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Chinese Journal of Biotechnology ; (12): 290-300, 2021.
Article in Chinese | WPRIM | ID: wpr-878562


For improving epitope immunogenicity and achieving the co-immunization, late protein 1 (L1) of HPV type 16 (HPV16L1) was selected as the vector to carry the dominant epitope of Toxoplasma gondii because of the shared common population between Toxoplasma gondii and human papillomavirus (HPV). RSepitope-HPV16L1 (RSepitope fused at the "N-terminus" of HPV16L1) and HPV16L1-RSepitope (RSepitope fused at the "C-terminus" of HPV16L1) chimeras were constructed. After transfection of COS-7 cells with the recombinants, Western blot, RT-PCR, and immunofluorescence experiments confirmed that RSepitope-HPV16L1 could successfully express the corresponding mRNA and protein of RSepitope and HPV16L1, but the HPV16L1-RSepitope construct could not. A "prime-boost" immunization program was applied in mice to further evaluate the immune response elicited by the constructs, and the RSepitope-HPV16L1 immunization group produced the most significantly increased humoral and cellular immune responses (the highest RSepitope-specific IgG antibody level and the highest IFN-γ production, respectively), in which both elevated Th1 and Th2 immune responses were obtained. Moreover, the advantage of HPV16L1 as an epitope carrier was remarkable for RSepitope-HPV16L1, which induced a more prominent immunological response than RSepitope alone (without fusion with HPV16L1). Our research indicated that the N-terminus of HPV16L1 could be a better insertion site for enhancing target epitope immunogenicity, and our study offers a design for epitope vaccine of reasonable combination.

Animals , Antibody Formation , Epitopes , Immunization , Mice , Mice, Inbred BALB C , Toxoplasma , Vaccination , Vaccines, DNA
Article in Korean | WPRIM | ID: wpr-190352


A number of recombinant proteins isolated from cell sources are being produced for biopharmaceuticals. Although most biopharmaceuticals are highly purified, there is a safety concern that such recombinant products could be contaminated with impurities including adventitious virus, mycoplasma, endotoxin and oncogenic DNA. Residual DNA in recombinant biopharmaceuticals is a potential risk factor and must be evaluated and removed to meet the regulatory guidelines. Recombinant HPV type 16 L1 VLPs, recombinant protein produced in Spodoptera frugiperda (Sf) 9 insect cells, is a HPV subunit vaccine candidate which has been studied as a preventive vaccine of cervical cancers. In this study, we performed detection and quantification of residual cellular DNA in the production of recombinant HPV type 16 L1 VLPs. HPV-16 L1 VLPs were purified by processes including detergent lysis, sonication treatment, sucrose cushion centrifugation, CsCl equilibrium density centrifugation, and DNase treatment which was added to inactivate residual cellular DNA after CsCl centrifugation step. We have developed a precise assay based on a dot-blot hybridization using digoxigenin random primed labeling DNA probes for the detection and quantification of residual cellular DNA during the purification process and final products. Detection limit of residual cellular DNA was 0.1 ng in this assay and the amount of residual cellular DNA in the final product was 0.5 ng~1 ng per 100 microgram of protein. This study describes safer and more sensitive methods alternative to radioactive techniques employed for residual cellular DNA quantification of biopharmaceuticals produced by recombinant protein technology and presents method validation data demonstrating precision and reproducibility.

Centrifugation , Deoxyribonucleases , Detergents , Digoxigenin , DNA Probes , DNA , Human papillomavirus 16 , Insecta , Limit of Detection , Mycoplasma , Recombinant Proteins , Risk Factors , Sonication , Spodoptera , Sucrose
Article in Korean | WPRIM | ID: wpr-24708


Insect cell-derived biotechnological products have a potential for viral contamination from cell line sources themselves or from adventitious introduction of virus during production. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs) using Japanese encephalitis virus (JEV) and bovine viral diarrhea virus (BVDV) as relevant viruses. The downstream process for the production of recombinant HPV-16 L1 VLPs was sequentially carried out employing detergent lysis (NP-40/PBS), sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. Recombinant HPV-16 L1 capsid protein (56 kD) expressed in Sf9 cell culture was clearly detected by SDS-PAGE and Western blotting analysis. Each purification step was evaluated to determine reduction factor for viral clearance by infectivity assay. In individual purification steps, detergent treatment (0.50% v/v, NP-40/PBS) and CsCl equilibrium density centrifugation were found to be effective in JEV and BVDV clearance. Overall cumulative reduction factors of JEV and BVDV infectivity titer for the purification procedure implemented in this study were 12.53 and 10.05 log TCID(50)/pool, respectively. The results suggest that the purification procedure employed in this study for the HPV-16 L1 VLPs produced from recombinant baculovirus-infected Sf9 cells will be effective over 10 log TCID(50)/pool reduction factor in the clearance of enveloped, adventitious viruses with a buoyant density lower than approximately 1.23 g/ml.

Blotting, Western , Capsid Proteins , Cell Line , Centrifugation , Cesium , Detergents , Diarrhea , Electrophoresis, Polyacrylamide Gel , Encephalitis Virus, Japanese , Human papillomavirus 16 , Humans , Insecta , Sf9 Cells , Sonication , Sucrose