BmMP54基因在家蚕组织的表达模式及与BmNPV多角体蛋白基因的融合表达分析  

Expression Pattern of Bm MP54 Gene in Silkworm Tissues and Its Fusion Expression with Bm NPV Polyhedrin Gene

作　　者：严婷婷[1,2] 于滨[1,2] 潘海涛[1,2] 舒建洪[1,2] 张耀洲[1,2] 

机构地区：[1]浙江理工大学生命科学学院,杭州310018 [2]浙江省家蚕生物反应器和生物医药重点实验室,杭州310018

出　　处：《蚕业科学》2017年第4期587-593,共7页ACTA SERICOLOGICA SINICA

基　　金：国家高技术研究发展计划"863"项目(No.2007AA021703);"十二五"国家科技重大新药创制专项(No.2012ZX09102301-9)

摘　　要：从家蚕蛹c DNA文库中筛选到一个403 bp的未知功能基因,将其命名为Bm MP54(Gen Bank登录号:DY231399.1)。该基因序列包含312 bp的开放阅读框,编码由103个氨基酸残基组成的蛋白质,生物信息学预测其含有2个跨膜区域,等电点及分子质量分别为9.90和10.463 k D。将Bm MP54与家蚕核型多角体病毒(Bm NPV)的多角体蛋白基因(Polh)融合后分别连接到原核表达载体p GEX-4T-1和家蚕杆状病毒表达系统转移载体p Fast BacTMHTB中构建重组质粒,并分别在大肠埃希菌BL21(DE3)和家蚕卵巢培养细胞(Bm N)中表达。原核表达的融合蛋白通过调节p H值、阴离子交换层析等纯化获得了65 k D的重组融合蛋白Polh-Bm MP54,通过Western blotting可在重组杆状病毒感染的Bm N细胞中检测到42 k D左右的Bm MP54重组蛋白,表明已按设计要求高效表达并纯化获得目的蛋白质。实时荧光定量PCR分析显示Bm MP54在家蚕5龄幼虫的气门和脂肪体组织中表达量最高,在表皮和头部的表达量最低。上述研究结果有利于后续探讨Bm MP54在家蚕生长发育过程中的生物学功能。A 403 bp unknown functional gene sequence was screened from the c DNA library of silkworm（ Bombyx mori）pupae and named Bm MP54（ Gen Bank accession number： DY231399.1）. This gene sequence contains a 312 bp ORF and encodes a protein of 103 amino acid residues. Bioinformatic analysis indicated that it contains two transmembrane regions,has an isoelectric point of 9. 90 and a molecular mass of 10. 463 k D. Fusion gene of Bm MP54 and Bm NPV polyhedrin gene（ Polh） was cloned into prokaryotic expression vector p GEX-4T-1 and Bombyx mori baculovirus expression transfer vector p Fast BacTMHTB for construction of recombinant plasmids,which were then expressed in E.coli BL21（ DE3） and silkworm ovarian cultured cells（Bm N） respectively. A 65 k D recombinant fusion protein Polh-Bm MP54 expressed in E. coli BL21（ DE3） was obtained through adjusting p H value and employing anion exchange chromatography. And recombinant Bm MP54 protein was detected by Western blotting in Bm N cells infected with recombinant baculovirus,indicating that the target protein had been highly expressed and purified.Real-time fluorescence quantitative PCR analysis showedthat Bm MP54 had the highest expression level in spiracle and fat body of the 5th instar silkworm larvae,and had the lowest expression in epidermis and head. The above results provide a good basis for follow-up exploration on biological functions of Bm MP54 in silkworm growth and development.

关 键 词：家蚕MP54基因 跨膜蛋白 家蚕核型多角体病毒多角体蛋白基因 融合表达 实时荧光定量PCR 表达谱 

分 类 号：S884.51[农业科学—特种经济动物饲养]