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

发布时间：2018-05-04 16:13

本文选题：家蚕MP基因 + 跨膜蛋白　；参考：《蚕业科学》2017年04期

【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。
[Abstract]:A 403bp unknown functional gene was screened from the c DNA library of silkworm pupae and named Bm MP54(Gen Bank accession number: DY231399.1. The gene sequence contains an open reading frame of 312bp and encodes a protein composed of 103 amino acid residues. Bioinformatics predicts that it contains two transmembrane regions with isoelectric point and molecular weight of 9.90 and 10.463 KD, respectively. The recombinant plasmids were constructed by fusion of Bm MP54 with polyhedrosis protein gene of Bombyx mori nuclear polyhedrosis virus (BmNPV) into prokaryotic expression vector p GEX-4T-1 and Bombyx mori baculovirus expression system transfer vector p Fast BacTMHTB, respectively. It was also expressed in Escherichia coli BL21DE3 and silkworm ovary culture cells BmNs, respectively. The prokaryotic expression fusion protein regulates the pH value, 65 KD recombinant fusion protein Polh-Bm MP54 was purified by anion exchange chromatography. The recombinant protein Bm MP54 was detected by Western blotting in BmN cells infected with recombinant baculovirus. The results show that the target protein has been expressed and purified according to the design requirements. Real-time fluorescence quantitative PCR analysis showed that the expression of BM MP54 was the highest in the fifth instar larvae of Bombyx mori and the lowest in the epidermis and head. These results are helpful to further study the biological function of Bm MP54 during the growth and development of Bombyx mori (Bombyx mori).
【作者单位】：浙江理工大学生命科学学院;浙江省家蚕生物反应器和生物医药重点实验室;
【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)
【分类号】：S884.51

【相似文献】