and purified using protein electroelution purification. (2) Immunized BALB/c mice with the recombinant human ppGalNAc-T2 protein, the MAb against ppGalNAc-T2 was prepared using hybridoma technique. The positive hybridoma was selected by ELISA and Flow Cytometry. Ascites MAb were obtained after inoculation of isolated hybridoma into BALB /c mice. (3) The MAb was used for ELISA, Western Blot(fusion protein and eukaryotic protein from transgenic L929-ppGalNAc-T2 cells), Flow Cytometry(Jurkat,HepG2) and immunofluorescence(HepG2,LSC).Results: Hybridoma LW-5F3 cell line was obtain, It’s IgMκisotype, mAb can specifically recognize human ppGalNAc-T2 protein in both fusion and native formats by Western blot, Flow cytometry and immunofluorescent staining. PartⅡ. The Effect of ppGalNAc-T2 Gene Expression Mediated by Lentivirus on the Malignant Phenotype of Jurkat Cell LineObjective: ppGalNAc-T2 expression pattern has been described in many human tumors, and our lab has demonstrated that it’s expression was also changed during 1,25(OH)2D3 induced differentiation in several leukemia cell lines. Based on these studies, we construct the recombinant lentivirus, in ordor to study the effection of ppGalNAc-T2 expression on Jurkat malignant phenotypes in vitro.Methods: (1) The cDNA were obtained from the human 293T cells, the full length of human ppGalNAc-T2 gene were amplified by RT-PCR. Then it was recombined into the lentiviral vector pRRL venus, After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, the recombinant lentivirus were packaged and then purified. (2) The RNAi and negative control shRNA were designed and synthesisd, then were inserted into lentivirus vector YH1. After enzyme digestion and sequencing confirmation, each of the recombinant vectors and packaging vectors were cotransducted into 293T cells, and the recombinant lentivirus were packaged and then purified. (3) Jurkat cells were infected by purified lentivirus, and then sorted the YFP positive cells into 96-well plate(1cell/well) by High-speed cell sorter (Beckman Coulter), the ppGalNAc-T2 mRNA and protein expression levels were analyzed using RT-PCR and Western Blotting. (4) The malignant phenotype of Jurkat cells were analyzed using MTT, cell aggregation and Transwell assays.Results: Lentiviral vector-based over-expression and siRNA expression plasmids against human ppGalNAc-T2 gene have been successfully constructed and identified by DNA sequencing. And stable trangenic Jurkat cells (over-expression and RNAi) were obtained. Over-expression of ppGalNAc-T2 could promote cell proliferation, aggregation and migration of acute lymphocytic leukemia Jurkat cells.
【Key words】 O-glycosylation; ppGalNAc-T2; monoclonal antibody; lentivirus; leukemia;