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

摘要/Abstract

摘要： 【目的】克隆家蚕（Bombyx mori）蛋白酶O基因Cathepsin O（BmCatO），分析其mRNA表达特征，通过原核表达、蛋白纯化和多次免疫新西兰大白兔获得多克隆抗体，为进一步探究组织蛋白酶O在家蚕变态发育中的功能奠定基础。【方法】利用RACE技术克隆获得家蚕组织蛋白酶O的全长cDNA序列,然后采用RT-PCR对其时空表达特征进行研究。从NCBI下载其他物种组织蛋白酶O基因序列，利用Clustalx和MEGA 6.0软件进行同源比对和进化分析。选取特异性较高的片段设计特异性引物，将PCR扩增产物连接至PET32a质粒，转化至E. coil表达菌株Rosetta(DE3)，经IPTG诱导获得组织蛋白酶O重组蛋白，然后利用Ni+亲和层析方法对重组蛋白进行纯化，最后多次免疫新西兰大白兔获得多克隆抗体。【结果】家蚕组织蛋白酶O基因存在于家蚕第14号染色体上，scaffold为nscaf2943，基因编号BGIBMGA009231。该基因有两种剪切形式，剪切体1开放阅读框（ORF）全长为1 071 bp，编码356个氨基酸，预测蛋白分子量36 kD,等电点8.594；剪切体2 ORF全长为942 bp，编码313个氨基酸，预测蛋白分子量40 kD，等电点7.951。两种剪切体均有6个外显子和5个内含子构成，剪切体2的第六外显子出现部分缺失。两种剪切体编码的蛋白结构相似，均含有信号肽、Inhibitor129和Pept-C1结构域。进化分析结果表明，无脊椎动物组织蛋白酶单独聚为一类，且家蚕组织蛋白酶O与同为鳞翅目成员的二化螟（Chilo suppressalis）组织蛋白酶O最为接近。RT-PCR结果显示该基因特异性持续表达于幼虫血细胞，剪切体1的表达水平明显高于剪切体2，但这两种剪切体在幼虫血细胞发育时期表达趋势一致。构建家蚕组织蛋白酶O原核表达系统，利用亲和层析纯化获得高纯度的家蚕组织蛋白酶O重组蛋白，并利用纯蛋白免疫新西兰大白兔制备多克隆抗体，ELISA显示其效价高达到1﹕1 280 000。Western印迹结果表明，该抗血清可以特异性识别家蚕组织蛋白酶O重组蛋白。【结论】获得了家蚕组织蛋白酶O基因的完整cDNA序列及表达特征，经原核表达、纯化获得高纯度的融合蛋白，通过多次免疫新西兰大白兔成功获得抗体。

关键词: 家蚕, 蛋白酶O基因, 克隆, 时空表达, 抗体制备

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

苏晶晶，陈思源，张奎，余霜，李钰添，梁航华，赵羽卒，晁会娟，崔红娟. 家蚕组织蛋白酶O（BmCatO）基因鉴定及表达分析[J]. 中国农业科学, 2015, 48(22): 4564-4573.

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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