发菜NdhK的抗体制备与蛋白表达

发菜是一种陆生蓝藻，分布于一些干旱和半干旱区域。其NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体，参与CO₂吸收、围绕光系统Ⅰ的循环电子传递和细胞呼吸。为研究该物种中ndhK基因的功能，本研究利用特异性引物，通过PCR方法从发菜中扩增ndhK基因并克隆到原核表达载体pET-32a上，得到表达载体pET-32a-ndhK，将其转入大肠杆菌BL21(DE3)，经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达，得到分子量大小为43 kDa的融合蛋白NdhK。随后，采用亲和层析，对融合蛋白进行纯化回收，并以此回收蛋白作为抗原进行免疫，制备NdhK的多克隆抗体。最后，利用Western blot蛋白免疫印迹对所得抗体的特异性进行验证。从而为进一步探索发菜ndhK基因的功能以及发菜中NDH-1复合体各亚基的作用进行前期准备。

Antibody Preparation and Protein Expression of NdhK in Nostoc flagelliforme

Nostoc flagelliforme,a terrestrial cyanobacterium,is distributed in arid and semi-arid steppes of the west and northwest of China.Cyanobacterial NADPH dehydrogenase(NDH-1) is an important photosynthetic membrane protein complex,and is essential to CO2 uptake,cyclic electron transport around photosystem Ⅰ and cellular respiration.However,little is know about the function roles of NdhK subunit in cyanobacteria.In this study,the ndhK gene was PCR amplified from the N.flagelliforme,and the expression plasmid pET32a-ndhK was generated and transformed into BL21(DE3)pLysS,and the expression of NdhK protein was induced by IPTG.After purification,the fusion protein pET-NdhK was used to immunize Japanese white rabbit to obtain the polyclonal antibody.The titer of the polyclonal antibody was detected and analyzed by Western blotting,the result showed that the polyclonal antibody possessed a high specificity.Therefore,the antibody of NdhK obtained in this study will further help us to reveal the functional roles of cyanobacterial NdhK subunit.