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Objective To develop a relative fluorescent quantitative PCR method for mitochondrial antiviral signaling protein (MAVS) SYBR Green I in Guinea pigs infected with avian influenza virus (AIV) subtype H5N1, and determine the expression levels of MAVS in lung tissue of Guinea pigs before and after infection. Methods RNA was extracted from the lung tissue of Guinea pigs and reversely transcribed into cDNA, which was 10-fold serially diluted to 9 concentrations (1. 00 E + 10 ∼ 1. 00 E + 02 copies/ μl) for PCR amplification. A standard curve was plotted using β-actin as an internal reference. The amplification efficiencies of two genes were compared, and the method was verified for specificity, reproducibility and sensitivity. The expression levels of MAVS in lung tissue of Guinea pigs before and after challenge with H5N1 AIV. Results The dilution range of cDNA template was 1. 00 E + 09 ∼ 1. 00 E + 04. The difference between slopes of standard curves of MAVS and β-actin genes was less than 0. 1, with a R2 value of 0. 999, while the amplification efficiencies were 100. 2% and 100. 3% respectively. Either of the melting curves of the two genes showed a single peak, and clear target fragments were amplified, no non-specific amplification was observed. Both the coefficients of variation (CV) of Ct values of the two genes were less than 10%. The sensitivity of the developed method was 1. 00 E + 03 copies/μl. The expression level of MAVS in lung tissue of Guinea pigs infected with H5N1 AIV was down-regulated. Conclusion A relative fluorescent quantitative PCR method for MAVS SYBR Green I in Guinea pigs infected with AIV subtype H5N1 was successfully developed, and infection with H5N1 AIV decreased the expression level of MAVS in Guinea pigs.


He W.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2014

Objective: To study the effect of recombinant endostatin (rhES) on endothelial cells by atomic force microscope (AFM). Methods: The proliferative activities of human umbilical vein endothelial ECV304 cells treated with rhES at various concentrations (0.05-2.4 μg / ml) were determined by MTT assay. ECV304 cells were treated with 0.8 and 2 μg / ml rhES respectively, then observed for overall morphology by AFM, and for local morphology on surface by SPI 3800 New DFM. Results: The rhES inhibited the proliferation of ECV304 cells significantly (P < 0.001), and decreased the thickness of adherent ECV304 cells, both in dose-dependent modes. The surface of cells after treatment with rhES changed from smooth to rough, on which some small processes were formed. However, the surface structure of ECV304 cells after treatment with rhES showed an irregular change. Conclusion: The samples for atomic force microscopy were easy to prepare, while the resolving power was high, indicating that the method was suitable for in situ observation of adherent cells.


Cao Y.-F.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013

Objective: To express fusion human insulin-like growth factor-1 (hIGF-1) in prokaryotic cells, and purify and identify the expressed product. Methods: The sequence of natural hIGF-1 gene was subjected to samesense mutation according to the E. coli preferred codons, synthesized amplified by PCR and cloned into vector pET48b(+). The construct recombinant plasmid pET48b-Trx A-hIGF-1 was transformed to E. coli BL21 (DE3) for expression under induction of IPTG. The expressed fusion protein Trx A-hIGF-1 was purified by nickel ion affinity chromatography, digested with enterokinase and identified by SDS-PAGE. Results: Recombinant plasmid pET48b-Trx A-hIGF-1 was constructed correctly as proved by colony PCR and sequencing. The expressed fusion protein, with a relative molecular mass of about 26 000, contained about 40% of total somatic protein and mainly existed in a soluble form, of which the proportion of soluble protein to total target protein was not less than 90%. The fusion protein reached a purity of not less than 90% and a concentration of 0.5 mg/ml, which was digested with enterokinase into hIGF-1 with a relative molecular mass of about 8 000 and Trx A with a relative molecular mass of about 18 000. The hIGF-1 showed specific binding to rabbit anti-hIGF-1 polyclonal antibody. Conclusion: The hIGF-1 fusion protein was successfully expressed, which laid a foundation of study on biological activity and large-scale production of hIGF-1.


Xia Q.-J.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013

Objective: To prepare the immunoglobulin of yolk(IgG) against hepatitis A virus (HAV) and develop a double antibody sandwich ELISA method for HAV antigen content in HA vaccine. Methods: IgY against HAV was prepared by immunizing healthy laying hens with purified HAV, purified by several steps, then determined for protein content by ultraviolet spectrophotometry, for relative molecular mass and purity by reduced SDS-PAGE, and for titer, specificity as well as heat, acid and base stabilities by indirect ELISA. A double antibody sandwich ELISA method was developed using the purified IgY as coating antibody, and HRP-labeled monoclonal antibody against HAV as the secondary antibody, and used for determination of HAV antigen content in HA vaccine during production, and the results were compared with those by commercial ELISA kit. Results: The IgY concentration after purification by affinity chromatography was 3.67 mg/ml. The relative molecular masses heavy and light chains of IgY were 66 000 and 27 000 respectively, while the purity was 96.78%, and the titer was 1:16 000 at most. The purified IgY showed only positive reaction with HAV, while showed no reactions with poliovirus or enterovirus 71 (EV71). Treatment at 25-70°C for 15 min or with Tris-HCl buffer at pH 3.0-10.0 for 2 h showed no significant influence on the titer of IgY. The log of HAV concentration at a range of 15.62-2 000.00 ng/ml was linearly related to the A450 value, with a R2 value of 0.935. The minimum detection limit of the developed ELISA method was 7.81 ng/ml, while the coefficient of relationship of detection result was 0.952 7 to that by commercial kit. Conclusion: The prepared IgY showed high concentration, purity, titer, specificity and stability, by which the developed double antibody sandwich ELISA method might be used for determination of HAV antigen content in HA vaccine during production.


Zhao D.-P.,Changchun Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013

Objective: To evaluate the safety and immunogenicity of influenza A H1N1 virus split vaccine in animals. Methods: Abnormal toxicity test was performed according to the requirements in Chinese Pharmacopoeia (Volume III, 2005 edition). Kunming mice were randomly divided into seven groups, ten for each. The mice in three test groups were injected i. m. with influenza A H1N1 virus split vaccine at hemagglutinin (HA) contents of 15, 30 and 45 μg/0.5 ml, while those in three parallel control groups with seasonal influenza H1N1 virus split vaccine at the corresponding HA contents, and those in negative control group with PBS, respectively. The vaccines were given at a dosage of 0.1 ml for 2 times at an interval of 21 d. Venous blood samples were collected 0, 21, 28, 35 and 42 d after the first immunization, from which sera were separated and determined for hemagglutination inhibition (HI) antibody level. Results: The abnormal toxicity test result met the requirements in Chinese Pharmacopoeia (Volume III, 2005 edition). The serum antibody levels in three test groups 21, 28, 35 and 42 d after the first immunization increased significantly as compared with those in negative control group, which reached a high level on 21 d and a peak value on 28 d (each P < 0.01). However, the antibody levels in test groups 0, 21, 28 and 42 d were higher than those immunized with the control vaccine at the corresponding HA contents (P > 0.05). The serum HI antibody positive rates of mice 21 d after immunization with test vaccine at HA contents of 15, 30 and 45 μg/0.5 ml were 80%, 90% and 90%, while the protection rates were 100%, 90% and 90%, respectively, which showed no significant difference with those in parallel control groups (each P < 0.05). The HI antibody positive rates of mice 28, 35 and 42 d after immunization with 15 and 45 μg/0.5 ml were more than 80%, while that with 30 μg/0.5 ml was slightly low. However, the protection rates of HI antibody in three test groups were 90%-100%. Conclusion: Influenza A H1N1 virus split vaccine showed good safety and immunogenicity in Kunming mice.

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