PubMed | University of Sichuan, Chengdu Institute of Biological Products Co. and Chengdu Medical College
Type: | Journal: Protein expression and purification | Year: 2015
Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli.
Xie M.-C.,Chengdu Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2012
Objective: To prepare Pseudomonas aeruginosa - IATS (International Antigen Typing System) 3 O-specific polysaccharide-tetanus toxoid conjugate vaccine and analyze its immune characteristics. Methods: Lipopolysaccharide (LPS) was isolated from P. aeruginosa IATS 3 by hot phenol method, in which the lipid A was removed. The obtained O-specific polysaccharide (O-SP) was purified and ac-tivated with CDAP, then conjugated to tetanus toxoid (TT) in mediation of EDAC using adipic acid dihydrazide (ADH) as a linker. The prepared conjugate vaccine was analyzed for physic-chemical property, immunogenicity and immune protection. Results: The yield of O-SP was equivalent to 30% of hydrolyzed LPS. Both the protein and nucleic acid contents in the obtained O-SP was less than 1.0%. The derivation of O-SP was 3.83%, while the binding rate to TT was about 40%, and the ratio (w/w) of polysaccharide to protein was 1:2.18. O-SP-TT vaccine induced high titer IgG in mice, which was significantly higher than that induced with O-SP (P < 0.05). The conjugate vaccine protected mice against the challenge with live bacteria at dosages of 5 and 10 LD50. Conclusion: The prepared O-SP-TT conjugate showed high immunogenicity and immune protection, which might be used as an effective vaccine for prevention and treatment of infection with P. aeruginosa IATS 3.
Duan W.,CAS Institute of Biophysics |
Zhou J.,CAS Institute of Biophysics |
Zhou J.,Chengdu Institute of Biological Products Co. |
Li W.,CAS Wuhan Institute of Virology |
And 6 more authors.
Protein and Cell | Year: 2013
The activation and deactivation of Ca2+- and calmodulindependent neuronal nitric oxide synthase (nNOS) in the central nervous system must be tightly controlled to prevent excessive nitric oxide (NO) generation. Considering plasma membrane calcium ATPase (PMCA) is a key deactivator of nNOS, the present investigation aims to determine the key events involved in nNOS deactivation of by PMCA in living cells to maintain its cellular context. Using time-resolved Förster resonance energy transfer (FRET), we determined the occurrence of Ca2+-induced protein-protein interactions between plasma membrane calcium ATPase 4b (PMCA4b) and nNOS in living cells. PMCA activation significantly decreased the intracellular Ca2+ concentrations ([Ca2+]i), which deactivates nNOS and slowdowns NO synthesis. Under the basal [Ca2+]i caused by PMCA activation, no protein-protein interactions were observed between PMCA4b and nNOS. Furthermore, both the PDZ domain of nNOS and the PDZ-binding motif of PMCA4b were essential for the protein-protein interaction. The involvement of lipid raft microdomains on the activity of PMCA4b and nNOS was also investigated. Unlike other PMCA isoforms, PMCA4 was relatively more concentrated in the raft fractions. Disruption of lipid rafts altered the intracellular localization of PMCA4b and affected the interaction between PMCA4b and nNOS, which suggest that the unique lipid raft distribution of PMCA4 may be responsible for its regulation of nNOS activity. In summary, lipid rafts may act as platforms for the PMCA4b regulation of nNOS activity and the transient tethering of nNOS to PMCA4b is responsible for rapid nNOS deactivation. © 2013 Higher Education Press and Springer-Verlag Berlin Heidelberg.
Liu J.,Chengdu Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To remove the residual Vero cell DNA in freeze-dried rabies vaccine for human use by combination of various methods. Methods: The residual Vero cell DNA in rabies virus liquid cultured in Vero cells was removed by the combination of clarification with filter element (filter plate), treatment with protamine, treatment with endonuclease and Sepharose 4FF chromatography or the combination of above-mentioned procedures except treatment with protamine, based on which the residual Vero cell DNA content, antigen content, residual endonuclease content and vaccine potency were determined. Results: The loss rate of antigen in virus liquid clarified with 0.45 μm UB glass fiber was low. However, the loss rate of antigen in virus liquid in which Vero cell DNA was removed by protamine was high. The residual Vero cell DNA content in virus liquid after treatment with endonuclease was still more than 1 ng / ml. Sepharose 4FF chromatography removed the endonuclease and Vero cell DNA in virus liquid after concentration and endonuclease treatment. After removal of residual Vero cell DNA by combination of various methods, the residual Vero cell DNA content was less than 100 pg / ml, while the vaccine potency of not less than 4. 0 IU / ml, which met the requirements in Chinese Pharmacopoeia (Volume III, 2010 edition). Conclusion: The residual Vero cell DNA content in freeze-dried rabies vaccine for human use was removed effectively by combination of various methods, and the vaccine potency met the requirements in Chinese Pharmacopoeia (Volume III, 2010 edition).
Yang H.-Q.,Chengdu Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To analyze the stabilities of gene and amino acid sequences of E protein of 23 batches of live attenuated Japanese encephalitis (JE) vaccine exported to Korea in 2003-2011. Methods: Viral RNAs were extracted from 23 bathes of live attenuated JE vaccine exported to Korea as well as master and working seeds for production, from which E gene fragments were amplified by RT-PCR and sequenced. The amino acid sequences at 8 key sites of E gene were analyzed and compared with those of attenuated strain SA14-14-2 in GenBank by using AlignX software. The 23 bathes of vaccine as well as master and working seeds for production of the vaccine were determined for titers. Results: The E gene sequences of 23 batches of vaccine as well as master and working seeds for production were completely consistent with those of SA14-14-2 strain in GenBank, which consisted of 1 500 nucleotides and encoded 500 amino acids, indicating a homology of 100%. However, none of the amino acids at 8 key sites closely related to attenuated virulence showed change, indicating consistencies of 100% to those of master and working seeds for vaccine production as well as attenuated JE virus strain SA14-14-2 (D90195) in GenBank.. The titers of master and working seeds for production of the 23 batches of vaccine were 6.72-7.17 lgPFU/ml, while those of the vaccine were 7.02-7.61 lgPFU/ml. Conclusion: The 23 batches of JE live attenuated vaccine exported to Korea produced during 2003-2011 showed high genetic stability and consistency, which proved that the vaccine was safe and reliable.
Yang H.-Q.,Chengdu Institute of Biological Products Co.
Chinese Journal of Biologicals | Year: 2013
Objective: To investigate the genetic stability of measles virus Shanghai-191 strain after continuous subculture in chick embryo fibroblasts. Methods: The seeds from primary seed lot of measles virus Shanghai-191 strain (P25) strain was subcultured in chick embryo fibroblasts for two passages (P27) and used as mater seed lot, for further three passages (P30) as working seed lot, and further subcultured to passages 33 (P33) and 35 (P35). The genome of various passages were sequenced, while the titers were determined. Results: The titer of Shanghai-191 strain from P25 to P35 were maintained at 5.0 ∼ 5.875 lgCCID50/ml. No any change of nucleotides or amino acids were observed in genomes P25, P27, P30, P33 and P35. Conclusion: The titer of measles virus Shanghai-191 strain was stable after continuous subculture in chick embryo fibroblasts, while the genome sequence showed no change, indicating high genetic stability of the strain.
Xu H.,Sichuan University |
Zhang F.,Chengdu Institute of Biological Products Co. |
Shang H.,Sichuan University |
Li X.,Sichuan University |
And 3 more authors.
Journal of Bioscience and Bioengineering | Year: 2013
Based on the strategy of changing pH-stability profiles by altering p. Ka values of catalytic residues, rational protein engineering was applied to improve alkalophilic adaptation of Aspergillus niger endoxylanase XynB. Computational predictions and molecular modeling were carried out using PROPKA server and SWISS-MODEL server, respectively. Three endoxylanase mutant of S108V, N151E, and Q178R, in which the p. Ka values of either catalytic glutamate residues shifted, were generated. In agreement with expectation, the variant of Q178R improved alkalophilic performances. The mutant Q178R raised the optimum pH of XynB from 5.5 to 6.0 and retained 37% of the maximum activity at pH 8.0. Interestingly, the p. Ka values of Glu84 and Glu175 in Q178R are 7.91 and 6.32, respectively. The p. Ka of Glu175 is lower than that of Glu84, as opposed to the fact that the p. Ka of Glu84 is lower than that of Glu175 in other GH11 xylanases. It indicated that Glu175 may convert into a nucleophile residue and Glu84 into an acid/base residue. © 2012 The Society for Biotechnology, Japan.
PubMed | University of Sichuan, Chengdu Institute of Biological Products Co. and University of Chinese Academy of Sciences
Type: Journal Article | Journal: Luminescence : the journal of biological and chemical luminescence | Year: 2015
Isoenzyme c of horseradish peroxidase (HRP-C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP-C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H2O2). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H2O2. Compared with HRP-C, the JcGP1-induced reaction was enhancer independent, which made the enzyme-linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long-term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H2 O2, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long-term stable CL signal combined with enhancer-independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection.
PubMed | Chengdu Institute of Biological Products Co., Food Republic and China National Biotech Group Co.
Type: Journal Article | Journal: Archives of virology | Year: 2016
To develop a potential dengue vaccine candidate, a full-length cDNA clone of a novel chimeric virus was constructed using recombinant DNA technology, with Japanese encephalitis virus (JEV) vaccine strain SA14-14-2 as the backbone, with its premembrane (prM) and envelope (E) genes substituted by their counterparts from dengue virus type 1 (DENV1). The chimeric virus (JEV/DENV1) was successfully recovered from primary hamster kidney (PHK) cells by transfection with the in vitro transcription products of JEV/DENV1 cDNA and was identified by complete genome sequencing and immunofluorescent staining. No neuroinvasiveness of this chimeric virus was observed in mice inoculated by the subcutaneous route (s.c.) or by the intraperitoneal route (i.p.), while some neurovirulence was displayed in mice that were inoculated directly by the intracerebral route (i.c.). The chimeric virus was able to stimulate high-titer production of antibodies against DENV1 and provided protection against lethal challenge with neuroadapted dengue virus in mice. These results suggest that the chimeric virus is a promising dengue vaccine candidate.
PubMed | China National Biotech Group, Chengdu Institute of Biological Products Co. and National Institutes for Food and Drug Control
Type: Journal Article | Journal: Viruses | Year: 2017
The attenuated Japanese encephalitis virus (JEV) strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E) protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439). Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.