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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

Author: LiuChunLiang
Tutor: WuShiLiang
School: Suzhou University
Course: Biochemistry and Molecular Biology
Keywords: O-glycosylation ppGalNAc-T2 monoclonal antibody lentivirus leukemia
CLC: R733.7
Type: Master's thesis
Year: 2011
Downloads: 20
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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.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia
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