Han Xiaodong, Jia Tianjun, Jia Xiaohui, Li Ting, Zhao Xia
Institute of Pathogen Biology and Immunology, School of Laboratory Medicine, Hebei North University, Zhangjiakou 075000, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 May;31(5):689-92, 696.
To express CUB1 and CUB2 functional domain proteins of mannan-binding lectin-associated serine protease-2 (MASP-2) by prokaryotic expression system, and further prepare and identify the polyclonal antibodies against these two domains.
The pGEX-6P-2 prokaryotic vector carrying the target gene CUB1 and CUB2 was used to prepare the recombinant protein GST-CUB1 and GST-CUB2. These two fusion proteins were purified with Glutathione SepharoseTM4B beads and then combined with Freund's adjuvant as antigens to immunize the BALB/c female mice aged 5 weeks for generating polyclonal antibodies. Subsequently, the specificity of the polyclonal antibodies was detected by Western blotting, and the titer was determined by indirect ELISA.
The fusion proteins GST-CUB1 and GST-CUB2 were successfully expressed and purified. Their polyclonal antibodies were gained from the BALB/c mice immunized with these two proteins. The polyclonal antibodies showed high specificity with no cross-reaction with other proteins. Indirect ELISA indicated that the titer of anti-CUB1 antibody was more than 1:32 000 and anti-CUB2 antibody was more than 1:16,000.
The polyclonal antibodies against GST-CUB1 and GST-CUB2 fusion proteins were obtained respectively with high titers and strong specificity.
利用原核表达系统表达甘露聚糖结合凝集素相关丝氨酸蛋白酶-2（MASP-2）的CUB1和CUB2功能域蛋白，并进一步制备和鉴定针对这两个结构域的多克隆抗体。
使用携带目标基因CUB1和CUB2的pGEX-6P-2原核载体，制备重组蛋白GST-CUB1和GST-CUB2。用谷胱甘肽琼脂糖凝胶4B珠对这两种融合蛋白进行纯化，然后与弗氏佐剂混合作为抗原，免疫5周龄的BALB/c雌性小鼠以产生多克隆抗体。随后，通过蛋白质印迹法检测多克隆抗体的特异性，通过间接酶联免疫吸附测定法测定抗体效价。
成功表达并纯化了融合蛋白GST-CUB1和GST-CUB2。用这两种蛋白免疫BALB/c小鼠后获得了它们的多克隆抗体。多克隆抗体显示出高特异性，与其他蛋白无交叉反应。间接酶联免疫吸附测定法表明，抗CUB1抗体效价大于1:32 000，抗CUB2抗体效价大于1:16 000。
分别获得了针对GST-CUB1和GST-CUB2融合蛋白的高滴度、强特异性的多克隆抗体。
