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A Seamless Cloning Approach for Porcine Reproductive and Respiratory Syndrome Virus Expression Vector Construction
Published on: May 17, 2024
Cloning, expression and immunogenicty analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003
Zhijuan Mao1, Lian Yu, Zhenqiang You
1College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou, Zhejiang Province 310027, China.
Abstract:
Genes of five outer membrane proteins of Vibrio parahaemolyticus zj2003, including OmpW, OmpV, OmpK, OmpU and TolC, were cloned and expressed as N-terminal His(6)-tagged proteins in Escherichia coli. The recombinant fusion proteins were purified with nickel chelate affinity chromatography. To analyze the immunogenicity of these proteins, large yellow croaker (Pseudosciaena crocea) were immunized by intraperitoneal injection. Antibody response was assessed by method of enzyme-linked immunosorbent assay. Titres to all five recombinant proteins increased during 4 to 8 weeks post immunization, within the range of log 2 values of 5.75 to 10.8. Recorded relative survival percent (RPS) of the vaccinated groups varied from 80% to 90%, while 10 fish in control group all died. Western blot tests were undertaken with the serum of survival fish after experimental infection. Except for recombinant TolC, the other four recombinant proteins were recognized by the serum. It is indicated that four outer membrane proteins of V. parahaemolyticus zj2003, including OmpW, OmpV, OmpU and OmpK, are immunogenic during in vivo infection, which would be of some significance in developing efficient vaccine in aquaculture. This is the first report of successful vaccination against V. parahaemolyticus with purified recombinant outer membrane proteins.
Insights
Four outer membrane proteins of Vibrio parahaemolyticus were found to be immunogenic in fish, showing potential for developing effective aquaculture vaccines. This study marks the first successful vaccination against this pathogen using purified recombinant proteins.
Area of Science:
- Aquatic animal health
- Bacteriology
- Vaccinology
Background:
- Vibrio parahaemolyticus is a significant pathogen causing disease in aquaculture.
- Outer membrane proteins (OMPs) are key targets for vaccine development.
Purpose of the Study:
- To clone, express, and purify five OMPs (OmpW, OmpV, OmpK, OmpU, TolC) from Vibrio parahaemolyticus zj2003.
- To evaluate the immunogenicity and protective efficacy of these recombinant OMPs in large yellow croaker (Pseudosciaena crocea).
Main Methods:
- Genes encoding five OMPs were cloned and expressed in Escherichia coli as His-tagged fusion proteins.
- Recombinant proteins were purified using nickel chelate affinity chromatography.
- Immunization of fish, antibody response assessment (ELISA), and challenge tests were performed.
Main Results:
- All five recombinant OMPs elicited antibody responses in fish.
- Vaccinated fish showed high relative survival percentages (80-90%) after challenge, while control fish died.
- Western blot analysis confirmed that four OMPs (OmpW, OmpV, OmpK, OmpU) were recognized by serum from infected fish.
Conclusions:
- Four outer membrane proteins of Vibrio parahaemolyticus zj2003 (OmpW, OmpV, OmpK, OmpU) are immunogenic and can induce protective immunity.
- These findings are significant for developing effective vaccines against Vibrio parahaemolyticus in aquaculture.
- This study reports the first successful vaccination against V. parahaemolyticus using purified recombinant outer membrane proteins.
