Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).
Masoumeh BagheriHamzeh AlipourTahereh KaramzadehMarzieh Shahriari-NamadiAbbasali RazKourosh AziziJavad Dadgar PakdelMohammad Djaefar Moemenbellah-FardPublished in: Genetica (2022)
The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.
Keyphrases
- nucleic acid
- amino acid
- escherichia coli
- wound healing
- binding protein
- aedes aegypti
- cell migration
- protein protein
- drosophila melanogaster
- single molecule
- molecular docking
- genome wide
- cell free
- stem cells
- single cell
- dna methylation
- circulating tumor
- cross sectional
- small molecule
- endothelial cells
- crispr cas
- cystic fibrosis
- vascular endothelial growth factor
- genome wide identification
- data analysis
- heat shock protein
- highly efficient
- molecular dynamics simulations
- label free