Chen L.,Shanghai Zerun Biotechnology Co.
Bing du xue bao = Chinese journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] | Year: 2012
The recombinant plasmid carrying the gene encoding 3C protease of Enterovirus 71 (EV71) was constructed, the recombinant protein was then expressed and purified, the functional activity was also measured. Firstly, the 3C protease gene was inserted into pET28a vector, the constructed recombinant plasmid was transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The expressed protein was purified by affinity chromatography (Ni-NTA) and the N-terminus His-tag was cleaved by enterokinase from 3C protease. The activity of 3C protease was evaluated with fluorescent peptide substrates. It was verified by restriction analysis and sequencing that recombinant plasmid pET28a-3C was constructed correctly and functionally expressed in E. coli BL21 (DE3) resulting in the production of recombinant 3C protease with a size of 22kD. Both His-tag and non-His-tag (cleaved by enterokinase) 3C protease exhibited similar enzyme activity to 3B-3C fluorescent peptide with Km, Vmax and Kcat values of 22 microM, 434nM. Min(-1) and 0.0669 Min(-1), respectively. The optimial pH and temperature were 7.0 and 30-37 degrees C, respectively. The acquirement of recombinant purified 3C protease with high activity has paved the way of further studies on anti-viral inhibitors, structural protein assembly, vaccine development and detection methods of EV71.
Zhou C.-M.,Shanghai Zerun Biotechnology Co.
Electrophoresis | Year: 2013
CIEF with whole-column imaging detection (WCID) can be a useful tool for the characterization and identification of human papillomavirus (HPV). This article is the initial report of the determination of the pI of HPV by CIEF-WCID method. In this study, components of the assay selected for optimization were ampholytes, additives, methylcellulose concentration, HPV concentration, salt concentration, and focusing time. Then the optimization CIEF-WCID method was validated for HPV 16L1 and HPV 18L1. As a result, a precise method to analyze the pI values of HPVs was achieved with RSD < 1.0%. The HPV peak pattern was reproducible. CIEF-WCID had great potential for HPV quality control, as WCID eliminated the mobilization step required by the conventional single-point detection. In the example, the five HPVs displayed pI values of 8.43 ± 0.06 (n = 10; HPV 6L1), 8.70 ± 0.04 (n = 10; HPV 11L1), 7.94 ± 0.05 (n = 18; HPV 16L1), 7.57 ± 0.04 (n = 18; HPV 18L1), and 8.45 ± 0.05 (n = 10; HPV 58L1). This CIEF-WCID platform could be a powerful analytical tool for characterization, process development support, release testing, and stability study in pharmaceutical industry. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Shanghai Zerun Biotechnology Co. | Date: 2014-06-05
The present invention discloses a codon-optimized gene encoding major capsid protein L1 of human papilloma virus, which is capable, after transduced into a yeast cell, of efficiently expressing the major capsid protein L1 of human papilloma virus. The present invention also discloses an immunogenic macromolecule which is essentially produced by expression of said codon-optimized gene encoding the major capsid protein L1 of human papilloma virus in a yeast cell. The present invention further discloses the use of said immunogenic macromolecule and a composition comprising said immunogenic macromolecule.
Ma X.-X.,Shanghai Zerun Biotechnology Co. |
Chen H.-Y.,Shanghai Zerun Biotechnology Co. |
Zeng X.-F.,Shanghai Zerun Biotechnology Co.
Chinese Journal of Biologicals | Year: 2015
Objective To develop a method for in vitro detection of cellular immunity with macrophages differentiated from THP-1 cells. Methods Reference and sample of bulk of therapeutic HPV vaccine were diluted with RPMI1640 medium containing phorbol myristate acetate (PMA), then added with THP-1 cells, and incubated at 37 °C in a 5% carbon dioxide incubator, using those without addition with THP-1 cells as control. The supernatant was collected and determined for tumor necrosis factor-α (TNF-α) content, based on which the in vitro relative potency was calculated. The cell density (2.0 × 105, 5.0 × 105 and 10.0 × 105 cells/ml), final PMA concentration (2.8, 8.3, 25, 75 and 225 ng/ml) and incubation time (2, 3 and 4 d) were optimized, and the method was verified for effectiveness by ELISPOT method with the bulk of prophylactic HPV vaccine. Results The optimal cell density, final PMA concentration and incubation time for the developed method were 5.0 × 105 cells/ml, 25 ng/ml and 3 d respectively. Verification result showed that the method was effective and feasible. Conclusion A method for in vitro detection of cellular immunity with macrophages differentiated from THP-1 cells was developed, which provided a platform for in vitro detection of cellular immunity of therapeutic vaccines.
Wang G.,Shanghai Zerun Biotechnology Co. |
Lv P.,Shanghai Zerun Biotechnology Co. |
Zhu H.,Shanghai Zerun Biotechnology Co. |
Wang H.,Shanghai Zerun Biotechnology Co.
Journal of Biomedicine and Biotechnology | Year: 2011
Human papillomavirus (HPV) L1 virus-like particles (VLPs) were proven an effective vaccine candidate to prevent against HPV-16 and -18 infections. In order to evaluate the potency of our produced HPV-16 and -18 L1 VLPs-based vaccine candidates, also to quantify neutralizing antibodies induced by them, a 2-plex Luminex-based competitive immunoassay was developed. Unlike the published paper, the no-biotin conjugated neutralizing mAbs spiked normal human serum (NHS) was used for standard curve preparation, while phycoerythrin (PE) was not labeled directly to neutralizing mAbs for signaling. After the coupling optimization of VLPs to microspheres and the neutralizing mAbs biotinylation, the 2-plex standard curve was prepared with good fit and high dynamic range. In addition, no cross-reactivity was also confirmed. The 2-plex Luminex-based immunoassay represents good potential not only for vaccine candidate's evaluation but also for its further clinical use. Copyright © 2011 Pin Lv et al.