Identification, expression, and functional analysis of Cathepsin L in silkworm (Bombyx mori).
Objective: The objective of this study is to identify and clone the BmCathepsin L (BmCat L) from silkworm (Bombyx mori), investigate its sequence features and expression profiles, and to clarify the response of BmCat L under E. coli stimulate in B. mori. Results of the study will provide a theoretical basis for further studying the function of BmCat L in B. mori. Method: The primers was designed based on the sequence of BGIBMGA006893 in SilkDB database. The full length cDNA of BmCat L was acquired by RACE (rapid-amplification of cDNA ends) technology. The protein structure and molecular weight was predicted online. About the homologous sequences of Cathepsin L from other species were downloaded from NCBI, and the deduced amino acid sequence of putative BmCat L was aligned using the Clustal X (1.83) software, phylogenetic tree was constructed using MEGA 6.0. Its expression profile was performed by RT-PCR and qRT-PCR. Specific primers were designed to amplify the high specificity fragment, and then the PCR product was ligated to the PET-28 vector, which was transformed into Rosetta E. coli. The recombinant protein was induced by IPTG and purified by Ni+ affinity chromatography. Finally, the immune response was challenged by E. coli. Its relative mRNA level was detected by qRT-PCR. Result: BmCat L was clustered on nscaf2860 which was located on chromosome 10, and the gene number in silkDB was BGIBMGA006893. Full length of its ORF is 687 bp, encoding 228 amino acids, there is a conservative Pept_C1 domain structure. The molecular weight and isoelectric point of the deduced protein is 26.132 kD and 4.57, respectively. Phylogenetic analysis showed that it is close to its homologous protein from Spodoptera frugiperda and Danaus plexippus. RT-PCR results showed that BmCat L was specifically and highly expressed in haemocyte. The recombinant protein of BmCat L was produced and purified, and finally verified by His-antibody. The immune challenge experiments results revealed the relationship between Cathepsin L gene and its immune system in B. mori. Conclusion: The Bmcat L cDNA was cloned. It was specific highly expressed in haemocytes. The recombinant proteins were obtained through prokaryotic expression and protein purification. More importantly, the expression of BmCat L was significantly increased after treating with E. coli in haemocytes. It is speculated that BmCat L might be involved in the immune response of B. mori.