Volume 32, Issue 217 (1-2023)
J Mazandaran Univ Med Sci 2023, 32(217): 16-31 |
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Tirbakhsh Gouran S, Doosti A, Jami M. Expression of Brucella abortus Omp25 Protein in Lactococcus lactis Probiotic Bacteria. J Mazandaran Univ Med Sci 2023; 32 (217) :16-31
URL: http://jmums.mazums.ac.ir/article-1-18746-en.html
URL: http://jmums.mazums.ac.ir/article-1-18746-en.html
Abstract: (651 Views)
Background and purpose: The sequence of Omp25 is conserved in all Brucella species. The high antigenicity of the product of this gene stimulates the host’s immune system. Using engineered probiotic bacteria is an appropriate method for vaccine transport. The aim of this study was to express the Omp25 of the Brucella abortus pathogenic bacterium in Lactococcus lactis probiotic bacterium.
Materials and methods: In this experimental study, the required vector was designed and synthesized to include the gene of interest and a signal peptide (pNZ8148-Usp45-Omp25). E. coli strain TOP10F was transformed using the pNZ8148-Usp45-Omp25 expression vector based on induction by nisin. The recombinant plasmid was extracted from the transformed bacteria using a plasmid extraction kit. The L. lactis was transformed by pNZ8148-Usp45-Omp25 vector using electroporation. Evaluation of the expression of Omp25 gene at the RNA level was assessed by reverse transcription method and confirming the presence of recombinant Omp25 protein in the engineered bacteria using SDS-PAGE method.
Results: Successful expression of B. abortus Omp25 in L. lactis was verified by RT-PCR. Subsequently, the proteins were separated based on molecular weight using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein expression analysis showed the expression of Omp25 as a 25kDa extra band in transformed L. lactis compared to the L. lactis receiving the vector lacking the target gene.
Conclusion: This study shows that Omp25 is expressed in L. lactis transformed via pNZ8148-Usp45-Omp25 by electroporation. Transformed L. lactis can be successfully used as a subunit oral vaccine in prevention of Brucellosis.
Materials and methods: In this experimental study, the required vector was designed and synthesized to include the gene of interest and a signal peptide (pNZ8148-Usp45-Omp25). E. coli strain TOP10F was transformed using the pNZ8148-Usp45-Omp25 expression vector based on induction by nisin. The recombinant plasmid was extracted from the transformed bacteria using a plasmid extraction kit. The L. lactis was transformed by pNZ8148-Usp45-Omp25 vector using electroporation. Evaluation of the expression of Omp25 gene at the RNA level was assessed by reverse transcription method and confirming the presence of recombinant Omp25 protein in the engineered bacteria using SDS-PAGE method.
Results: Successful expression of B. abortus Omp25 in L. lactis was verified by RT-PCR. Subsequently, the proteins were separated based on molecular weight using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The protein expression analysis showed the expression of Omp25 as a 25kDa extra band in transformed L. lactis compared to the L. lactis receiving the vector lacking the target gene.
Conclusion: This study shows that Omp25 is expressed in L. lactis transformed via pNZ8148-Usp45-Omp25 by electroporation. Transformed L. lactis can be successfully used as a subunit oral vaccine in prevention of Brucellosis.
Type of Study: Research(Original) |
Subject:
Biotechnology
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