Identification of In-Vitro Red Fluorescent Protein with Antipathogenic Activity from the Midgut of the Silkworm (Bombyx Mori L.)

Background: The midgut of silkworm (Bombyx mori L.) plays an important role as a natural barrier and source of innate immunity. We had purified the novel red fluorescent protein (RFP) from the midgut of the silkworm Bombyx mori L. and bioassay studies confirmed RFPs possess antiviral, antifungal and antibacterial properties. N-terminal sequence of RFP analysis predicted chbp gene and it belongs to lipocalin gene family and is known to involve in anti-pathogenic activities. Objective: The main objective of this study was to purify RFP from the midgut of Kolar Gold silkworm and confirm its antimicrobial activity. Methods: For isolation of RFP, midgut juice was collected by brief exposure to chloroform vapours to fifth instar Kolar Gold silkworm larvae. Juice was purified by 40 % ammonium sulfate precipitation and purified by gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected. Molecular weight and purity of RFP was identified using PAGE, MALDI-TOF and HPLC. Antimicrobial property of purified RFP against BmNPV, Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii was performed. N-terminal sequencing of RFP was performed using Edman degradation method. Using ten amino acid sequence, using default parameter BLAST search was performerd. From the fifth day old fifth instar silkworm midgut mRNA was isolated and cDNA was synthesized using oligo-dt primer and amplification of ChBP gene was carried out by using cDNA as the template and ChBP gene specific primers. chbp protein sequence as a input built the homology model by using SWISS-MODEL. Results: RFP was purified by 40 % ammonium sulfate precipitation and gel filtration chromatography (GFC) and fractions with fluorescence red under Ultra violet (UV) were collected and SDS - PAGE revealed a size of 40 kDa. RFP purified by GFC was further reconfirmed by HPLC with a single peak with a retention time of 8.755 min. MALDI-TOF produced a peak at a molecular mass of 40 kDa. RFP from the midgut juice showed antiviral activity against the silkworm virus BmNPV, antibacterial activity against Escherichia coli, Klebsiella pneumonia, Bacillus subtilis and Phytophthora meadii. N-terminal sequencing of RFP by Edman degradation method sequenced TQTIETDYWV amino acids and BLAST analysis predicted the Chlorophyllide-a Binding Protein (chbp) with B. mori. PCR product was sequenced and obtained 911bp nucleotides encoding 302 amino acid residues and deposited with the accession number KX186723 in NCBI. Sequence analysis revealed Chbp belongs to lipocalin gene family and known to involve in antiviral, antifungal and anti-bacterial properties. Chbp gene homology model was predicted using crystal structure of insecticyanin A from the tobacco hornworm as a template. Conclusion: Our results indicated RFP present in midgut juice of 5th instar larvae of kolar gold silkworm. We have purified novel RFP with molecular mass of 40 kDa and showed its antipathogenic activities. Chbp gene synthesises RFP and further it could be utilized for agriculture and pharmaceutical industry. Keywords: Bombyx mori, midgut, RFP, chbp, in-silico analysis, antipathogenic, bioprospecting.