Dataset Information

Cloning, expression and molecular analysis of recombinant Netrin-A protein of Lucilia sericata Meigen (Diptera: Calliphoridae) larvae.

ABSTRACT:

Objectives

Lucilia sericata (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from Lucilia sericata larvae by the baculovirus expression vector system in the Sf9 insect cell line.

Methods

The coding sequence of Netrin-A was cloned, amplified in the pTG19 vector, and then cloned in the pFastBac HTA vector. It was then transformed into DH10Bac, and the recombinant Bacmid was subsequently transfected into Sf9 cells. The recombinant Netrin-A was purified by Ni-NTA agarose. The evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay.

Results

The molecular weight of this protein was 52 kDa with 404 amino acids. The signal peptide was located between amino acids 24 and 25. The concentration of Netrin-A was calculated to be 48.8 μg/ml. It reaffirmed the characterized gene codes of Lucilia sericata Netrin-A in a previous study.

Conclusions

The generation of recombinant Netrin-A could be used in larval therapy, and as a biomarker in certain diseases. The netrin-A of Lucilia sericata was unprecedentedly cloned and expressed in a eukaryotic cell line. Given that this larva is FDA-approved, and non-pathogenic, it conduces to research on the development of maggot therapy in future.

SUBMITTER: Moemenbellah-Fard MD

PROVIDER: S-EPMC10826387 | biostudies-literature | 2024

REPOSITORIES: biostudies-literature

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Publications

Cloning, expression and molecular analysis of recombinant Netrin-A protein of <i>Lucilia sericata</i> Meigen (Diptera: Calliphoridae) larvae.

Moemenbellah-Fard Mohammad Djafar MD Bagheri Masoumeh M Bonyani Mehdi M Sedaghat Hamed H Raz Abbasali A Azizi Kourosh K Soltani Abouzar A Alipour Hamzeh H

SAGE open medicine 20240129

<h4>Objectives</h4><i>Lucilia sericata</i> (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from <i>Lucilia sericata</i> larvae by the baculovirus expression vector system in the Sf9 insect cell line.<h4>Methods</h4>The coding sequence of Netrin-A was cloned, amplified in the pTG1 ...[more]

PMID: 38292417

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