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Li X.-J.,Chengdu Ronsen Pharmaceuticals Co.
Chinese Journal of Biologicals | Year: 2015

Objective: To investigate the immune effect of AD2 site I (AD-2S1) of human cytomegalovirus (HCMV) glycoprotein B (gB) in mice. Methods: AD-2 and 4 × AD-2S1 nucleic acid sequences were cloned into plasmid pET42b-FljB containing flagellin gene. The constructed recombinant plasmids pET/FljB-AD-2 and pET/FljB-4 × AD-2S1 were transformed to E. coli Rosetta 2(DE3) for expression under induction of IPTG. The lysate supernatant of E. coli containing FljB-AD-2 and precipitate containing FljB-4 × AD-2S1 were purified by Superdex-200 gel filtration chromatography and, according to their protein contents and purities, prepared into adjuvant-free antigens FljB-AD-2 and FljB-4 × AD-2S1 as well as adjuvant-containing antigens FljB-AD-2 + Al (OH)3 and FljB-4 × AD-2S1 + A1(OH)3. BALB/c mice were immunized s. c. with the prepared antigens on days 0 and 21, using PBS as control. Serum samples were collected one week after the second immunization and determined for specific IgG, IgGl and IgG2a levels against AD-2S1 by ELISA, and for neutralizing antibody level against HCMV by neutralization test. Results: The purified FljB-AD-2 antigen, with a relative molecular mass of about 54 000, reached a purity of 88. 2% and a protein content of 0. 91 mg/ml. The relative molecular mass, purity and protein content of purified FljB-4 × AD-2S1 were about 50 000, 96. 8% and 0. 45 mg/ml respectively. As compared with those in control group, specific IgG antibodies against AD-2S1 were induced in immunized mice. The IgG levels were low in FljB-AD-2 and FljB-AD-2 + Al (OH)3 groups and high in FljB-4 × AD-2S1 and FljB-4 × AD-2S1 + Al(OH)3 groups, which showed significant difference in FljB-4 × AD-2S1 and FljB-AD-2 groups (P < 0. 05). FljB-4 × AD-2S1 induced IgGl and IgG2a in mice, while the level of the latter was significantly higher than that of the former (P < 0. 05). However, the IgGl and IgG2a levels induced by FljB-4 × AD-2S1 + Al (OH);, showed no significant difference (P > 0. 05). All the sennn neutralizing antibody levels of mice in test groups were not more than 1:4. Conclusion: Both FljB-4 × AD-2S1 and FljB-AD-2 induced specific IgG against AD-2S1, while induced no complement-independent neutralizing antibody in mice. Source

Song T.,Sichuan University | Cao Y.,Sichuan University | Xu H.,Sichuan University | Zhang W.,Sichuan University | And 3 more authors.
Journal of Bioscience and Bioengineering | Year: 2014

Agar is a polysaccharide polymer material, generally extracted from seaweed. Most agar degradation strains were isolated from seawater. In order to find new species resources and novel agarase from soil, an agar-degrading bacterium Paenibacillus sp. SSG-1 was isolated from soil. Agarase SSG-1a was purified to homogeneity by 30.2 fold with a yield of 4.8% through ammonium sulfate precipitation, DEAE FF chromatography and native-PAGE separation. The tandem mass spectrometry (MS/MS) results indicated that purified SSG-1a should be a novel β-agarase. The molecular mass of SSG-1a was estimated to be 77kDa. The optimal temperature and pH for SSG-1a were 50°C and pH 6.0, respectively. Moreover, SSG-1a was stable in pH range of 4.0-10.0 and at temperature up to 40°C. It could hydrolyze the β-1,4 linkage of agarose to produce neoagarohexaose (95mol%) and neoagarooctaose (5mol%). Metal ion Mn2+ and reducing reagents (β-Me and DTT) could increase its activity by 150% and 60%, respectively. © 2014 The Society for Biotechnology, Japan. Source

Zheng C.-G.,Chengdu Ronsen Pharmaceuticals Co. | Xu T.,Chengdu Ronsen Pharmaceuticals Co. | Rong X.-Z.,Chengdu Ronsen Pharmaceuticals Co. | Huang Y.,Chengdu Ronsen Pharmaceuticals Co.
Chinese Journal of Biologicals | Year: 2013

Objective: To develop, verify and preliminarily apply a fluorescent quantitative PCR (QPCR) method for porcine parvovirus (PPV). Methods: Primers were designed according to VP2 region of PPV NADL-2 strain in GenBank for PCR amplification using the extracted PPV nucleic acid as a template. The reaction system and condition were optimized, and the developed method was verified for specificity, reproducibility and sensitivity. The efficacies of removal of PPV in products by caprylic acid precipitation and by nanofilters manufactured by manufacturers L, P and S were determined by the developed method. Results: The optimized QPCR system consisted of pre-mixture of Eva Green, 0.5 μl of upstream and downstream primers and 1 μl of template DNA, supplemented with dH2O to a total volume of 15 μl. The reaction condition was optimized as follows: 95°C 3 min, 95°C 10 s and 60°C 10 s, 40 cycles in total. The log of template concentration showed good relationship to the cycle number (R2 = 0.999). The amplification efficacy was 102.5%. PPV was amplified specifically by the developed method, while no Pseudorabies virus (PRV), bovine parvovirus(BPV), minute virus of mice (MVM) or porcine kidney cell PK-15 were amplified. The CVs of intra- and inter-assays were 0.79%-2.81% and 1.23%-2.21% respectively, both of which were less than 5%, while the minimum detection limit wsa 102 copies / μl. Caprylic acid precipitation decreased the PPV concentration in test samples by 5 Logs. Both the filtration volume and efficacy of 20 nm membrane filter manufactured by various manufacturers showed significant difference. However, the filter manufacturer manufactured by manufacturer S showed satisfactory filtration efficacy, which decreased the PPV titer in test samples by 4 Logs. Conclusion: The fluorescent QPCR method for PPV was developed successfully, which laid a foundation of rapid and accurate evaluation of removal efficacy of PPV by virus removal procedure. Source

Liu L.-J.,Chengdu Ronsen Pharmaceuticals Co. | Yang C.,Chengdu Ronsen Pharmaceuticals Co. | Qin T.-T.,Chengdu Ronsen Pharmaceuticals Co. | Liu R.-X.,Chengdu Ronsen Pharmaceuticals Co. | And 3 more authors.
Chinese Journal of Biologicals | Year: 2011

Objective: To develop a method for detection of neutralizing antibody titer against human cytomegalovirus (HC MV). Methods: Serially diluted test samples were mixed with HCMV at an equal volume respectively for neutralization for 1 h, inoculated to MRC-5 cells and incubated at 37°C overnight, then added with anti-HCMV IE1 McAb, Biotin-SP-Goat anti Mouse IgG, Peroxidase-Labeled Streptavidin and substrate in turn. The nuclei of cells infected with HCMV were stained specifically and observed for location. The infected cells in each well were counted by microscopy. The neutralizing titers of test samples were calculated by Reed-Muench method. The developed method was verified for precision, serviceability and specificity. Results: MRC-5 cells infected with HCMV showed typical CPEs, of which the nuclei were stained specifically, while no stained particles were observed in uninfected cells. The variation coefficient of detection result by the developed method in reproducibility test was 3.26%, while that in intermediate precision test was 1.05%. After the test samples were neutralized with an equal volume of neutralizing virus liquid at 37°C for 55, 60 and 65 min, the neutralizing antibody titers showed no significant difference (P > 0.05). No specifically stained particles were observed in the MRC-5 cells infected with varicella virus, or in the plasma, IVIG or CMV-IVIG samples after co-incubation with MRC-5 cells. Conclusion: A method for detection of neutralizing antibody titer against HCMV was developed, which was rapid, objective, and of high precision, serviceability and specificity, and might be used for the detection of neutralizing antibody titer against HCMV in plasma and IVIG. Source

Qin T.-T.,Chengdu Ronsen Pharmaceuticals Co. | Liu B.,Chengdu Ronsen Pharmaceuticals Co. | Liu R.-X.,Chengdu Ronsen Pharmaceuticals Co. | Yang S.,Chengdu Ronsen Pharmaceuticals Co. | And 3 more authors.
Chinese Journal of Biologicals | Year: 2015

Objective To optimize the condition for culture of human cytomegalovirus (HCMV) and develop a method for rapid detection of HCMV titer. Methods Human embryo lung diploid cell line MRC-5 was used for vims subculture and determination of vims titer, and the culture conditions including MOI, temperature, protective agent were optimized. The developed micro-neutralization assay-infected nuclei staining test was used for the determination of vims titer, and compared with plaque formation test and micro-CPE test. The vims titer was calculated by Behrens-Karher method. Results Obvious CPE was observed in the HCMV-infected MRC-5 cells after culture at 37°C. The vims titer basically readied a platform when the MOI was 1.0, which was stable if 1% human serum albumin or 10% fetal bovine semm (FBS) as a protective agent was added during the harvest process. The vims titer of the same sample determined by plaque formation test, micro-CPE test and infected nuclei staining were 5.707 lgPFU/ml, 5.633 lgCCID50/ml and 5.667 lgCCID50/ml, which showed no significant difference (P > 0.05). Conclusion The optimal condition for culture of HCMV in MRC-5 cells was as follows: HCMV was inoculated at a MOI of 1.0 and incubated at 37°C, using 1% human semm albumin or 10% FBS as a protective agent. The developed infected nuclei staining method may be used for the rapid detection of HCMV titer, which provides a novel technical tool for development and effect evaluation of anti-vims material. Source

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