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Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus

RT-PCR amplification product for the mature catalytic domain of TsMP (mTsMP) cDNA (A) and restriction map of the pGEX-mTsMP recombinant plasmid by 1% agarose gel electrophoresis (B). Lane 1, DNA marker (100 bp DNA Ladder); lane2, RT-PCR amplification fragment of mTsMP; lane 3, control group to confirm the absence of any contaminated DNA in the RNA sample by preparing reactions without reverse-transcriptase during synthesis of the first strand cDNA; lane 4, λDNA /EcoR I + Hind III marker; lane 5, pGEX-mTsMP recombinant plasmid; lane 6, pGEX-mTsMP recombinant plasmid cut by Sal I and Not I; lane 7, pGEX-6P-1 empty plasmid cut by Sal I and Not I
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Figure 1: RT-PCR amplification product for the mature catalytic domain of TsMP (mTsMP) cDNA (A) and restriction map of the pGEX-mTsMP recombinant plasmid by 1% agarose gel electrophoresis (B). Lane 1, DNA marker (100 bp DNA Ladder); lane2, RT-PCR amplification fragment of mTsMP; lane 3, control group to confirm the absence of any contaminated DNA in the RNA sample by preparing reactions without reverse-transcriptase during synthesis of the first strand cDNA; lane 4, λDNA /EcoR I + Hind III marker; lane 5, pGEX-mTsMP recombinant plasmid; lane 6, pGEX-mTsMP recombinant plasmid cut by Sal I and Not I; lane 7, pGEX-6P-1 empty plasmid cut by Sal I and Not I

Mentions: Total RNA was isolated from fresh intact Taenia solium metacrestode. After reverse transcription, PCR of the putative mature catalytic domain of TsMP resulted in an amplification fragment with a length of 717 bp (Fig. 1A). For the recombinant pGEX-mTsMP plasmid cut by Sal I and Not I, the results on 1% agarose gel revealed two bands of approximately 5 kb and 0.7 kb (Fig. 1B). The empty vector pGEX-6P-1 showed only one band demonstrating that the recombinant plasmid, pGEX-mTsMP, had an inserted fragment in it. DNA sequencing result showed that the inserted fragment completely matched the TsMP complementary DNA.


Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis.

Zhang Y, Bae YA, Zong HY, Kong Y, Cai GB - Iran J Parasitol (2016 Jan-Mar)

RT-PCR amplification product for the mature catalytic domain of TsMP (mTsMP) cDNA (A) and restriction map of the pGEX-mTsMP recombinant plasmid by 1% agarose gel electrophoresis (B). Lane 1, DNA marker (100 bp DNA Ladder); lane2, RT-PCR amplification fragment of mTsMP; lane 3, control group to confirm the absence of any contaminated DNA in the RNA sample by preparing reactions without reverse-transcriptase during synthesis of the first strand cDNA; lane 4, λDNA /EcoR I + Hind III marker; lane 5, pGEX-mTsMP recombinant plasmid; lane 6, pGEX-mTsMP recombinant plasmid cut by Sal I and Not I; lane 7, pGEX-6P-1 empty plasmid cut by Sal I and Not I
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4835468&req=5

Figure 1: RT-PCR amplification product for the mature catalytic domain of TsMP (mTsMP) cDNA (A) and restriction map of the pGEX-mTsMP recombinant plasmid by 1% agarose gel electrophoresis (B). Lane 1, DNA marker (100 bp DNA Ladder); lane2, RT-PCR amplification fragment of mTsMP; lane 3, control group to confirm the absence of any contaminated DNA in the RNA sample by preparing reactions without reverse-transcriptase during synthesis of the first strand cDNA; lane 4, λDNA /EcoR I + Hind III marker; lane 5, pGEX-mTsMP recombinant plasmid; lane 6, pGEX-mTsMP recombinant plasmid cut by Sal I and Not I; lane 7, pGEX-6P-1 empty plasmid cut by Sal I and Not I
Mentions: Total RNA was isolated from fresh intact Taenia solium metacrestode. After reverse transcription, PCR of the putative mature catalytic domain of TsMP resulted in an amplification fragment with a length of 717 bp (Fig. 1A). For the recombinant pGEX-mTsMP plasmid cut by Sal I and Not I, the results on 1% agarose gel revealed two bands of approximately 5 kb and 0.7 kb (Fig. 1B). The empty vector pGEX-6P-1 showed only one band demonstrating that the recombinant plasmid, pGEX-mTsMP, had an inserted fragment in it. DNA sequencing result showed that the inserted fragment completely matched the TsMP complementary DNA.

Bottom Line: After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed.The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates.TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Genetics, Wuhan University School of Basic Medicial Sciences, Wuhan 430071, China.

ABSTRACT

Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite's evasion from the host's immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis.

Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis.

Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity.

Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis.

No MeSH data available.


Related in: MedlinePlus