| Objective:NHL (Non Hodgkin’s lymphoma) is the most common malignant tumor in the immune system, and its incidence and mortality rates increased year by year. Human lymphocyte differentiation antigen CD20 is B cell transmembrane protein, and is specifically expressed in patients with NHL, not expressed in normal tissues. Thus, in the treatment of B cell lymphoma, CD20 is a ideal target antigen. The purpose of this project is to use site-specific integration technology to construct eukaryotic expression vector of anti-CD20 chimeric antibody, to integrate anti-CD20 gene in specific site of FRTCHO cell lines. To filter stability, high expression of anti-CD20monoclonal CHO cell lines by detecting the expression and biological activity of anti-CD20 monoclonal antibody, lay the foundation for large-scale industrial production.Methods:(1) Construction eukaryotic expression vector of chimeric anti-CD20 monoclonal antibody:Using 32#-2, BF27-L and BF27-H plasmid as template, PCR amplified the fragment of sp163-Lv, κC-κT-CMV, sp163-Hv, Hc-Fc; respectively. And then through overlap PCR ligated to generate destination fragment form sp163-Lv-κC-κT-CMV and sp163-Hv-Hc-Fc. To digest sp163-Lv-κC-κT-CMV fragment by the restriction enzyme by Hind III, EcoR V, then cloned into the eukaryotic expression vector pcDNA5/FRT to construct a pcDNA5/FRT-spl63-Lv-KC-KT-CMV. sp163-Hv-Hc-Fc by EcoR V, Xho I double digestion was cloned into vector pcDNA5/FRT-sp163-Lv-KC-KT-CMV verified as positive, after identification to determine the positive recombinant pcDNA5/FRT-sp163-Lv-KC-KT-CMV-sp163-Hv-Hc-Fc.(2) Construction Site-specific integration FRTCHO Clones:By liposome mediated, linear pFRT/lacZeo-dhfr plasmid transfection CHO/dhfr- cells, and then resistant clones were screened by Zeocin antibiotic. By β-Gal detection kit to screening higher expression cell clones of β-galactosidase, MTX gradient pressure and β-Gal detection filter the highest levels of β-galactosidase expression site-specific integration of monoclonal cell lines FRTCHO.(3) Stable, high expression of anti-CD20 monoclonal CHO cell lines constructed:The recombinant expression vector pcDNA5/FRT-spl63-Lv-κC-κT-CMV-sp163-Hv-Hc-Fc and pOG44 vector (mixed according to the ratio of 1:9), in liposome mediated co-transfected FRTCHO engineered cells, with the hygromycin B selected resistant cell clones, the purpose of initial screening higher levels of protein expression clones by Elisa. Then detect the Zeocin sensitization, MTX gradient pressure, Elisa detection of protein expression screening the highest amount of expression of anti-CD20 monoclonal site-specific integration of the cell clones CHO-CD20, and then domesticated by the serum-free suspension cultured engineering cell line, supernatant of enlarged cultivation was purified by ProteinA affinity chromatography, UV absorption measured to detect the expression of the target protein.(4) The quality of protein research:using SDS-PAGE and SEC-HPLC identification of the molecular weight and purity of the target protein, N-terminal sequencing identified the initial purpose of protein structure, the last living cells using indirect immunofluorescence (FACS) and the CDC method to detect the expression products of biological functions.Results:(1) The project using PCR and overlap PCR technical successfully obtained target gene, and with the directed cloning, successfully constructed the site-specific integration eukaryotic expression vector pcDNA5/FRT-sp 163-Lv-κC-κT-CMV-sp 163-Hv-Hc-Fc.(2)After linearization pFRT/lacZeo-dhfr plasmid transfect CHO/dhfr-cells, through Zeocin antibiotics selected, β-Gal Detection Kit to detect the β-galactosidase expression of each hole, which numbered as 12 cells Hole β-galactosidase expression was highest, through the limited dilution to obtained 20 monoclonal cell lines, then MTX gradient pressure, through the detection of β-galactosidase expression, the higher expression of the fixed-point monoclonal integration cell FRTCHO, found that after 500nM pressurized, the highest level expression of β-galactosidase cell line was 12-A15. Therefore, selected the 12-A15 cell line to construct engineerd cell lines.(3)After vector βcDNA5/FRT-sp163-Lv-κC-κT-CMV-sp163-Hv-Hc-Fc and pOG44 FRTCHO engineered cells co-transfection, selection by hygromycin B, Elisa to detect the protein expression of pore volume, which numbered CD9 holes expression the highest protein, and cultured by limiting dilution obtained 31 monoclonal cell lines, and then Elisa detect the protein expression, get the high expression of anti-CD20 monoclonal site-specific integration of cells clones CHO-CD20, respectively, CD9-6, CD9-11, CD9-17, CD9-20, CD9-27. After Zeocin sensitization test, then MTX gradient pressure, Elisa screening, ultimately get the number of CD9-17 protein expression highest 1.55mg/ml. Serum-free culture after acclimation to enlarge, supernatant was purified by ProteinA affinity chromatography, elution and then measured UV absorption, calculated the protein expression was 1.13mg/ml.(4)After SDS-PAGE, the molecular of protein is about 150kDa under the condition of non-reducing, reducing conditions the heavy chain and light chain is approximately 50kDa and 27kDa; SEC-HPLC detection of the target protein purity is 96%; FACS detection of expression of the antigen-binding antibody capacity is consistent with the reference substance; CDC antibodies detected by the purpose of the median effective concentration EC50 values is 238.8ng/ml.Discussion:The subject was successfully constructed stable CHO-CD20 engineering cell lines which can high expression of anti-CD20 monoclonal antibody. It can effectively kill CD20+ B-lymphocytes to CD20 as a target antigen. This subject provides a feasible treatment to tumor immunotherapy and targeted therapy of B lymphocytes. |
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