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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

SDS-PAGE analysis of expressed gene productColumn1:DH5α before expressing by IPTG (control)Column2: pc-DNA3.1 before expressing by IPTG (control)Column3: pc-LACK before expressing by IPTG (control)/Column4: Pre stain protein ladderColumn5: pc-LACK 1h after expressing by IPTGColumn6: pc-LACK 2h after expressing by IPTGColumn7: pc-LACK 3h after expressing by IPTGColumn8: pc-LACK 4h after expressing by IPTGColumn9: pc-LACK 5h after expressing by IPTG
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Figure 5: SDS-PAGE analysis of expressed gene productColumn1:DH5α before expressing by IPTG (control)Column2: pc-DNA3.1 before expressing by IPTG (control)Column3: pc-LACK before expressing by IPTG (control)/Column4: Pre stain protein ladderColumn5: pc-LACK 1h after expressing by IPTGColumn6: pc-LACK 2h after expressing by IPTGColumn7: pc-LACK 3h after expressing by IPTGColumn8: pc-LACK 4h after expressing by IPTGColumn9: pc-LACK 5h after expressing by IPTG

Mentions: A 36 KD band was recognized by Leishmania antibody-positive [polyclonal] dog sera in protein extracts of the cells transfected with pc-LACK. In Western blot LACK protein was not detected in the non-transfected control cells. (Fig. 5, 6)


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)

SDS-PAGE analysis of expressed gene productColumn1:DH5α before expressing by IPTG (control)Column2: pc-DNA3.1 before expressing by IPTG (control)Column3: pc-LACK before expressing by IPTG (control)/Column4: Pre stain protein ladderColumn5: pc-LACK 1h after expressing by IPTGColumn6: pc-LACK 2h after expressing by IPTGColumn7: pc-LACK 3h after expressing by IPTGColumn8: pc-LACK 4h after expressing by IPTGColumn9: pc-LACK 5h after expressing by IPTG
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Related In: Results  -  Collection

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Figure 5: SDS-PAGE analysis of expressed gene productColumn1:DH5α before expressing by IPTG (control)Column2: pc-DNA3.1 before expressing by IPTG (control)Column3: pc-LACK before expressing by IPTG (control)/Column4: Pre stain protein ladderColumn5: pc-LACK 1h after expressing by IPTGColumn6: pc-LACK 2h after expressing by IPTGColumn7: pc-LACK 3h after expressing by IPTGColumn8: pc-LACK 4h after expressing by IPTGColumn9: pc-LACK 5h after expressing by IPTG
Mentions: A 36 KD band was recognized by Leishmania antibody-positive [polyclonal] dog sera in protein extracts of the cells transfected with pc-LACK. In Western blot LACK protein was not detected in the non-transfected control cells. (Fig. 5, 6)

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