Immunogenicity of Escherichia coli Expressing Streptococcus equi subsp. equi Recombinant SeM

Abstract The Equine Strangles, caused by Streptococcus equi subs. equi, is a contagious disease, causing high rates of morbidity been responsible for important economic losses. The M protein synthesized by S. equi plays an important role in the pathogenesis and is a promising candidate for a vaccine antigen. The innate immune system is responsible for the first immune response against microorganisms, this response is mediated by receptors that detect PAMPs and their activation trigger crucial modulation of the adaptative immune response. This work describes the immune response of S. equi subs. equi. recombinant SeM protein, using Escherichia coli BL21 (DE3) as an expression and delivery vaccine system. To characterize and to determine the vaccine efficacy, mice were vaccinated as followed: 1. Recombinant E. coli expressing rSeM protein; 2. The same recombinant E. coli, inactivated adsorbed in Alumen; 3. Purified rSeM protein adsorbed in Alumen; 4. Inactivated S. equi whole cells adsorbed in Alumen; 5. Control group. All vaccinated mice developed protective response against S. equi infection, however the groups that received the E. coli expressing rSeM presented significant higher IgG level than other vaccinated groups. The recombinant E. coli delivery vaccine system also induced a highest IgG response than inactivated S. equi or purified rSeM vaccines in horses. This study evidence that the recombinant E. coli, live or inactivated, enhanced the humoral response, reaching significant higher antibodies levels than those obtained in the vaccination with the bacterin or purified antigen, showing the feasibility of producing low-cost vaccines against strangles.

Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture

Aims@#Hyaluronic acid (HA) is a high molecular weight polymer and a major component of mucoid capsule in bacteria and extracellular matrix (ECM) of vertebrate tissue. Due to its unique characteristics, HA is used extensively in medical and cosmetic field. However, because of the exotoxins production from animal tissues extraction and Streptococcus zooepidemicus, HA production by recombinant microorganisms has gained interest. The present study was aimed at cloning of hasA gene in Escherichia coli and optimization of the medium components for HA production. @*Methodology and results@#A fragment of an approximate size of 1.5 kb that encodes the hyaluronan synthase (hasA) gene from S. zooepidemicus ATCC 39920 was amplified by PCR using hasA-specific primers. The hasA gene was ligated into the bacterial expression vector pLbADH and transformed into the expression host, Escherichia coli BL21 strain. Then, genetically engineered E. coli strain BL21 was used for the production of HA by fermentation using different glucose concentration (10-50 g/L) and different IPTG concentration (0.1, 0.5 and 1.0 mM) in shake flask culture. Amongst varying glucose concentrations, results showed that 50 g/L glucose with nutrient rich media containing nitrogen source was able to produce the highest HA concentration (0.115 ± 0.002 g/L). With addition of 1.0 mM IPTG, HA production reached a peak 0.532 ± 0.026 g/L which is around fivefold higher compared to without IPTG. @*Conclusion, significance and impact of study@#The hasA gene was cloned from S. zooepidemicus and successfully expressed in recombinant E. coli BL21 cells. This low molecular weight HA is gaining more importance in medical and cosmetic application due to possess pronounced free radical scavenging and antioxidant activities.

Response surface methodology of nitrilase production by recombinant Escherichia coli

Growth and nitrilase production by recombinant Escherichia coli cells harbouring pET 21 (b) plasmid, for the expression of Pseudomonas putida nitrilase were improved using response surface methodology. Central composite design was used for obtaining ideal concentration of critical medium components which include fructose, tryptone, yeast extract and lactose. The optimal values for the concentration of fructose, tryptone, yeast extract and lactose were found to be 1.13, 2.26, 3.25 and 0.9 percent (w/v), respectively. Here, fructose served as carbon source for the growth while lactose was preferably used as inducer for the expression of foreign protein. Yeast extract in the medium was used as a growth promoter while tryptone was added as a major nitrogen source. Using this optimized medium, an experimental growth of 6.67 (OD at 600 nm) and nitrilase activity of 27.13 U/ml was achieved. This approach for medium development led to an enhancement of the growth and enzyme activity by 1.4 and 2.2 times, respectively, as compared to the un-optimized medium.

Optimization of Fermentation and Induction Conditions of Recombinant E.coli BL21(DE3)/pET30a(+)hrpNEcc / 中国生物工程杂志

In order to obtain high yield of the HrpNEcc protein with a lower total cost,fermentation and lactose induction conditions for recombinant E.coli BL21(DE3)/pET30a(+)hrpN Ecc were optimized in flasks and the recombinant E.coli was fermentated in 7L fermenter.The optimized incoulum concentration was 5% and the optimized nutrient medium was TB medium.The HrpNEcc protein yield reached 417.60mg/L by adding 3g/L exogenous inducer lactose in the growth prophase of log-phase for the recombinant E.coli.The HrpNEcc protein yield was higher 36.73% than that of the case of no any inducer,and was higher 16.85% than that of the case of adding IPTG.The wet weight of cell pellet of the recombinant E.coli reached 57.24g/L after fermentation in 7L fermenter,the HrpNEcc protein reached 3.29g/L,about 50.2% of total cellular protein.

Effective activation of CD11c and anti-tumor immune in immunized mice with recombinant E.coli. LLO/OVA / 第三军医大学学报

Objective To investigate the anti-tumor pathway and the effect of recombinant E.coli. LLO/OVA in C57BL/6 mice. Methods Magnetic sorting of splenic CD11c, CD4+ and CD8+ T cell from immunized mice with E.coli. LLO/OVA and E.coli. OVA vaccination were performed. In addition, the lymphocytes mixed reaction and IL-2 and IFN-? in the supernatant were detected. The percentage of OVA257-264 SIINFEKL specific CD8+ T cell were checked by flow cytometry. B16-OVA melanoma protection and inhibition response in E.coli. vaccinated mice were compared. Results Compared to E.coli. OVA, E.coli. LLO/OVA-vaccinated splenic CD11c cells stimulated proliferation of autogenic CD4+ T cells and IL-2 production of these cells. CD11c cells induced autogenic CD8+T cells proliferation and IFN-? secretion. A great number of OVA257-264 SIINFEKL-specific splenic CD8+ T cells were induced and the number of lung tumor nodules was significantly reduced in E.coli. LLO/OVA vaccinated mice. Conclusion Recombinant LLO, as an effective adjuvant of E.coli. OVA, can induce murine splenic CD11c cells activation and play potential roles on CD4+ and CD8+ T cells proliferation and IL-2 and IFN-? secretion of these cells, induce more OVA-specific CD8+ T cells and stimulate stronger tumor inhibition in vivo of vaccinated C57BL/6 mice.

Effect of Temperature on Lycopene Prodmction by Recombinant E.coli and Control Strategy / 微生物学通报

Batch fermentation of lycopene by recombinant E. c ol i ZYL-2 at various temperatures ranging between 23℃～37℃ were studied in 7L fermentor. At 33℃, cell specific growth rate in earlier stage of culture was higher, and the time obtained maximum cell dry weight was shorter at other tempe rature. while after 9 h, lycopene specific production rate was higher at 28℃. Based on these results, a two-stage temperature control strategy was developed in which 33℃ was used for fermentation for the first 9 h and the temperaturew as switched to 28℃ after 9h. Using this temperature-shift strategy, the maxim al lycopene content and productivity reached 605.25?g L-1 and 28.82?g L-1h-1. The lycopene fermentation level obtained by the strategy was higher than those in single temperature-control experiments.

Study on the Recombinant Human ApoA-I_Milano High Density and High Expression by Two Temperature-Shifted Induction in Escherichia coli / 微生物学通报

The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.