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Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain.

Shirali S, Haddadzadeh H, Mohebali M, Kazemi B, Amini N - Iran J Parasitol (2015 Apr-Jun)

Bottom Line: The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector.Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction.The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Faculty of Basic science, Ahvaz branch, Islamic Azad University, Ahvaz, Iran ; Dept. of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

ABSTRACT

Background: There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum.

Methods: Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

Results: The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein.

Conclusion: We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.

No MeSH data available.


Related in: MedlinePlus

Electrophoresis of T/A-LACK and pc- LACK recombinant plasmids and pcDNA3 plasmid were loaded on a 1% agarose gel./ Column1: T/A-LACK recombinant plasmid/Column2: pc- LACK recombinant plasmid/Column3: The band of pcDNA3
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Figure 1: Electrophoresis of T/A-LACK and pc- LACK recombinant plasmids and pcDNA3 plasmid were loaded on a 1% agarose gel./ Column1: T/A-LACK recombinant plasmid/Column2: pc- LACK recombinant plasmid/Column3: The band of pcDNA3

Mentions: Leishmania infantum Genomic DNA was extracted and LACK gene amplified by PCR reaction. Then gene was cloned into PTZ57R/T vector and after confirmation plasmid by colony PCR and restriction analysis, recombinant plasmid was digested by BamHI and sub cloned into pcDNA3.1 expressing vector (Fig. 1). Recombinant plasmid was confirmed by colony PCR (Fig. 2) and Restriction analysis (Fig. 3).


Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain.

Shirali S, Haddadzadeh H, Mohebali M, Kazemi B, Amini N - Iran J Parasitol (2015 Apr-Jun)

Electrophoresis of T/A-LACK and pc- LACK recombinant plasmids and pcDNA3 plasmid were loaded on a 1% agarose gel./ Column1: T/A-LACK recombinant plasmid/Column2: pc- LACK recombinant plasmid/Column3: The band of pcDNA3
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522291&req=5

Figure 1: Electrophoresis of T/A-LACK and pc- LACK recombinant plasmids and pcDNA3 plasmid were loaded on a 1% agarose gel./ Column1: T/A-LACK recombinant plasmid/Column2: pc- LACK recombinant plasmid/Column3: The band of pcDNA3
Mentions: Leishmania infantum Genomic DNA was extracted and LACK gene amplified by PCR reaction. Then gene was cloned into PTZ57R/T vector and after confirmation plasmid by colony PCR and restriction analysis, recombinant plasmid was digested by BamHI and sub cloned into pcDNA3.1 expressing vector (Fig. 1). Recombinant plasmid was confirmed by colony PCR (Fig. 2) and Restriction analysis (Fig. 3).

Bottom Line: The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector.Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction.The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biology, Faculty of Basic science, Ahvaz branch, Islamic Azad University, Ahvaz, Iran ; Dept. of Parasitology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

ABSTRACT

Background: There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum.

Methods: Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

Results: The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein.

Conclusion: We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.

No MeSH data available.


Related in: MedlinePlus