Volume 11, Issue 42 (3-2021)
NCMBJ 2021, 11(42): 83-94 |
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Saroughi S, Zare M, Kazemi R, Motamedi M, Amani J. PCR and cloning of the lcs chimeric gene in pcDNA3.1 vector and its expression in the cell line. NCMBJ 2021; 11 (42) :83-94
URL: http://ncmbjpiau.ir/article-1-1383-en.html
URL: http://ncmbjpiau.ir/article-1-1383-en.html
Department of Genetics, Payam Noor University, Tehran, Iran
Abstract: (2029 Views)
Aim and Background: Diarrhea is the second most common cause of under-5 mortality. The most important strains of Entrotoxigenic Escherichia coli causing Lt and St toxin cause diarrhea and Entrohemoragic E.coli causing Shiga-like toxin secretion. Chlorine enterotoxin B subunit (Ctx) plays a key role in the development of diarrhea in Vibrio cholerae. More specific antibodies could be developed to counter these toxins by combining the CtxB, LtB and StB (LSC) epitopes and the production of trivalent vaccine. The aim of this study was to cloning lsc gene into pcDNA3.1 to design a vaccine DNA.
Materials and Methods: The lsc gene sequence was transferred to pcDNA3.1(+) vector after primer design and amplification by PCR. The pcDNA3.1(+) vector and the PCR product were digested using HindIII and EcoRI enzymes. Cloning of lsc gene was performed in pcDNA3.1(+) vector and PCR. The clones were digested enzymatically. To ensure expression of lsc gene, it was transferred to HEK-293T cell and confirmed by Western blotting.
Results: The lsc gene was confirmed by PCR and cloning in pcDNA3.1(+) vector using enzymatic digestion and a fragment length of 933 bp was detected and confirmed. Transfection kit was then transferred to HEK-293T cell and expression of the recombinant protein was confirmed by Western blotting and the protein was 39 kDa.
Conclusion: The results of the chimeric gene are well expressed in the cell line and confirmed by Western blotting that can be a good candidate for the fight against bacterial infection.
Materials and Methods: The lsc gene sequence was transferred to pcDNA3.1(+) vector after primer design and amplification by PCR. The pcDNA3.1(+) vector and the PCR product were digested using HindIII and EcoRI enzymes. Cloning of lsc gene was performed in pcDNA3.1(+) vector and PCR. The clones were digested enzymatically. To ensure expression of lsc gene, it was transferred to HEK-293T cell and confirmed by Western blotting.
Results: The lsc gene was confirmed by PCR and cloning in pcDNA3.1(+) vector using enzymatic digestion and a fragment length of 933 bp was detected and confirmed. Transfection kit was then transferred to HEK-293T cell and expression of the recombinant protein was confirmed by Western blotting and the protein was 39 kDa.
Conclusion: The results of the chimeric gene are well expressed in the cell line and confirmed by Western blotting that can be a good candidate for the fight against bacterial infection.
Type of Study: Research Article |
Subject:
Cellular and molecular
Received: 2020/01/25 | Accepted: 2021/05/11 | Published: 2021/06/21
Received: 2020/01/25 | Accepted: 2021/05/11 | Published: 2021/06/21
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