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Updated: Jul 18, 2026
Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay
Published on: February 22, 2018
Cloning and characterization of Bombyx mori PP-BP a gene induced by viral infection
Zhi-Gang Hu1, Ke-Ping Chen, Qin Yao
1Institute of Life Sciences, Jiangsu University, Zhenjiang 212013, China.
Abstract:
The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12(782)) was obtained from the hemolymph RNA pools. Using 5'-RACE with a specially designed primer based on the FDD study, a 1,401 bp cDNA clone was obtained containing a 1,311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.
Insights
The study identifies a novel protein, Bombyx mori paralytic peptide-binding protein (PP-BP), in silkworms. Its expression significantly increases in hemolymph after Bombyx mori nuclear polyhedrosis virus (BmNPV) infection, suggesting a role in cellular immunity.
Area of Science:
- Insect molecular biology
- Immunology
- Biochemistry
Background:
- The ENF peptide family plays roles in insect defense and homeostasis.
- Bombyx mori paralytic peptide (BmPP) and Pseudaletia separata growth-blocking peptide (PsGBP) are ENF family members involved in immune responses.
- Growth-blocking peptide-binding protein (GBP-BP) can attenuate PsGBP functions.
Purpose of the Study:
- To identify genes differentially expressed in silkworm hemolymph following Bombyx mori nuclear polyhedrosis virus (BmNPV) infection.
- To characterize a novel binding protein homologous to GBP-BP and investigate its role in the silkworm immune response.
Main Methods:
- Fluorescent differential display (FDD) to screen for differentially expressed genes.
- 5'-RACE to obtain the full-length cDNA sequence.
- Bioinformatics analysis for gene structure.
- RT-PCR and real-time quantitative PCR to analyze gene expression patterns.
Main Results:
- A differentially expressed gene, G12(782), was identified and its cDNA sequence (1,401 bp) obtained.
- The deduced protein showed high homology to GBP-BP and was named Bombyx mori paralytic peptide-binding protein (PP-BP).
- The B. mori PP-BP gene is expressed primarily in hemolymph and its mRNA levels significantly increase upon BmNPV infection.
Conclusions:
- Bombyx mori PP-BP is a novel protein involved in the cellular immune response of silkworms.
- The increased expression of PP-BP suggests its critical role in combating BmNPV invasion in the hemolymph.
