人血管生成素相关蛋白2单克隆抗体的制备与鉴定
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国家自然科学基金(30171029).


Preparation and identification of monoclonal antibody against angiopoietin-related protein 2
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Supported by National Natural Science Foundation of China(30171029).

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    摘要:

    目的:制备人血管生成素相关蛋白2(angiopoietin-related protein 2,ARP2)的单克隆抗体.方法:用RT-PCR方法获取人脾脏组织ARP2基因序列,构建pET32a-ARP2,经电穿孔导入大肠杆菌诱导表达,获得可溶蛋白样品和包涵体蛋白样品,回收、纯化蛋白,免疫BALB/c小鼠,采用杂交瘤技术建立产生ARP2抗杂交瘤细胞株,Western印迹鉴定.结果:获得融合蛋白的相对分子质量为570 000,与理论计算值相符,纯化后蛋白浓度>90﹪,杂交瘤细胞培养上清抗体效价为1:104,腹水抗体效价为1:107~1:108,抗体亚型为IgG2a类,Western印迹示目的蛋白相对分子质量为570 000,免疫组化显示该抗体能特异结合人ARP2.结论:制备的抗ARP2单克隆抗体可用于ARP2蛋白鉴定.

    Abstract:

    Objective:To prepare a monoclonal antibody against human angiopoietin-related protein 2(ARP2). Methods: Human spleen ARP2 gene was obtained by RT-PCR. A pET32a-ARP2 plasmid was constructed and was incorporated into E. coli. The products were purified and were used to immunize 6-week-old BALB/c female mice. Hybridoma secreting antiARP2 monoclonal antibody was obtained by standard procedure. Mass production was carried out after specificity identification with Western blotting. Results: The fusion protein obtained by pET32a system had a relative molecular weight of about 570 000, which was in accordance with the theoretical value. The purity of the protein was more than 90% after purification. The antibody titer was 1 : 104 in the hybridoma culture supernatant and 1 : 107-10s in the ascites. The IgG2a type antibody had a relative molecular weight of about 570 000 by Western blot analysis. Immunohistochemistry method showed that the antibody bond with human ARP2. Conclusion: The prepared anti-human ARP2 monoclonal antibody in this study can be used for identification of ARP2 protein.

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  • 收稿日期:2005-12-19
  • 最后修改日期:2006-04-24
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  • 在线发布日期: 2006-07-20
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