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Human/Mouse CXCR3 Gene Transfected Cell Line Establishment and Mouse Anti-human CXCR3 Monoclonal Antibody Research and Its Function

Author: SunJie
Tutor: QiuYuHua
School: Suzhou University
Course: Immunology
Keywords: CXCR3 chemokine stable expression Monoclonal antibody cell migration
CLC: R392
Type: Master's thesis
Year: 2010
Downloads: 67
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Abstract


CXC Chemokine Receptor 3——CXCR3(CD183)is a 7-time transmembrane G protein couple receptor, mainly present at activated and (or) memory T cell, NK cell and Macrophage cell surface. Loetscher initiatively cloned human CXCR3 gene from human CD4+ cell in 1996. CXCR3 gene coding region consists of 1,104 bp coding 368 amino acids of protein molecular weight 40.659 kD. It is known existing three splice variant, CXCR3-A (namely CXCR3), CXCR3-B and CXCR3-alt respectively, its chemotatic factor ligands are CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC accordingly. The recent research shows that CXCR3 is highly expressed on malignant tumor cell surface and mediates tumor transfer and invasion through receptor-ligand interaction. Therefore, the study aims to clone human and mouse CXCR3 gene and establish gene transfection cell lines L929-huCXCR3 and L929-mCXCR3 with stably expression of human/mouse CXCR3 molecule, further investigates migration of L929-huCXCR3 and L929-mCXCR3 under the effect by CXCR3. We used L929-huCXCR3 as immunogen and screening cell to prepare hybridoma cell line which can secrete mouse anti-human CXCR3 mAb continuously, investigated how CXCR3 signal path affects tumor migration and growth. PartⅠHuman/Mouse CXCR3 Gene Transfected Cell Line Establishment and IdentificationObject: Clone human/mouse CXCR3 coding region gene with whole length and obtain gene transfection cell line L929-huCXCR3 and L929-mCXCR3 with stable expression of human/mouse CXCR3 molecule. Investigate migration effect of L929-huCXCR3 and L929-mCXCR3 induced by CXCR3 ligands.Method: Amplify human CXCR3 gene from activated human peripheral blood T cells through RT-PCR, In the same way, amplify mouse CXCR3 gene from mouse spleen and thymus cells undergone Con A stimulation in vivo and IL-2 induction in vitra. Then we inserted the above gene into retroviral vector pEGZ-term. Through lipoplast method, cotransfected incasing cell 293 T with recombinate retrovirus vectors, pEGZ-term/huCXCR3 and pEGZ-term/mCXCR3, and two helper virus vectors. Then we collected cultivation supernatant of 293 T cells which contained complete retrovirus particles and repeatedly infected L929 cell three times. We obtained gene transfection cell lines L929-huCXCR3 and L929-mCXCR3 with stable expression of human/mouse CXCR3 molecule via Zeocin screening. We used immunofluorescence and RT-PCR for verification of L929-huCXCR3 and L929-mCXCR3, further investigate its migration effect by Mig, IP-10 and I-TAC through Transwell isolation cabin system.Result: We obtained gene transfection cell lines L929-huCXCR3 and L929-mCXCR3 with stable expression of human/mouse CXCR3 membrane molecule. CXCR3 positive expression rate on the surface and GFP expression in the cells of the two cell lines are both over 95.0%. Both L929-huCXCR3 and L929-mCXCR3 can migrate oriented, but differs from initiative concentration. And also migration rates were different for the three kinds of ligand of same concentration, L929-mCXCR3 can migrate oriented under the effect of 50 ng/mL mouse IP-10. Mouse CXCR3 antibody can inhibit L929-mCXCR3 migration.Conclusion: Establishment of human/mouse gene transfection cell lines L929-huCXCR3 and L929-mCXCR3 has laid material and laboratory foundation for investigation of CXCR3 signal transduction biological function, establishment of tumor migration model and preparation of CXCR3 mAb.Part II Mouse Anti-human CXCR3 mAb preparation and its biological functionObjective: Obtain hybridoma cell line with continue expression of mouse anti human CXCR3 mAb, investigate expression characteristics of human CXCR3 and how CXCR3 signal transduction function on L929-huCXCR3 and colon carcinoma cell lines transfer and growth.Method: Taking L929-huCXCR3 cell with high expression of human CXCR3 membrane molecule as immunogen to immunize BALB/c mouse, we fused immunized mouse spleen cell with myeloma cell SP2/0 of its same germ line, then used L929-huCXCR3 as screening cell and empty vector transfected cell L929-mock as negative control. Obtained hybridoma cell line with continue secretion of anti human CXCR3 mAb through flow cytometry. We used cell nucleus chromosome count, Ig subclass type rapid qualitation indicator paper method, indirect immunofluorescence and Western blot to identify obtained hybridoma cell line and mAb. Used indirect immunofluorescence to analyze CXCR3 expression on tumor cell surface, Transwell isolation cabin to assess effect on L929-huCXCR3 and colon carcinoma cell line Colo205, HCT116 and HT29 migration by mAb, MTT method to analyze how mAb function on colon carcinoma cell line Colo205 proliferation.Result: Obtained a hybridoma cell line with continue secretion of mouse anti-human CXCR3 mAb, named 9B5. According to rapid qualitation test paper analysis, light chain of the mAb isκchain and heavy chain is IgG1 subclass. Indirect immunofluorescence and flow cytometry results shows that the mAb can recognize CXCR3 molecules on activate T lymphocyte and colon carcinoma cell line Colo205, HCT116 and HT29 cell surface and can be used for Western blot detection. mAb 9B5 can inhibit L929-huCXCR3 cell and colon carcinoma cell line Colo205, HCT116 and HT29 oriented migration and Colo205 growth promotion effect by IP-10.Conclusion: We successfully obtain a hybridoma cell line with continues secretion of mouse anti-human CXCR3 mAb, which has laid material foundation on investigation on CXCR3 expression characteristics and how CXCR3 signal transduction function on tumor growth and migration. It is prospective to create a new thought and a new drug for treatment of tumor migration.

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