家蚕V型ATP酶B亚基的克隆及表达特征

Gene cloning and expression characteristics of vacuolar-type ATPase subunit B in Bombyx mori

摘要:

V型ATP酶 (Vacuolar-type ATPase) 是一种定位于细胞膜和细胞器膜上的氢离子转运酶。它利用ATP水解的能量将氢离子转运到液泡、囊泡或者胞外，从而维持细胞内正常的酸碱环境。V型ATP酶B亚基 (V-ATPase B) 作为ATP的催化位点，也有着非常重要的作用。为了探讨家蚕V-ATPase B (BmV-ATPase B) 的功能，首先从家蚕五龄幼虫的中肠cDNA中克隆了BmV-ATPase B基因并构建原核表达载体进行原核表达，获得了重组蛋白，经质谱鉴定正确后，通过镍柱亲和层析的方法纯化了该蛋白并制备了多克隆抗体；最后分析了该蛋白在家蚕丝腺中的表达特征并利用免疫荧光对其在丝腺中的表达位置进行了定位。结果显示BmV-ATPase B基因序列全长1 473 bp，预测蛋白分子量55 kDa，预测等电点5.3。通过Western blotting对家蚕5龄第3天和上蔟第1天幼虫丝腺的不同区段进行BmV-ATPase B蛋白的表达特征分析，发现在两个时期该蛋白均在前部丝腺高量表达，而在中部丝腺和后部丝腺表达量相对较低。进一步对两个时期丝腺的不同区段进行免疫荧光定位，发现该蛋白在两个时期的前部丝腺、中部丝腺和后部丝腺均定位于细胞层。利用激光共聚焦显微镜对该蛋白进行进一步的定位，发现该蛋白主要在丝腺的细胞膜表达。研究结果明确了该蛋白在丝腺中的表达模式，为深入研究该蛋白在蚕丝纤维形成中的作用奠定了基础。

Abstract:

Vacuolar-type ATPase (V-ATPase), located in the membrane and organelle membrane, is one of important H+-transporting proteins. It keeps the proton balance by transporting H+ into vacuole, vesicle, or extracellular using the energy from ATP hydrolysis. The subunit B of the vacuolar-type ATPase (BmV-ATPase B) contains the ATP catalytic site, and plays an important role in this process. To study the function of V-ATPase B in Bombyx mori (BmV-ATPase B), we cloned its coding gene from the midgut of the 5th instar silkworm larvae. Then we constructed prokaryotic expression vector and produced the recombinant protein in E. coli. The recombinant protein was identified as BmV-ATPase B by mass spectrometry and puri?ed using Ni-NTA affinity chromatography. This purified protein was used to immunize rabbit to generate polyclonal antibodies of BmV-ATPase B. Finally, the expression patterns of BmV-ATPase B in the silk gland were analyzed by western blotting and immunofluorescence. The full length CDS sequence of BmV-ATPase B was 1 473 bp. BmV-ATPase B was 55 kDa with a PI of 5.3. We analyzed the expression patterns of BmV-ATPase B in different sections of silk gland from the silkworm on the 3rd day of 5th instar and 1st day of wander stage by western blotting. BmV-ATPase B was expressed in all sections of the silk gland and it was abundant in the anterior silk gland (ASG) both in these two developmental stages. Furthermore, immunofluorescence indicated that BmV-ATPase B was located in the silk gland cells. Laser confocal scanning microscopy analysis revealed that BmV-ATPase B was mainly expressed in the cytomembrane of silk gland cells. These data elucidated the expression patterns of BmV-ATPase B in the silk gland of silkworm, which provides a good basis for further studies on the function of V-ATPase B in silk fiber formation.