Liu C.,Jilin University |
Liu C.,Changchun Institute of Biological Products |
Yang G.,Shanghai JiaoTong University |
Wu L.,Jilin University |
And 4 more authors.
Protein and Cell | Year: 2011
The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/T560W was generated, which had the highest catalytic efficiency (kcat/Km) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes. © 2011 Higher Education Press and Springer-Verlag Berlin Heidelberg.
Liang X.-F.,Centers for Disease Control and Prevention |
Wang H.-Q.,Centers for Disease Control and Prevention |
Wang J.-Z.,National Institute for the Control of Pharmaceuticals and Biological Products |
Fang H.-H.,National Institute for the Control of Pharmaceuticals and Biological Products |
And 19 more authors.
The Lancet | Year: 2010
Background: The current influenza pandemic calls for a safe and effective vaccine. We assessed the safety and immunogenicity of eight formulations of 2009 pandemic influenza A H1N1 vaccine produced by ten Chinese manufacturers. Methods: In this multicentre, double-blind, randomised trial, 12 691 people aged 3 years or older were recruited in ten centres in China. In each centre, participants were stratified by age and randomly assigned by a random number table to receive one of several vaccine formulations or placebo. The study assessed eight formulations: split-virion formulation containing 7·5 μg, 15 μg, or 30 μg haemagglutinin per dose, with or without aluminium hydroxide adjuvant, and whole-virion formulation containing 5 μg or 10 μg haemagglutinin per dose, with adjuvant. All formulations were produced from the reassortant strain X-179A (A/California/07/2009-A/PR/8/34). We analysed the safety (adverse events), immunogenicity (geometric mean titre [GMT] of haemagglutination inhibition antibody), and seroprotection (GMT ≥1:40) of the formulations. Analysis was by per protocol. Two sites registered their trial with ClinicalTrials.gov, numbers NCT00956111 and NCT00975572. The other eight studies were registered with the State Food and Drug Administration of China. Findings: 12 691 participants received the first dose on day 0, and 12 348 participants received the second dose on day 21. The seroprotection rate 21 days after the first dose of vaccine ranged from 69·5% (95% CI 65·9-72·8) for the 7·5 μg adjuvant split-virion formulation to 92·8% (91·9-93·6) for the 30 μg non-adjuvant split-virion formulation. The seroprotection rate was 86·5% (796 of 920; 84·1-88·7) in recipients of one dose of the 7·5 μg non-adjuvant split-virion vaccine compared with 9·8% (140 of 1432; 8·3-11·4) in recipients of placebo (p<0·0001). One dose of the 7·5 μg non-adjuvant split-virion vaccine induced seroprotection in 178 of 232 children (aged 3 years to <12 years; 76·7%, 70·7-82·0), 211 of 218 adolescents (12 years to <18 years; 96·8%, 93·5-98·7), 289 of 323 adults (18-60 years; 89·5%, 85·6-92·6), and 118 of 147 adults older than 60 years (80·3%, 72·9-86·4), meeting the European Union's licensure criteria for seroprotection in all age-groups. In children, a second dose of the 7·5 μg formulation increased the seroprotection rate to 97·7% (215 of 220, 94·8-99·3). Adverse reactions were mostly mild or moderate, and self-limited. Severe adverse effects occurred in 69 (0·6%, 0·5-0·8) recipients of vaccine compared with one recipient (0·1%, 0-0·2) of placebo. The most common severe adverse reaction was fever, which occurred in 25 (0·22%; 0·14-0·33) recipients of vaccine after the first dose and four (0·04%; 0·01-0·09) recipients of vaccine after the second dose compared with no recipients of placebo after either dose. Interpretation: One dose of non-adjuvant split-virion vaccine containing 7·5 μg haemagglutinin could be promoted as the formulation of choice against 2009 pandemic influenza A H1N1 for people aged 12 years or older. In children (aged <12 years), two 7·5 μg doses might be needed. Funding: Sinovac Biotech, Hualan Biological Bacterin, China National Biotec Group, Beijing Tiantan Biological Products, Changchun Institute of Biological Products, Changchun Changsheng Life Sciences, Jiangsu Yanshen Biological Technology Stock, Zhejiang Tianyuan Bio-Pharmaceutical, Lanzhou Institute of Biological Products, Shanghai Institute of Biological Products, and Dalian Aleph Biomedical. © 2010 Elsevier Ltd. All rights reserved.
Guo X.,Changchun Institute of Biological products
Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology | Year: 2011
To express St. Louis encephalitis virus-like particles in mammalian cells, it will provide a prerequisite for further immunological diagnostic studies. 293T-cell were transiently transfected with recombinant PreM-E plasmid. Expression and antigenicity of the purified protein were determined by transmission electron microscope (TEM), Western-Blot, immunofluorescence assay and ELISA. Recombinant subviral particles, about 50 nm in diameter, were observed by TEM in the supernatant of transfected cells. The results of Western-Blot, IFA and ELISA showed the recombinant proteins retained immunoreactivity similar to those of native virus proteins. St. Louis encephalitis virus-like particles has good antigenicity and physical appearance. It will provide a prerequisite for further immune diagnostic reagent.
Chen W.-J.,Changchun Institute of Biological Products
Chinese Journal of Biologicals | Year: 2010
Objective: To investigate the effect of boiling chick eggs on the activity of specific IgY and lay a foundation of prevention and treatment of infectious diseases in digestive tract by edible specific IgY. Methods: Specific IgYs were prepared by immunizing hens with Shigella flexneri and rotavirus Wa strain respectively. The eggs containing specific IgYs were boiled for various minutes and observed for the characters of egg white and yolk. Specific IgY was extracted from the eggs boiled for 4 and 6 min and those stored at 4°C as control respectively, and determined for activity by agglutination test, inhibition test in suckling mice and ELISA. Results: The egg white and yolk of eggs boiled for 4 min were easily to be separated, in which the activity of specific IgY showed no significant difference with that in the eggs stored at 4°C. However, after being boiled for 6 min, all the egg white were coagulated, and the activity of specific IgY decreased significantly. Conclusion: Boiling for 4 min showed no significant effect on the activity of specific IgY in eggs which might be eaten directly to prevent and treat the infectious diseases in digestive tract.
Ren J.,Jilin University |
Sun L.,Jilin University |
Yang L.,Jilin University |
Wang H.,Jilin University |
And 6 more authors.
Vaccine | Year: 2010
In order to develop novel canine CpG ODNs as adjuvant for rabies vaccine of dog use, a panel of CpG ODNs containing different CpG motifs was designed and screened for their ability to induce the proliferation of canine splenocytes. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, were outshined with stronger ability to activate canine immune cells. The CpG ODNs were tested for their adjuvant activity for rabies vaccine in mice and dogs. It was found that YW07 could facilitate the rabies vaccine to induce more vigorous and long-lasting specific antibody response in mice and dogs, respectively. These findings suggest that YW07, a canine favored CpG ODN, could be used as a novel adjuvant for developing more efficient rabies vaccine of dog use. © 2010 Elsevier Ltd. All rights reserved.
Zhuang M.-X.,Changchun Institute of Biological Products
Chinese Journal of Biologicals | Year: 2010
Objective: To observe the safety and immunogenicity of domestic influenza A H1N1 virus cleavage vaccine. Methods: A clinical trial was performed according to a random, control and blind principle. Each of the aged, adolescent and children groups consisted of 220 subjects of whom 110 were inoculated with 15 μg of influenza A H1N1 virus cleavage vaccine and the other 110 with 30 μg, for 2 times by a schedule of 0 and 21 d. Adult group consisted of 330 subjects of whom 110 were inoculated with 15 μg of influenza A H1N1 virus cleavage vaccine, 110 with 30 μg, and 110 with placebo, for 2 times by a schedule of 0 and 21 d. Another 220 adults were inoculated with 15 and 30 μg of influenza A H1N1 virus cleavage vaccine, 110 for each, by a schedule of 0 and 28 d. The total, systemic and local adverse rates, HI antibody positive conversion rates, protective rates as well as GMT level and increasing folds of HI antibody in various groups after inoculation were observed. Results: The total adverse reaction rate of the 1210 subjects was 21.8%, most of which were of degree 1, no adverse reactions of degree 3 or above, other abnormal reactions or severe adverse events were observed. The HI antibody positive conversion rate and protective rate induced by 30 μg of vaccine showed no significant difference with those induced by 15 μg. No significant differences were observed in the HI antibody positive conversion rate, protective rate, GMT levels and increasing folds after inoculation with the first dose of vaccine at the same dosage by different schedule. Conclusion: Domestic influenza A H1N1 virus cleavage vaccine showed good safety and immunogenicity. The inoculation with one dose of 15 μg vaccine by a schedule of 0 and 21 d induced satisfactory immune effect in the population aged 12-60 years.
Yang Y.,Changchun Institute of Biological Products
Chinese Journal of Biologicals | Year: 2011
Objective: To prepare rabies vaccine for human use with Vero cells in basket bioreactor. Methods: Rabies vaccine virus strain aGV was cultured in Vero cells in 14 L bioreactor (500 g of disk carriers in basket) by perfused culture. The continuously harvested virus liquid was concentrated by ultrafiltration, inactivated, and purified by column chromatography, then added with stabilizer and prepared into freeze-dried vaccine. The prepared vaccine was determined for titers before and after lyophilization and after storage at 37°C for 28 d. Results: The cell density reached 1. 0 × 107 cells/ml at most, and the titer of harvested virus liquid was 7. 5 × 106-3. 4 × 108 FFU/ml. After purification, the total protein content, residual Vero cell protein content, residual Vero cell DNA content and vaccine titer were less than 80 μg/0. 5 ml, less than 3. 5 μg/0. 5 ml, less than 100 pg/0. 5 ml and more than 5. 0 IU/0. 5 ml. Conclusion: Basket bioreactor may be used for preparation of rabies vaccine for human use with high quality.
Yu Z.,Chinese Academy of Sciences |
Yu Z.,Military Veterinary Research Institute of Academy of Military Medical science |
Cheng K.,Military Veterinary Research Institute of Academy of Military Medical science |
Cheng K.,Shandong Academy of Agricultural Sciences |
And 18 more authors.
Veterinary Microbiology | Year: 2014
Avian influenza viruses (AIVs) are globally important contagions. Several domestic mammals can be infected with AIVs and may play important roles in the adaptation and transmission of these viruses in mammals, although the roles of wild mammals in the natural ecology of AIVs are not yet clear. Here, we performed a serological survey of apparently healthy Plateau pikas at Qinghai Lake in China to assess the prevalence of exposure to AIVs. Ninety-two of 293 (31%) of wild Plateau pikas possessed serum antibodies against a lowly pathogenic avian influenza (LPAI) H9N2 virus. Experimental inoculation of Plateau pikas with a LPAI H9N2 virus resulted in productive viral replication in respiratory tissues without prior adaptation. Our findings suggest that Plateau pikas represent a natural mammalian host to H9N2 AIVs and may play a role in the ongoing circulation of H9N2 viruses at Qinghai Lake in China. Surveillance for AIV infection in Plateau pika populations and other mammals that have close contact with the Plateau pikas should be considered. © 2014 Elsevier B.V.
PubMed | Changchun Institute of Biological products
Type: Journal Article | Journal: Zhonghua shi yan he lin chuang bing du xue za zhi = Zhonghua shiyan he linchuang bingduxue zazhi = Chinese journal of experimental and clinical virology | Year: 2012
To express St. Louis encephalitis virus-like particles in mammalian cells, it will provide a prerequisite for further immunological diagnostic studies.293T-cell were transiently transfected with recombinant PreM-E plasmid. Expression and antigenicity of the purified protein were determined by transmission electron microscope (TEM), Western-Blot, immunofluorescence assay and ELISA.Recombinant subviral particles, about 50 nm in diameter, were observed by TEM in the supernatant of transfected cells. The results of Western-Blot, IFA and ELISA showed the recombinant proteins retained immunoreactivity similar to those of native virus proteins.St. Louis encephalitis virus-like particles has good antigenicity and physical appearance. It will provide a prerequisite for further immune diagnostic reagent.
PubMed | Jilin University, Changchun Institute of Biological Products and Wuhu Kang Wei Biological Technology
Type: | Journal: Analytical biochemistry | Year: 2014
Egg allergy is an important public health and safety concern, so quantification and administration of food or vaccines containing ovalbumin (OVA) are urgently needed. This study aimed to establish a rapid and sensitive magnetic particles-chemiluminescence enzyme immunoassay (MPs-CLEIA) for the determination of OVA. The proposed method was developed on the basis of a double antibodies sandwich immunoreaction and luminol-H2O2 chemiluminescence system. The MPs served as both the solid phase and separator, the anti-OVA MPs-coated polyclonal antibodies (pAbs) were used as capturing antibody, and the horseradish peroxidase (HRP)-labeled monoclonal antibody (mAb) was taken as detecting antibody. The parameters of the method were evaluated and optimized. The established MPs-CLEIA method had a linear range from 0.31 to 100ng/ml with a detection limit of 0.24ng/ml. The assays showed low reactivities and less than 5% of intraassay and interassay coefficients of variation (CVs), and the average recoveries were between 92 and 97%. Furthermore, the developed method was applied in real samples analysis successfully, and the correlation coefficient with the commercially available OVA kit was 0.9976. Moreover, it was more rapid and sensitive compared with the other methods for testing OVA.