Yin Wei, Wei Xiaoming, Jiang Junbing, Fan Kuohai, Zhao Junxing, Sun Na, Wang Zhiwei, Sun Yaogui, Ma Haili, Zhao Xin, Li Hongquan
College of Animal Science and Veterinary Medicine, Shanxi Agriculture University, Taigu, 030801, Shanxi, People's Republic of China.
Immunol Res. 2016 Aug;64(4):1025-32. doi: 10.1007/s12026-016-8792-z.
Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8 kDa and 30 kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes.
灵长类补体受体1（CR1）蛋白是一种单链跨膜糖蛋白，在免疫黏附及清除补体调理的免疫复合物中发挥重要作用。在此，对猪灵长类样补体受体（CR1样）基因的mRNA进行了分析，并将两个具有潜在功能的结构域序列克隆到pwPICZalpha载体中，以便在毕赤酵母中表达。重组蛋白通过蛋白纯镍-亚氨基二乙酸树脂和强阴离子交换树脂进行纯化。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、蛋白质印迹法和补体受体测定来评估纯化后重组蛋白的活性。结果表明，CR1样蛋白的两个结构域，即CCP36和CCP811，分子量分别为29.8 kDa和30 kDa，已在毕赤酵母中成功表达。这两种重组蛋白具有灵长类CR1蛋白的某些功能。利用这两种蛋白结合抗体阻断技术，我们还证明了CR1样蛋白在天然猪红细胞上表达。
