家蚕丝氨酸蛋白酶抑制剂20的表达特征分析
Expression of serine protease inhibitor 20 in the silkworm,Bombyx mori
张学义** 于 皓 张小娟 朱保建 魏国清钱 岑 刘朝良 王 磊***
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DOI：10.7679/j.issn.2095-1353.2015.112
作者单位：安徽农业大学生命科学学院，合肥 230036
中文关键词：家蚕，丝氨酸蛋白酶抑制剂，诱导表达，表达分析
英文关键词：Bombyx mori, serine protease inhibitor, induced expression, expression analysis
中文摘要：
【目的】 原核表达家蚕Bombyx mori丝氨酸蛋白酶抑制剂20（Serine proteinase inhibitor 20，BmSerpin-20），分析BmSerpin-20基因和蛋白组织表达分布特点，以及不同外源病原物刺激家蚕后BmSerpin-20基因的表达模式。【方法】 利用pET-28a表达载体在大肠杆菌Transetta(DE3)中融合表达BmSerpin-20蛋白，利用纯化的重组蛋白作为抗原免疫新西兰大白兔制备多克隆抗体，利用实时定量PCR技术及Western blot方法分别分析BmSerpin-20基因和蛋白的组织表达水平，以及病原物免疫刺激后的表达模式。【结果】 在大肠杆菌中正确表达和纯化了融合BmSerpin-20蛋白，并制备了多克隆抗体；BmSerpin-20基因和蛋白在脂肪体、中肠、血细胞、马氏管、表皮、睾丸、卵巢中均有表达，且在表皮中表达量最高；家蚕5龄幼虫经核型多角体病毒（Nuclear polyhedrosis viruses）、藤黄微球菌Micrococcus luteus、大肠杆菌Escherichia coli、白僵菌Beauveria bassiana处理后，BmSerpin-20基因表达量先下调而后上调, 但不同病原物在不同时间的影响不尽相同。【结论】 研究了BmSerpin-20基因和蛋白的表达模式，为下一步研究其在家蚕免疫系统中的作用奠定了基础。
英文摘要：
[Objectives] To examine the expression of the Bombyx mori serine proteinase inhibitor 20 gene (BmSerpin-20) in Escherichia coli; and more specifically, to analyze its mRNA and protein expression levels in tissues, and its mRNA expression patterns in fat bodies after exogenous microorganism injection. [Methods] The BmSerpin-20 gene was inserted into the pET-28a vector. Recombinant plasmids were transfected into E. coli Transetta (DE3), and expression of the BmSerpin-20 protein induced with IPTG. The recombinant BmSerpin-20 protein was employed as an antigen to immunize New Zealand rabbits and to prepare a polyclonal antibody serum. The expression patterns of BmSerpin-20 were analyzed using real-time quantitative PCR and the Western blot technique. [Results] The BmSerpin-20 protein was successfully expressed in E. coli cells, purified and a polyclonal antibody against BmSerpin-20 prepared. The BmSerpin-20 gene and protein were expressed in the fat body, midgut, hemocyte, malpighian, integument, testis, ovary, with the highest expression in the integument. After treatment of B. mori larvae with nuclear polyhedrosis viruses, Micrococcus luteus, E. coli or Beauveria bassiana, the expression patterns of the BmSerpin-20 gene in the fat body was first down-regulated and then up-regulated. The expression level of BmSerpin-20 differed depending on the microorganism injected and injection time. [Conclusion] The expression pattern of BmSerpin-20 was successfully studied and may lay a foundation for further research on the function of this gene in the immune system of B. mori.