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Cloning and Sequence Analysis of Recombinant Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-142 kDa) In pTZ57R/T Vector.

Mirahmadi H, Spotin A, Fallahi S, Taghipour N, Turki H, Seyyed Tabaei SJ - Iran J Parasitol (2015 Apr-Jun)

Bottom Line: Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-142 kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-133 kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-119 kDa). 2 out of 38 mutations were found as to be novel haplotypes.High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran ; Infectious Diseases and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.

ABSTRACT

Background: Carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 is considered as an important antigen in blood stage. Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.

Methods: In the present study, the genetic diversity of cloned PvMSP-142 kDa gene from an Iranian patient is analyzed. Parasite DNA was extracted from a P. vivax - infected patient in Iran. The region of PvMSP-142 kDa was amplified by PCR, cloned into pTZ57R/T vector and then sequenced.

Results: Sequencing of cloned PvMSP-142 kDa gene clearly has a high degree of homology (95%) with reference Sal-I sequence and also with the homogeneous sequences from some studied countries (97%). Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-142 kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-133 kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-119 kDa). 2 out of 38 mutations were found as to be novel haplotypes.

Conclusion: High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.

No MeSH data available.


A: Lane1: Intact plasmid pTZ57R, Lane2: Digested plasmid pTZ57R by EcoRV, Lane3: Size marker: 1kb. B: Lane1: Recombinant plasmid, Lane2: Intact Plasmid, Lane3: Size marker1kb. C: Lane1: (a: Separated PCR product from digested recombinant plasmid), Lane2: Intact recombinant plasmid, Lane3: Size marker1kb
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Figure 1: A: Lane1: Intact plasmid pTZ57R, Lane2: Digested plasmid pTZ57R by EcoRV, Lane3: Size marker: 1kb. B: Lane1: Recombinant plasmid, Lane2: Intact Plasmid, Lane3: Size marker1kb. C: Lane1: (a: Separated PCR product from digested recombinant plasmid), Lane2: Intact recombinant plasmid, Lane3: Size marker1kb

Mentions: Digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing: 25 μl plasmid DNA (2μg), 4 μl of 10X EcoR V buffer, (10 unit) 1 μl of EcoR V (Fermentase Co.) and dd H2O up to 40μl. Then it was mixed by gentle vortex, centrifuged and incubated at 37 °C for 2 hours. In the next step, 2 μl of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed. Thereafter it was incubated to 65 °C by water bath for 10 min at the end of digestion (Fig. 1A). Blunt-ended plasmid DNA was purified by electrophoresis on 1% agarose gel.


Cloning and Sequence Analysis of Recombinant Plasmodium vivax Merozoite Surface Protein 1 (PvMSP-142 kDa) In pTZ57R/T Vector.

Mirahmadi H, Spotin A, Fallahi S, Taghipour N, Turki H, Seyyed Tabaei SJ - Iran J Parasitol (2015 Apr-Jun)

A: Lane1: Intact plasmid pTZ57R, Lane2: Digested plasmid pTZ57R by EcoRV, Lane3: Size marker: 1kb. B: Lane1: Recombinant plasmid, Lane2: Intact Plasmid, Lane3: Size marker1kb. C: Lane1: (a: Separated PCR product from digested recombinant plasmid), Lane2: Intact recombinant plasmid, Lane3: Size marker1kb
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4522295&req=5

Figure 1: A: Lane1: Intact plasmid pTZ57R, Lane2: Digested plasmid pTZ57R by EcoRV, Lane3: Size marker: 1kb. B: Lane1: Recombinant plasmid, Lane2: Intact Plasmid, Lane3: Size marker1kb. C: Lane1: (a: Separated PCR product from digested recombinant plasmid), Lane2: Intact recombinant plasmid, Lane3: Size marker1kb
Mentions: Digesting plasmids were performed in a 0.5 ml microcentrifuge tube containing: 25 μl plasmid DNA (2μg), 4 μl of 10X EcoR V buffer, (10 unit) 1 μl of EcoR V (Fermentase Co.) and dd H2O up to 40μl. Then it was mixed by gentle vortex, centrifuged and incubated at 37 °C for 2 hours. In the next step, 2 μl of the mixture was taken and run on a 1% agarose gel to make sure the digestion was completed. Thereafter it was incubated to 65 °C by water bath for 10 min at the end of digestion (Fig. 1A). Blunt-ended plasmid DNA was purified by electrophoresis on 1% agarose gel.

Bottom Line: Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-142 kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-133 kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-119 kDa). 2 out of 38 mutations were found as to be novel haplotypes.High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Parasitology and Mycology, Shahid Beheshti University of Medical Sciences, Tehran, Iran ; Infectious Diseases and Tropical Medicine Research Center, Zahedan University of Medical Sciences, Zahedan, Iran.

ABSTRACT

Background: Carboxy-terminal 42 kDa region of Plasmodium vivax merozoite surface protein-1 is considered as an important antigen in blood stage. Since, this region has been observed to be polymorphic among isolates of P. vivax, it is significant to survey on different regions of this antigen in various areas of the world.

Methods: In the present study, the genetic diversity of cloned PvMSP-142 kDa gene from an Iranian patient is analyzed. Parasite DNA was extracted from a P. vivax - infected patient in Iran. The region of PvMSP-142 kDa was amplified by PCR, cloned into pTZ57R/T vector and then sequenced.

Results: Sequencing of cloned PvMSP-142 kDa gene clearly has a high degree of homology (95%) with reference Sal-I sequence and also with the homogeneous sequences from some studied countries (97%). Thirty eight SNPs (single nucleotide polymorphism) were identified in cloned PvMSP-142 kDa gene which the mutations had localized in the 33 kDa fragment (PvMSP-133 kDa), while there was nearly no variation in the 19 kDa fragment (PvMSP-119 kDa). 2 out of 38 mutations were found as to be novel haplotypes.

Conclusion: High similarity of cloned PvMSP-142 kDa gene in comparison to reference sequence and other sequences could be beneficial as a remarkable molecular marker for serological diagnostic kits of P. vivax in malarious neighboring countries of Iran and around the world.

No MeSH data available.