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

Mohammad Djafar Moemenbellah-Fard; Masoumeh Bagheri; Mehdi Bonyani; Hamed Sedaghat; Abbasali Raz; Kourosh Azizi; Abouzar Soltani; Hamzeh Alipour

doi:10.1177/20503121231223607

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

SAGE Open Med. 2024 Jan 29:12:20503121231223607. doi: 10.1177/20503121231223607. eCollection 2024.

Authors

Mohammad Djafar Moemenbellah-Fard¹, Masoumeh Bagheri², Mehdi Bonyani³, Hamed Sedaghat⁴, Abbasali Raz⁵, Kourosh Azizi¹, Abouzar Soltani¹, Hamzeh Alipour¹

Affiliations

¹ Research Center for Health Sciences, Institute of Health, Department of Biology and Control of Disease Vectors, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
² Student Research Committee, Department of Biology and Control of Disease Vectors, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
³ Department of Clinical Nutrition, School of Nutrition and Food Sciences, Shiraz University of Medical Sciences, Shiraz, Iran.
⁴ Department of Chemistry, Faculty of Sciences, Persian Gulf University, Bushehr, Iran.
⁵ Malaria and Vector Research Group, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.

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.

Keywords: Arthropod; BEVS; Sf9; blowfly; gene; nucleotide; recombinant protein.