家蚕蜕皮液羧肽酶A的表征及免疫荧光定位分析

Characterization and immunofluorescence localization analysis of carboxypeptidase A in molt fluid of silkworm

摘要:

蜕皮是许多变态发育昆虫的一种重要生理现象，昆虫通过蜕皮液中的酶对新旧表皮进行分离。已有相关蛋白组学的研究证明，家蚕蜕皮液中具有一种含量丰富的羧肽酶A (Bombyx mori-carboxypeptidase A, Bm-CPA)，目前对其作用功能尚不清楚。为了更好地了解Bm-CPA在家蚕蜕皮发育过程的作用，本研究通过生物信息学分析、实时荧光定量PCR、抗体制备、免疫荧光染色和毕赤酵母表达等方法对Bm-CPA进行了研究。结果显示，Bm-CPA具有保守的M14锌羧肽酶结构域和糖基化位点，并且受蜕皮激素(20-hydroxyecdysone, 20E)调控，在眠期和上簇期的表皮中大量表达；免疫荧光染色显示Bm-CPA在眠期的表皮中富集，Bm-CPA抑制剂会导致幼虫因无法蜕皮而死亡；通过毕赤酵母表达系统在体外成功获得大量的重组Bm-CPA蛋白。这些结果为深入了解家蚕蜕皮发育过程提供了一定的参考。

Abstract:

Molting is an important physiological phenomenon of many metamorphosis insects, during which the old and new epidermis are separated by enzymes present in the molting fluid. Various proteomic studies have discovered the presence of Bombyx mori carboxypeptidase A (Bm-CPA) in the molting fluid of silkworm, but its function remains unclear. In order to better understand the role of Bm-CPA in the molting process of silkworm, Bm-CPA was analyzed by bioinformatics analysis, real-time fluorescence quantitative PCR, antibody preparation, immunofluorescence staining, and expression in Pichia pastoris. The results showed that Bm-CPA had a conserved M14 zinc carboxypeptidase domain and glycosylation site. Its expression was regulated by ecdysone 20E, and large expression was observed in the epidermis of the upper cluster stage. Immunofluorescence staining showed that Bm-CPA was enriched in the epidermis during the molting stage, and the inhibitor of Bm-CPA led to the larval death due to the inability to molt. We also successfully obtained a large number of recombinant Bm-CPA proteins by Pichia pastoris expression in vitro. These results may facilitate further understanding the molting development process of silkworm.