家蚕组织蛋白酶O（BmCatO）基因鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2015.22.016

Abstract

Abstract: 【Objective】The objectives of this study are to clone Cathepsin O gene (BmCatO) of silkworm (Bombyx mori), analyze its mRNA expression features, obtain polyclonal antibody through prokaryotic expression, protein purification and immune rabbit, and to provide a theoretical basis for further studying the function of Cathepsin O in B. mori. 【Method】 The full-length of BmCathepsin O cDNA was acquired by rapid-amplification of cDNA ends (RACE). And then its expression profiles were investigated by RT-PCR. Cathepsin O sequences from other species were downloaded from NCBI, the deduced amino acid sequences of putative BmCathpsin O were aligned using the Clustal X program, and the phylogenetic tree was constructed using MEGA 6.0 software. Specific primers were designed to amplify the high specificity fragment, and the PCR product was ligated to the PET32a vector, which was then transformed into Rosetta (DE3) E. coli to obtain recombinant protein by IPTG induction, BmCathepsin O recombinant protein was purified by Ni+ affinity chromatography, finally the recombinant protein was used to immue rabbit to get antibody. 【Result】 BmCathepsin O was clustered on nscaf2943 which was located on chromosome 14 in B. mori genome, and the gene number in SilkDB database was BGIBMGA009231. There are two spliced variants. Variant 1 has an ORF (open reading frame) of 1 071 bp, encodes 356 amino acids. The protein molecular weight is predicted to be 36 kD, and isoelectric point is 8.594. Variant 2 has an ORF of 942 bp, encodes 313 amino acids. The predicted protein molecular weight is 40 kD, isoelectric point is 7.951. Both the two variants are comprised of six exons and five introns, but partial deletion is occurred in the 6th exon of variant 2. Both variants contain a signal peptide, conserve Inhibitor I29 and Pept-C1 domain structure. Phylogenetic analysis showed that Cathepsin O from invertebrate was clustered alone, and BmCathepsin O was most closely related to Cathepsin O from Chilo suppressalis. RT-PCR results showed that Cathepsin O mRNA was specifically and continually expressed in B. mori hemocytes. The expression level of variant 1 was significantly higher than variant 2, but two variants had the same change tendency in hemocytes. BmCathepsin O recombinant protein was obtained by prokaryotic expression system and purified using Ni+ affinity chromatography, then the anti-rabbit polyclonal antibody was prepared by immuizing rabbit using purified protein. The titer of antiserum generated was 1﹕ 1 280 000 by ELISA. Western blot analysis showed that this antibody could bind with BmCathepsin O recombinant protein specifically.【Conclusion】BmCathepsin O was cloned and its expression patterns were investigated. The BmCathepsin O recombinant protein was prepared and purified, and finally anti-rabbit polyclonal antibody was acquired by immuizing rabbit.

Key words: Bombyx mori, BmCathepsin O gene, clone, expression analysis, antibody preparation

SU Jing-jing, CHEN Si-yuan, ZHANG Kui, YU Shuang, LI Yu-tian, LIANG Hang-hua, ZHAO Yu-zu, CHAO Hui-juan, CUI Hong-juan. Identification and Expression Analysis of Cathepsin O Gene in Silkworm (Bombyx mori)[J].Scientia Agricultura Sinica, 2015, 48(22): 4564-4573.

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Identification and Expression Analysis of Cathepsin O Gene in Silkworm (Bombyx mori)

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