ABSTRACT
Recombinant Fl-V (rFl-V) fusion protein is the main ingredient of the current candidate vaccine against Yersinia pestis infection, which has been under investigation in clinical trial in USA. We investigated the soluble expression conditions of rF1-V in Escherichia coli BL21 (DE3) that we constructed before. After scale-up and optimization of fermentation processes, we got the optimized fermentation process parameters: the culture was induced at the middle exponential phase with 50 µmol/L of IPTG at 25 °C for 5 h. Soluble rFl-V protein was isolated to 99% purity by ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and gel filter chromatography. The protein recovery was above 20%. Protein identity and primary structure were verified by mass spectrometry and Edman sequencing. Results of purity, quality and western blotting analysis indicated that the target protein is a consistent and properly folded product. Furthermore, the immunogenicity of various antigens formulated with aluminum hydroxide adjuvant was evaluated in mice. Serum antibody titers of 4 groups including 20 µg rFl, rV and rFl-V and 10 µg rFl+10 µg rV, were assayed by ELISA after 2 doses. The antibody titers of anti-Fl with 20 µg rFl-V were obviously higher than titers with other groups; meanwhile there were no significant difference of anti-V antibody titers among them. These findings confirm that rFl-V would be the active pharmaceutical ingredient of the plague subunit vaccine.
Subject(s)
Animals , Mice , Adjuvants, Immunologic , Antibodies, Bacterial , Blood , Antibody Formation , Antigens, Bacterial , Allergy and Immunology , Blotting, Western , Chromatography, Ion Exchange , Enzyme-Linked Immunosorbent Assay , Plague , Plague Vaccine , Allergy and Immunology , Recombinant Fusion Proteins , Allergy and Immunology , Vaccines, Subunit , Allergy and Immunology , Yersinia pestisABSTRACT
OBJECTIVE:To know the utilizatior of Shenfu injection(SFI)in our hospital,and provide reference for ratio-nal use of SFI. METHODS:In respective study,patients received SFI from the Hospital Information System(HIS)in surgery department in our hospital from Jan. 2013 to Dec. 2014 were analyzed statistically in aspects of general situation,indications, usage and dosage,course of treatment,drug combination,etc. And the incidence of ADR/ADE was monitored. RESULTS:The 1 247 surgery inpatients used SFI distributed in thyroid and galactophore department(634 cases),comprehensive minimal-ly invasive department (292 cases),gastrointestinal surgery department (250 cases),urology department (64 cases) and car-diothoracic surgery department(7 cases),including 466 male and 781 female,the ratio of male to female was 1∶1.68,aver-age age was 58.50 years;it mainly used in perioperative medication (78.11%) and treated for tumors (11.23%),treatment course concentrated on 2-7 d(71.45%),dosage mainly was 100 ml(70.01%);combination medication were mainly pantopra-zole sodium,Fructus bruceae injection,Xueshuantong for injection(lyophilized),etc. The nonstandard phenomenon was exist-ed in indications,dosage,solvent selection,medication frequency,drug combination,etc. There were no obvious ADR/ADE related to SFI. CONCLUSIONS:More attention must be paid to the irrational use of SFI in surgery clinic of our hospital,the safety of traditional Chinese medicine injection in clinical use should be correctly understood,achieve dialectical therapy and rational drug use.