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Designing and Construction Pcdna3.1 Vector Encoding Cfp10 Gene of Mycobacterium tuberculosis.

Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z - Jundishapur J Microbiol (2015)

Bottom Line: Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning.Colony PCR and restriction enzyme digestion confirmed the cloning.This recombinant vector can also be used in the construction of fusion proteins.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Resistance Research Center, Bu Ali Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran.

ABSTRACT

Background: Pathogenic mycobacteria are a major cause of human morbidity and mortality. Mycobacterium tuberculosis is an etiological agent of human tuberculosis (TB). Designing new vaccines, including DNA vaccines, may be a useful strategy for preventing TB.

Objectives: The purpose of this study was to design and construct an eukaryotic expression vector containing M. tuberculosis.

Materials and methods: Genomic DNA of M. tuberculosis H37Rv cultured on Lowenstein Jensen medium was extracted, and cfp10 was amplified by PCR. After digesting the PCR product and the plasmid, the cfp10 fragment was ligated into the vector pcDNA3.1 (+). Correct insertion was confirmed by colony PCR, restriction enzyme digestion, and sequencing.

Results: Electrophoresis of the PCR product on gel showed a 303-bp target fragment. Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning. Colony PCR and restriction enzyme digestion confirmed the cloning.

Conclusions: Cloning of cfp10 of M. tuberculosis into an eukaryotic expression vector was performed successfully. We propose this recombinant plasmid for inducing immunity in animal models in future studies. This recombinant vector can also be used in the construction of fusion proteins.

No MeSH data available.


Related in: MedlinePlus

Agarose Gel (1% w/v) With PCR Products Viewed Under UV Light and Stained With Ethidium BromideLanes 1 and 2 correspond to a 1-kbp DNA ladder and the PCR products of the CFP10-coding region, respectively.
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fig23108: Agarose Gel (1% w/v) With PCR Products Viewed Under UV Light and Stained With Ethidium BromideLanes 1 and 2 correspond to a 1-kbp DNA ladder and the PCR products of the CFP10-coding region, respectively.

Mentions: We amplified cfp10M. tuberculosis strain H37Rv using PCR. A 303-bp fragment was obtained following 1% w/v agarose gel electrophoresis (Figure 1).


Designing and Construction Pcdna3.1 Vector Encoding Cfp10 Gene of Mycobacterium tuberculosis.

Baghani A, Youssefi M, Safdari H, Teimourpour R, Meshkat Z - Jundishapur J Microbiol (2015)

Agarose Gel (1% w/v) With PCR Products Viewed Under UV Light and Stained With Ethidium BromideLanes 1 and 2 correspond to a 1-kbp DNA ladder and the PCR products of the CFP10-coding region, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644269&req=5

fig23108: Agarose Gel (1% w/v) With PCR Products Viewed Under UV Light and Stained With Ethidium BromideLanes 1 and 2 correspond to a 1-kbp DNA ladder and the PCR products of the CFP10-coding region, respectively.
Mentions: We amplified cfp10M. tuberculosis strain H37Rv using PCR. A 303-bp fragment was obtained following 1% w/v agarose gel electrophoresis (Figure 1).

Bottom Line: Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning.Colony PCR and restriction enzyme digestion confirmed the cloning.This recombinant vector can also be used in the construction of fusion proteins.

View Article: PubMed Central - PubMed

Affiliation: Antimicrobial Resistance Research Center, Bu Ali Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran.

ABSTRACT

Background: Pathogenic mycobacteria are a major cause of human morbidity and mortality. Mycobacterium tuberculosis is an etiological agent of human tuberculosis (TB). Designing new vaccines, including DNA vaccines, may be a useful strategy for preventing TB.

Objectives: The purpose of this study was to design and construct an eukaryotic expression vector containing M. tuberculosis.

Materials and methods: Genomic DNA of M. tuberculosis H37Rv cultured on Lowenstein Jensen medium was extracted, and cfp10 was amplified by PCR. After digesting the PCR product and the plasmid, the cfp10 fragment was ligated into the vector pcDNA3.1 (+). Correct insertion was confirmed by colony PCR, restriction enzyme digestion, and sequencing.

Results: Electrophoresis of the PCR product on gel showed a 303-bp target fragment. Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning. Colony PCR and restriction enzyme digestion confirmed the cloning.

Conclusions: Cloning of cfp10 of M. tuberculosis into an eukaryotic expression vector was performed successfully. We propose this recombinant plasmid for inducing immunity in animal models in future studies. This recombinant vector can also be used in the construction of fusion proteins.

No MeSH data available.


Related in: MedlinePlus