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The major challenge in the development of recombinant biologics lies in generating and isolating rare high-producing stable single clone in a short period of time. The selection marker is an essential component of the plasmid vector, it plays an important part in the generation and screening of producing cell lines. Engineering the selection marker to enhance the stringency of selection for high producing cells is one of the most effective approaches to improve the cell line development process. Here, using Chinese hamaster overy (CHO) cells as an example, we introduce the application of selection marker for generation of recombinant biologics producing mammalian cell lines, methods of engineering the selection markers to enhance the selection stringency, and propose considerations on cell substrate stability and selection marker safety, in order to provide references for high-efficiency development of recombinant biologics.
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Aim To establish a cell model which stably co-ex-press human kappa opioid receptor (hKOR) and enhanced green fluorescent protein ( EGFP) labeled catalytic domain of cAMP-dependent protein kinase A(PKAcat) fusion protein (PKAcat-EGFP) in Chinese hamster ovary(CHO) cells, laying the foun-dation for the high-throughput screening of hKOR drugs and drug molecular mechanisms in vitro. Methods Hygromycin B resist-ant hKOR recombinant plasmid [ pcDNA3.1/Hygro ( + ) -hKOR] was transfected into CHO cells stably expressing PKA-cat-EGFP by a lipofectin based method. Transfected cells were selected in culture medium containing hygromycin B. The posi-tive clones were selected by PKA redistribution assay. Z’ factor was used for evaluation and validation the reliability of the cell model. PKA redistribution assay and LANCE cAMP 384 Kit were used to test the function of the receptors in selected clone. Results CHO-PKAcat-EGFP/hKOR-13 cell model exhibited stable response in PKA redistribution assay and LANCE cAMP 384 Kit. Treated with 100 nmol·L-1U-50488 for 30 min, the average value of Z’ factor was 0.596, proving the reliability of the cell model. The hKOR expression in cell model remained stable after a few generations. Conclusion The CHO-PKAcat-EGFP/hKOR-13 cell model with stable co-expression of hKOR and PKAcat-EGFP has been successfully established.
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Objective@#To establish a functional antibody detection method for acellular pertussis vaccines in order to conveniently and effectively evaluate the production consistency and potency of acellular pertussis vaccine bulks and final products.@*Methods@#Chinese hamster ovary (CHO) cell clustering assay was optimized and used to measure titers of neutralizing antibodies against pertussis toxin in mouse immune serum samples.@*Results@#Vaccine samples were determined to be immunized intraperitoneally with 1/5 the human dose to ten female NIH mice (20-24 g, 5-week-old). Four weeks after immunization, blood samples were collected to isolate serum. Serially diluted serum samples were used to neutralize 0.1 IU/ml of pertussis toxin national reference product for 2 hours. Results of clustering were determined after 48 hours of incubation in pre-cultured CHO cell wells. The geometric mean of the serum dilution of the final unclustered wells was the neutralizing antibody titer of vaccine sample. There were significant differences in the titers of neutralizing antibodies elicited by acellular pertussis vaccines prepared with different manufacturing processes. Vaccine samples succeed or failed the modified intracerebral challenge assay (MICA) were easily distinguishable by neutralizing antibodies.@*Conclusion@#The method of detecting neutralizing antibodies to pertussis toxin greatly reduces the amount of animals used in research. CHO cell clustering assay that has better repeatability and precision can be used for monitoring and initial evaluation of the consistency and potency of the bulks and final products of pertussis vaccines prepared with different manufacturing processes.
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Objective To investigate the effect of different promoters on the expression level of transgene containing MAR expression vector in recombinant CHO cells.Methods The CMV promoter and 3-globin MAR were amplified by PCR,then CMV promoter was replaced the SV40 promoter in pCAT1 for constructing the expression vector droved by CMV promoter.The control vectors of pCAT1 and pCAT2 without containing MAR were simultaneously transfected into the CHO cells.Then the stably transfected cell line was screened by G418.The CAT gene expression level was analyzed by ELISA.Results The expression level of CAT enzyme in the cells transfected with MAR-containing vectors was increased compared with the cells transfected by pCATG and pCAT3 vectors without containing MAR,which were increased by 1.75 and 1.25 times respectively(P＜0.05);but CAT enzyme expression level in the pCAT1 transfected cells droved by SV40 promotor with the MAR-containing expression vectors was 1.4 times higher than that in the pCAT2 vector droved by the CMV promoter(P＜0.05).Conclusion MAR can enhance the transgene expression level in stably recombinant CHO cells,and the promoting efficiency of SV40 promoter and MAR combination is superior to that of CMV promoter and MAR combination.
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OBJECTIVE: To prepare the national standard substance for quantitative determination of residual DNA in CHO cells. METHODS: CHO cell DNA was prepared using QIAGEN Genomic-tip 500/G and genomic DNA purification reagents and analyzed for purity and concentration by UV spectrophotometry and agarose gel electrophoresis. Then the standard substance of CHO DNA was calibrated collaboratively, and evaluated for stability and applicability. RESULTS: The prepared national standard substance of CHO DNA was qualified as indicated by A260/A280 between 1.7 and 1.9 and a single specific band in agarose gel electrophoresis. The standard substance was calibrated for 90 times by six laboratories, and the results showed a geometric mean concentration of 93.63 μg · mL-1 (95% confidence interval 92.86-94.40 μg · mL-1). The 95% confidence interval of the geometric mean concentration in a single determination was 86.51-100.89 μg · mL-1. The mean confidence limit rate was 0.81%. The DNA concentration was stable after storage at-20, 4, 25 and 37°C for 4 months, but A260/A280 was decreased when stored at 37°C for 4 months. The electrophoresis results showed a single band after storage at -20, 4 and 25°C for 4 months, but showed degradation after storage at 37°C for 4 months. The long term stability test revealed that the DNA concentration and purity were stable after storage at -20°C for 12 months. In applicability studies using the CHO DNA standard substance, the fluorescence method showed good linearity (r > 0.9900) in the concentration range of 0.781-100 ng · mL-1, with RSD of less than 10%. The real-time PCR had high sensitivity up to 10-2 pg of DNA with good linearity (r > 0.9900) in the content range of 10-2-103 pg, and the melting curve showed a single peak. CONCLUSION: The prepared standard substance with batch number of 270026-201101 and DNA concentration of 93.63 μg · mL-1 is qualified in overall tests and may be used as national standard substance for residual DNA assay by fluorescence and real-time PCR methods. Copyright 2013 by the Chinese Pharmaceutical Association.
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Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.
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Objective To eonstruct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity, then check their expression in CHO cells. Methods With the available PUC-hGHR vector, two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions.Then with Lipofectamine2000, we trans-fected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western blotting were used to examine hGHR and STAT5-P. Results After sequencing, two mutations were introduced to hGHR, three eukaryotic expression vectors were identified. The transfected CHO cells expressed vd-hGHR or its mutants. Compared with hGH-wt, two mutate cells (E42K and H56R) had decreased phosphorylated STAT5 levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established, E42K and H56R partly interfered the phosphorylation of STAT5.
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Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.
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Objective To establish a stable cell line secreting human IgE Cε2-4 protein, and in-vestigate the binding capacity of receptor FcεR Ⅰ Methods The E24 gene was derived from SKO-O07 cell line, and was then cloned into pcDNA3.1 (+) (signal peptides were synthesized and fused at the 5'-end of E24 gene) or pCMV-L vector. After transient transfection into 293T cell, the secreted F24 protein was ana-lyzed by sandwich ELISA. The best vector was chosen to be transfected into CHO cells with LipofectAMI-NETM 2000 reagent. After being selected by G418 and subcloned three times by limited-dilution method, two stable cell lines were established. E24 gene was amplified by RT-PCR, and the E24 protein in the superna-tant was identified by ELISA. Besides, the binding capacity of FceR ⅠⅡ was analyzed by flow cytometry method. Results Three mammalian expression vector SP-E24-F3. 1, SP lI-E24-P3.1 and E24-PL were constructed and transient transfected to 293T cells. The output of E24 protein in the supernatant were 19.1, 19.4 and 8.7 μg/ml, respectively. Then the vector SP IX-E24-P3.1 was transfected into CHO cells. Final-ly, two single clones secreting E24 protein were stably obtained. The output of E24 were all at least 25 μg/ml. RT-PCR could detect the E24 gene from one of the two clones. Furthermore, flow cytometry results showed that E24 could bind the receptor in a dose-dependent manner. Conclusion Two stable cell line se- creting E24 protein were obtained, while E24 could specifically bind FcεR Ⅰ.
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Objective To explore immune effects of HB vaccine and relative strategies of controlling hepatitis B in adults of different ages, genders, and different anti-HBs levels before immunization. Methods In Shaoxing city, 1055 healthy adults aged 18 to 50 with HBsAg negative were randomly selected and inoculated with 10?g/ml indigenous CHO HB vaccine according to the programme of 0,1,6 months. Results 835 adults completed the whole programme, and the total immunization coverage rate was 85.12%. Before and after immunization,the positive rates of anti-HBs were 52.10% and 98.44% respectively, and anti-HBsGMT rose from 14.32mU/ml to 413.98mU/ml. For the two groups, anti-HBsGMT=0 and 0
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The intracellular hepatitis B surface antigen (HBsAg) content per cell was increased by 7.2-fold in the culture with 1.5% dimethyl sulfoxide (DMSO) compared with that in the control without DMSO, while the extracellular HBsAg production and specific productivity were only improved by 70% and 3.2-fold, respectively. Electron microscope has been employed to reveal large dilated structures within recombinant CHO cells in the presence DMSO. The dilated structures have a distribution within whole cytoplasm, and some dilated areas were engulfed in the nucleus. These large, dilated structures were not observed in the control. Immunogold labeling was used to discover the accumulated HBsAg was localized within these dilated areas, and some HBsAg-specific labels were detected in the nucleus membrane, owing to the encroachment of the dilated areas upon nucleus. The result could help to reveal the mechanism of intracellular HBsAg accumulation in the presence of DMSO.
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Objective To construct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity,then check their expression in CHO cells. Methods With the available PUC-hGHR vector,two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions. Then with Lipofectamine2000,we transfected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western blotting were used to examine hGHR and STAT5-P.Results After sequencing,two mutations were introduced to hGHR,three eukaryotic expression vectors were identified. The transfected CHO cells expressed wt-hGHR or its mutants. Compared with hGH-wt,two mutate cells (E42K and H56R) had decreased phosphorylated STAT5levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established,E42K and H56R partly interfered the phosphorylation of STAT5.
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Objective To obtain Chinese hamsterovary (CHO) cell line expressing human decay accelerating activity (hDAF) stably and to observe the protective effect of hDAF on heterologous cells under the circumstance of complement activation. Methods The eukaryotic expression vector DAF pcDNA3.1 was constructed and then transfected into CHO cells by lipofection. Monoclones of cells expressing hDAF stably were screened by the method of limiting dilution. hDAF expression was detected by flow cytometry. The decay accelerating activity of hDAF was determined by assay of C3 deposition and 51Cr release. Results The expression vector DAF pcDNA3.1 was successfully constructed, and monoclones of cells expressing hDAF were obtained. CHO cells expressing hDAF could decrease C3 deposition and attenuate the killing effect of activation of the complement system. Conclusion We have obtained CHO cell clones expressing hDAF stably, which is helpful for the further studies of the relationship of the structure with the functions of hDAF.
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Objective To investigate the transfection and expression of pcDNA3 1-VEGF 165 plasmid in mammalian cells. Methods pcDNA3 1-VEGF 165 plasmid was transfected into CHO cells by electroporation and the positive clones were screened by G418. The expression of VEGF 165 gene in the transfected CHO cells were detected by RT-PCR, ELISA and western blotting analysis. The biologic activities of VEGF 165 secreted by the transfected CHO cells were measured by Mile's assay and MTT method. Results G418-resistant CHO cell clones were obtained. RT-PCR, ELISA and western blotting analysis showed that there were the transcription and translation of VEGF 165 gene in anti-G418 CHO cells. The mile's assay showed that the expressed VEGF 165 could increase the vessel permeability, and MTT assay proved that it could promote the proliferation of endothelial cells. Conclusion pcDNA3 1-VEGF 165 plasmid could express VEGF 165 with biological activities in mammalian cells.
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Objective To recombine the tissue plasminogen activator (TPA) gene and establish the expressing model in vitro to provide both theoretical basic and technical guidance for gene therapy of ischemic heart disease and prevention of postoperative vessel re-stenosis. Methods Expression vector pcDNA3.1 TPA was constructed, and transfected into Chinese hamster ovary(CHO) cells. Then the exogenous TPA expression was observed. Results The expression quantity of TPA in transfected CHO cells was higher than that in non-transfected cells. The exogenous TPA activity was 12 296 IU/10 6cell/24hr in the transfected cells, when measured by chromogenic substrate assay, while that of non-transfected cells was 3 176IU/10 6cell/24hr. The TPA quantity of non-transfected cells was 9 608 ng/10 6cell/24hr, when measured by enzyme-linked immunosorbent assay (ELISA), while that of transfected cells was 586 172 ng/10 6 cell/24hr, and the latter was the 60 times of the former. Conclusions When pcDNA3 1(+)TPA was transfected into CHO cells, exogenous TPA can efficiently be expressed, which provides theoretical basic for TPA clinical gene therapy.
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BACKGROUND: Graves' disease and primary myxedema are thought to be caused by the action of TSH receptor autoantibodies(thyroid stimulating antibody; TSAb & thyroid stimulation blocking antibody; TSBAb). Thus, detection of these antibodies is crucial in diagnosis and in follow up of those patients. Recently, a sensitive method using human TSH receptor transfected Chinese Hamster Ovary(CHO) cells has been developed. However, the complexity of IgG purification procedure is considered as a limitation for its clinical application as a routine test. The aim of this study is to determine whether polyethylene glycol(PEG)-precipitated immunogiobuIin fraction could substitute for purified IgG. METHODS: We developed optimal conditions for TSAb and TSBAb assays using crude, PEG precipitated immunoglobulin fraction; and evaluated the correlation of TSAb and TSBAb activities between thase measured using crude immunoglobulin fraction and purified IgG to clarify the usefulness of PEG-precipitated immunoglobulin fraction. TSH receptor expressing wild type CHO cells were used in TSAb and CHO cells expressing chimeric TSH receptor(Mc2; 90-165 amino acid residues were substituted by those of rat LH/CG receptar) were used in TSBAb assay to minimize the possible disturbing effects of TSAb in serum. RESULTS: The optimal serum amount for TSAb and TSBAb assay using PEG-precipitated immunoglobulin fraction were 250mL serum equivalent/well and 50mL serum equivalent/well, respectively. The optimal incubation time for both assays were 2 homs, and aptimal ccrncentration of bTSH for TSBAb assay was 0.1U/L. TSAb activities measured with PEG-precipitated immunoglobulin were significantly correlated with those measured with purified IgG in 26 patients with Graves diseases(r=0.93, p<0.001). Although TSBAb activities measured using PEG-precipitated imrnunoglobulin were conelated with those measured using purified IgG in 20 patients with primary myxedema(r=0.86, p<0.001), the positive rate in TSBAb assay using PEG-precipitated immunoglobulin was lower than that of usmg purified IgG(20% v.s. 65%) because of negative conversion of TSBAb activities in samples with weakly positive TSBAb activities measured using purified IgG. CONCLUSION: PEG-precipitated immunoglobulin fraction could be used instead of purified IgG in TSAb assay using hTSHR-tranasfected wild type CHO cells with equal sensitivity and specificity. This simple and practical TSAb assay using PEG-precipitated immunoglobulin in hTSHR-transfected CHO cells would be useful in clinica1 practiee.
Subject(s)
Animals , Cricetinae , Humans , Humans , Rats , Antibodies , Asian People , CHO Cells , Cricetulus , Diagnosis , Graves Disease , Immunoglobulin G , Immunoglobulins , Myxedema , Polyethylene Glycols , Polyethylene , Receptors, Thyrotropin , Sensitivity and Specificity , Thyroid GlandABSTRACT
No abstract available.
Subject(s)
Animals , Cricetinae , Female , Humans , Asian People , CHO Cells , Cricetulus , Hepatitis B Surface Antigens , Hepatitis B Vaccines , OvaryABSTRACT
Aim To construct the eukaryotic expression vector of hIL-24 cDNA,and express it in CHO cells and detect its anti-tumor effect of recombinant hIL-24 protein.Methods Constructed pcDNA3-hIL-24 was identified by endonucleases digestion & PCR.The recombinant expression plasmids were transfected into CHO cells,human hIL-24 expressed in CHO cells was detected with RT-PCR.The apoptosis-inducing activities of recombinant protein hIL-24 was tested by MTT assay,Hoechst& FCM assay,and the expression of IL-6 and IFN-? from PBMC induced by rhIL-24 was tested by ELISA.Results The eukaryotic expression vector pcDNA3-hIL-24 was constructed correctly.Stable expression of human IL-24 in CHO cells was identified with RT-PCR.The apoptosis of A549 cells induced by hIL-24 was proved by Hoechst & FCM assay,and the expression of IL-6 and IFN-? from PBMC induced by rhIL-24 was identified with ELISA.Conclusion The successful stable expression & experimental study of apoptosis effect of human IL-24 gene lay the foundation for the further study of molecular mechanism of hIL-24 on anti-tumors and potential application.
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To establish a cell line expressing human MAdCAM-1 molecular.Methods: hMAdCAM-1 cONA was achieved from plasmid pUC21/hMAdCAM-1 by restriction enzyme digestion. Hie recombinant eukaryotic expression vector pCIneo-hMAd was constructed and then transfected into CHO cells with lipofectamine. Results: G418-resistant clones and their expression products were identified by immunofluo-rescence、immunoenzymatic histochemistry、 western blot and lymphocytes adhesion assay. hMAdCAM-1 with relative molecule mass of 63 000 could be demonstrated in cell lysates of the pCIneo-hMAd transfected cells. Conclusion: A stable transfectant expressing hMAdCAM-1 is successfully established, which might provide the basis for further studying the biologic functions and their mechanism.
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Objective: To amplify two human mutant CD59 eukaryotic expressing systems and investigate mutant CD59 functional activity. Methods: Mammalian expression vector PLATER of mutant CD59 cDNAs was transfected into CHO together with the pcDNA by lipofectamine,which confered resistance to G418(400 ?g/ml). The positive clones were tested by FIH. Activity of both mutants CD59 was determined by BCECF release assay. Results: Mutant CD59 cDNAs subcloned into the mammalian expression vector PLATER and transfected CHO together with the pcDNA,which confered resistance to G418. The positive clones were tested by FIH.Activity of both mutants CD59 before and after glycation was determined by BCECF release assay,both of them could restrict MAC formation ,and glycation could inhibit CD59. Conclusion: A eukaryotic system that expressing mutant CD59 cDNA was successfully set up.It was found that mutant CD59 could restrict MAC formation,and glycation could inhibit mutant CD59. These would be helpful for the furthur study of link mutant CD59 and the vascular proliferative of diabetes.