The Preparation Of The Polyclonal Antibody And Monoclonal Antibody Against Human DcR3 And Their Rudimental Application

Decoy receptor 3(DcR3/TR6)produced as a soluble protein is a new member of the tumor necrosis factor receptor(TNFR)superfamily.The DcR3 mRNA is 1 114 bp in length and contains one open reading frame which encodes 300 amino acids those of the first 29 constitutes the signal peptide sequence and its protein relative molecular weight is 35 000 approximately.DcR3 mRNA was detected weakly in normal human embryon lung, brain, liver,stomach, spinal cord, lymph node, trachea, spleen and colon while at high levels in malignant tumors tissue such as lung cancer, colon adenocarcinoma,esophageal carcinoma, gastric carcinoma, hepatoma, pancreatic carcinoma, EBV related lymphoma,neurospongioma and peripheral blood mononuclear cells(PBMC) of some autoallergic diseases SLE, silicosis patients by Northern blot and so on. DcR3 has multiple biological functions including inducing cells apoptosis blockage by competitive binding corresponding ligands FasL,LIGHT,TL1A with Fas,HVEM,DR3. In addition,it participates in immune response acting as a modulator in the immune system.It plays an important role in cell growth, differentiation,death,immunomodulation It correlates to malignant tumours and autoimmune diseases and graft rejection. We undertook below study in order to go futher into biological function ,to find the relation between DcR3 and diseases of respiratory system that refers to apoptosis abnormality and to approach clinical application value.1. Expression,purification, renaturation and detection of biologic activitySubcloning pET28a(+)/DcR3 was transformed competent E coli BL21.We explored the optimum condition of target protein expression at different temperature, with different final concentrations IPTG and for different time course. Soluble form of product were analyzed by supersound. Protein purified by Ni-NTA affinity chromatography and dialysed to anneal were analyzed by SDS-PAGE, identified by indirect ELISA and Western blot. We observed inhibition of FasL mediated apoptosis by DcR3 on NCI-H446 cells by flow cytometry (FCM) and indirectly judged biologic activity of target protein after renaturation. The sequencing of pET28a(+)/DcR3 proved that the sequence we had got was totally coincident with that of reported by Genebank.SDS-PAGE showed a new protein band in relative molecular weight of 33 000. The fusion protein was induced by IPTG and reached 38% of the total bacteria protein, The amount of expressed protein at 28℃was higher than at 37℃and reached a peak after addition of IPTG for 3 h,while there was no obviously distinction at different final concentration IPTG.The target protein after split by supersound were expressed mainly in the formof inclusion body.The protein purifity reached 98% after cleaning, denaturation, lysis and purification.The purified product showed good antigenicity with anti-human DcR3 monoclonal antibody (mAb) by indirect ELISA and Western blot. Apoptosis rate of NCI-H446 cells treated by the renaturated DcR3 protein and FasL was obviously lower than treated by FasL alone.Inhibitory action of apotosis tended to be manifest with increase of DcR3 final concentration,which showed that the renaturated protein possessed capability to bind FasL competitively and inhibit Fas/FasL mediated apoptosis. DcR3 fusion protein was successfully purified and renaturated ,which laid a foundation for the furth study of its function and application.2. The preparation and identification of the polyclonal antibody(pAb) and mAb against human DcR3The serum of rabbit immunized with purified fusion DcR3 protein were collected and identified.BALB/c mice were immunized with purified fusion DcR3 protein, spleen cells which were collected when the titer reach above 1:106 and myeloma cells Sp2/0 were fused by PEG4000. Hybridoma cells which excreted specific antibodies were obtained by selective culturing with HAT medium, screening by indirect ELISA,cloning by limiting dilution assay. Hybridoma cell strains were inoculated into cavum abdominis of homologous series mice to induce ascitic fluid. Purified mAbs by caprylic acid-saturated ammonium sulfate fractional precipitation and Protein G affinity chromatography were identified by their specificity, subtype, titers via ELISA and Western-blot. The results showed that the value of purified pAb against DcR3 reached 1.28×10-6 and the concentration of that was 9.0 g/L.The purified pAb could recognize DcR3 fusion protein, lysate and debris of SW480 cells.The cell clones were found after cell-fusion and HAT selective culturing for 3 d and the fusional rate was 93.0%(269/288), the positive cloning efficiency was 85.0%(246/288). Five hybridoma cell lines which named 1A9, 1B1,1F5,1G4,2H6 secreted mAb against human DcR3 were obtained. Five mAbs were determined as IgG1 subtype and ascites titers reached 1×10-5～ 1×10-7 after 2～3 subclones by limiting dilution assay. Five mAbs were proved to recognize DcR3 protein specifically by Western blot, one of which (1B1) went furth to be identified and could react with supernatant and lysate of SW480 cells. The expressions of DcR3 on membrane and endochylema of SW480 cells were average 22.8% and 34.2% by FCM. Five hybridoma cell strains kept excreting high-titer mAbs via freezing, reanimating repeatedly and serial subcultivation vitro for 3 months.The development of pAb and mAbs against DcR3 made up a domestic deficiency and set up the basement of the research for furth to study the function and purification of DcR3, expression and distributing of tissue or cells, detection in body fluid as well.3. The foundation of the assay for the quantitative determination of DcR3 and the rudimental clinical applicationSandwich BAS-ELISA which coated with pAb ,detected with biotinylated mAb 1B1 and employed biotin-avidin system(BAS) has been established to detect DcR3 in the serum from patients with the respiratory diseases involving abnormal apoptosis such as idiopathic fibrosis of the lung (IPF), chronic obstructive pulmonary disease (COPD) in the period of acute exacerbation, small cell lung cancer.DcR3 levels were compared with that of patients with bronchial asthma, pneumonia and healthy individuals. We established the Sandwich BAS-ELISA ELISA to detect serum DcR3 successfully and the assay had a detection limit of 86 pg/mL with a dynamic range of 0.15～10 ng/mL on the standard curve. Intra-and inter-assay average CVs were less than 10%. The recovery range was 95～114%. We discovered that an average levels (mean±S) of DcR3 from 22 cases of bronchial asthma and 25 cases of pheumonia were 0.86±0.57 ng/mL,1.02±0.37 ng/mL respectively, whch was no statistics difference with that of healthy individuals(0.91±0.43 ng/mL).The sera DcR3 levels of 27 cases of IPF(1.92±1.04 ng/mL) and 34 cases of small cell lung cancer(2.04±0.94 ng/mL)were significantly higher than that of control (p<0.01).Meanwhile 23 cases of COPD in the period of acute exacerbation had a higher DcR3 levels(1.42±0.77 ng/mL) than that of control (p<0.05).Taken together, we constructed pET28a(+)/DcR3 prokaryotic expression plasmid and prepared high purity DcR3 which renaturated successfully. Rabbit and BALB/c mice were immunized with purified DcR3,anti-DcR3 pAb and mAbs with high titer, better specificity were acquired. Sandwich BAS-ELISA which coated with pAb and detected with biotinylad mAb was sensitive and constant to quantify sera DcR3 levels.The findings which DcR3 levels in sera of patients with IPF, small cell lung cancer and COPD in the period of acute exacerbation was significantly higher than that of the normal control indicated that the assay will possess favourable application value and extensive clinical application perspective.In addition,we will further to explore that abnormal expression of DcR3 was the cause or result for the related disorders.The causality between DcR3 and diseases will contribute to investigating pathogenesy,thus provide a new therapeutical means.