家蚕组织蛋白酶O(BmCatO)基因鉴定及表达分析  

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

作　　者：苏晶晶[1] 陈思源[1] 张奎[1] 余霜[1] 李钰添 梁航华 赵羽卒 晁会娟 崔红娟[1] 

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716

出　　处：《中国农业科学》2015年第22期4564-4573,共10页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划("973"计划)(2012CB114600);国家自然科学基金(31172268)

摘　　要：【目的】克隆家蚕(Bombyx mori)蛋白酶O基因Cathepsin O(Bm Cat O),分析其m RNA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c DNA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 k D,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 k D,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【Objective】The objectives of this study are to clone Cathepsin O gene（Bm Cat O） of silkworm（Bombyx mori）,analyze its m RNA 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 Bm Cathepsin O c DNA was acquired by rapid-amplification of c DNA 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 Bm Cathpsin 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 PET32 a vector,which was then transformed into Rosetta（DE3） E. coli to obtain recombinant protein by IPTG induction,Bm Cathepsin O recombinant protein was purified by Ni＋ affinity chromatography,finally the recombinant protein was used to immue rabbit to get antibody. 【Result】 Bm Cathepsin O was clustered on nscaf2943 which was located on chromosome 14 in B. mori genome,and the gene number in Silk DB 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 k D,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 k D,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 Bm Cathepsin O was most closely related to Cathepsin O from Chilo suppressalis. RT-PCR results

关 键 词：家蚕 蛋白酶O基因 克隆 时空表达 抗体制备 

分 类 号：S881.2[农业科学—特种经济动物饲养]