Dissertation
Dissertation > Medicine, health > Basic Medical > Medical Immunology

1. TmTNF-α-mediated Apoptotic Signaling Via TNFR2 2. Antibody Preparation and Identification

Author XiaoLi
Tutor LiZhuoZuo
School Huazhong University of Science and Technology
Course Immunology
Keywords Transmembrane TNF-α Secreted TNF-α NF-κB TNFR2 Apoptosis Caspase tmTNF-α sTNF-α Monoclonal antibody IgY Polyclonal antibodies
CLC R392
Type Master's thesis
Year 2011
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Tumor necrosis factor-alpha(TNF-α)is a pleiotropic cytokine.It exists as a 26kDa transmembrane form (transmembrane TNF-α, mTNF-α) and a 17kDa soluble form (secretory TNF-α,sTNF-α). Preliminary work in our laboratory confirmed tmTNF-αhad a wider spectrum of killing tumor than sTNF-α.Most of the biological effects of TNF is mediated by TNFR1 which can induce apoptosis, or proliferate some cells by activation of nuclear transcription factor NF-κB .sTNF-αmainly combined with TNFR1 to produce a marked effect, and tmTNF-αplayed important roles by combined with TNFR1 and TNFR2 . TNFR2 signal transduction pathways of apoptosis activated by tmTNF-αhas not been reported. This study is using HepG2 cells to explore TNFR2 signal transduction pathways of apoptosis inducing by tmTNF-α.1. KnockdownTNFR1 by siRNA had no effect on cytotoxicity induced by tmTNF-αand proliferation mediated by sTNF-α:We found that tmTNF-αand sTNF-α-induced cellular cytotoxicity have no influence by blocking the HepG2 TNFR1 signaling pathway using TNFR1 siRNA. These data indicated that in HepG2 tumor cells TNFR2 signaling pathway is most important.2. tmTNF-αinactivated NF-κB, but sTNF-αactivated NF-κB: Immuno -precipitation showed that tmTNF-αinhibited the recruitment of IKKα,but sTNF-αenhanced to recruit this molecule.In plasmolysis, tmTNF-αsignificantly increased IκB-αrecruitment,but sTNF-αcompletely decreased the recruitment of above-mentioned molecule.3. tmTNF-αactivated caspase-8 and -3: Western blot showed that Caspase-8 was activated as early as 24 h by tmTNF-αstimulation. At the same time, Caspase-3 levels were contently decreased and PARP was activated. No other significant change in caspase levels. The results showed that tmTNF-α-mediated apoptosis is associated with Caspase-8 and Caspase-3.4. Inhibiting protein synthesis had opposite effects on the cytotoxicity of two forms of TNF-α:Cycloheximide which is inhibiting protein synthesis increased in sTNF-α-mediated cytotoxicity sensitivity. But significantly inhibited the tmTNFα-mediated cytotoxicity. All of these showed that sTNF-α-mediated apoptosis is associated with inhibiting protein synthesis,and tmTNF-α-mediated apoptosis is associated with protein synthesis.5. tmTNF-α-mediated-cytotoxicity is associated with the synthesis of pro-apoptotic molecule: Cycloheximide which is inhibiting protein synthesis can lead to decreased Caspase-3,suggesting that tmTNF-αmay reduced the cytotoxicity by CHX inhibiting the synthesis of Caspase-3. Tumor necrosis factor-α(TNF-α) is a pleiotropic cytokine with a variety of biological activities which is produced mainly by activated T cells, monocytes/macrophages and NK cells. TNF-αexists in two biological forms, a 26kD transmembrane TNF-α(tmTNF-α)and a 17kD secreted TNF-α(sTNF-α). Commercial TNF-αantibody combine with two types of TNF-αand can not distinguish between biological functions and pathological effects of two types of TNF-α. We have already cloned hybridoma clones C1 stably secreted tmTNF-αwhich is used for preparing and identifying tmTNF-αspecific monoclonal antibody. And we also have constructed pET expression vector which has already inserted sTNF-αcDNA used for preparing sTNF-αPolyclonal Antibody.1. Preparation and identification of tmTNF-αmonoclonal antibody Hybridoma clones C1 cells2~5×105 was administered to BALB/c mouse which was already injected incomplete Freund adjuvant. After two weeks, we collected 82ml ascites. Firstly we depurated ascites using caprylic acid-ammonium sulfate.Then we depurated McAb using Protein G affinity chromatograph.The results as follows:1.1 Quality identification of McAb: A total amount of about 150ml antibody was obtained,and the concentration was 3mg/ml We found that a color zone of 52kD heavy chain and 27 kD light chain were observed by SDS-PAGE.1.2 Flow Cytometry:McAbs were used in FACS to measure the expression of tmTNF-αon Raji,and the expression rate was higher than 70%.1.3 Immunoblotting detection:After administered BALB/C mice with 231. We detected tmTNF-αin tumors with the method of western blot, and a color zone of 26kD was found.2. Preparation and identification of sTNF-αpolyclonal antibodiesChicken egg yolk immunoglobulin (IgY) which was produced by hens immunized by specific antigens and transferred from serum to egg yolk, has been recognized as an excellent source of polyclonal antibodies. Due to the evolutionary difference between mammals and birds, IgY can react with more epitopes on a mammalian antigen, which will give an amplification of the signal.As a high-production and high-efficiency antibody,IgY has already used in more and more medical domain.2.1 Identification of sTNF-αpolyclonal antibodies from rabbits2.1.1 Total amount of antibody:A total amount of about 50ml antiserum was obtained from two rabbits immunized with TNF-α2.1.2 Titre detected by ELISA: Expression of sTNF-αdetected by double antibodies sandwich ELISA and the titer was 1:16000.2.1.3 Immunoblotting detection:With the method of western blot , a color zone of 22kD was found. by the rabbit antiserum..2.2 Identification of IgY polyclonal antibodies from hens2.2.1 Total amount of antibody:A total amount of about 150ml antibody was obtained from a Lai Hang hen,and the storage concentration was 2mg/ml2.2.2 Titre detected by ELISA:Expression of sTNF-αdetected by double antibodies sandwich ELISA and the titer was 1:1600at 30 days.2.2.3 Immunoblotting detection:The results showed that a color zone of 22kD was found. by IgY. using bacterial protein as an antigen2.2.4 Fluorescence confocal microscopy: Green fluorescent protein under laser confocal microscopy suggested FITC-labeled IgY. And red fluorescent protein under laser confocal microscopy suggested PE-labeled tmTNF-α. Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine which has immunomodulatory and anti-tumor effects,mainly produced by T cells, monocytes/macrophages and NK cells.The members of TNF superfamily share the highly conserved sequence in their primary structures. Therefore, we hypothesized that TNF superfamily members and highly conserved extracellular amino acid sequence are likely to be common features for their trimer formation.In this study, mutant TNF-αgenes were amplified through recombinant ploymerase chain reaction (PCR) from pcDNA3.0-wtTNF-αby site-directed deletion of 119-126. The major results in the present study are as follows:1.Construction and cloning of recombinant plasmid: Using the plasmid pcDNA3.0- wtTNF-αas a template, the gene mTNF-αwas amplified with recombinant ploymerase chain reaction (PCR) by site-directed deletion., The TNF-αfragment and empty vector of pcDNA3.0 were linked by T4 DNA ligase after digestion with endonucleases of BamHI and XhoI. Then the recombinant plasmid was transformed into E. coli DH5α. The positive clones were screened.2.Identification of positive clones: Firstly the positive clones were confirmed by colony PCR, followed by identification with endonucleases digestion.The results showed the cleaved fragment from the recombinant plasmid with the molecule weights of 702bp.And DNA sequence analysis proved that the recombinant contains sequences which sited mutated at 119-126, suggesting that expected mutant pcDNA3.0-TNF-αrecombinant plasmid was constructed successfully.

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