| 双峰驼血清IgG纯化及其抗血清制备 |
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| 引用本文：贾存瑜，韩熙梦，聂佳琪，张 欣，焦韵洁，刘宝元，周恩民，穆 杨.双峰驼血清IgG纯化及其抗血清制备[J].西北农业学报,2018,27(11):1584~1590 |
| DOI:10.7606/j.issn.1004-1389.2018.11.004 |
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| 基金项目:国家重点研发计划（2016YFD0500706）。 |
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| 中文摘要:纯化双峰驼血清 IgG并制备兔抗骆驼IgG抗血清，为制备针对特定蛋白的特异性纳米抗体储备材料，利用Protein G纯化双峰驼血清IgG，并用纯化的IgG免疫成年家兔后采用ELISA方法检测抗血清效价；采用间接ELISA方法，利用制备的抗血清检测PCV2 Cap蛋白免疫双峰驼血清中抗Cap蛋白的抗体水平，Western blot技术检测制备的抗血清与从噬菌体纳米抗体库中筛选到的针对不同蛋白的纳米抗体的反应性，以确定抗血清的适用性。结果显示，从双峰驼血清中纯化到骆驼IgG，SDS-PAGE分析显示纯化的骆驼IgG包括3条链，约50 ku的传统重链IgG1，约43 ku缺失CH1的重链IgG3和约30 ku的传统轻链；3次免疫后兔抗骆驼IgG抗血清效价达1∶512 000；应用制备的抗血清检测PCV2 Cap蛋白免疫5次的双峰驼血清中抗 Cap蛋白的抗体效价达1∶1 024 000；制备的抗血清可以与原核表达的针对不同病毒蛋白的纳米抗体反应；说明制备的兔抗骆驼IgG抗血清可用于免疫骆驼血清和表达的纳米抗体的检测。研究结果为目标蛋白免疫骆驼后抗体效价检测及纳米抗体文库构建提供关键材料。 |
| 中文关键词:骆驼 亲和层析 IgG 抗血清 纳米抗体 |
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| Purification of Camelus bactrianus Serum IgG and Preparation of the Antiserum |
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| Abstract:In order to purify IgG from Camelus bactrianus serum and product rabbit anti-camel IgG antiserum, and provide materials for the preparation of specific nanobodies against targeted proteins using phage display technology, IgG was purified from serum of Alashan Camelus bactrianus using Protein G and adult rabbits were immunized with purified IgG to product rabbit anti-camel IgG antiserum. The titers of antiserum were detected with enzyme linked immunosorbent assay(ELISA). Then the titer of antibody in porcine circovirus type 2(PCV2) Cap protein immunized camel serum was detected with indirect ELISA using the prepared antiserum and the reactogenicity of the prepared antiserum with nanobodies against different viral proteins was analyzed using western blot method. The results showed that camel IgG was purified from serum of Alashan Camelus bactrianus and SDS-PAGE analysis showed that the purified IgG includes 3 chains, including the conventional heavy chain IgG1 with molecular weight about 50 ku, the heavy chain IgG3 deleted CH1 with molecular weight about 43 ku, and the traditional light chain with molecular weight about 30 ku. The titer of rabbit anti-camel IgG antisera was 1∶512 000 after immunization 3 times. Antibody titer in serum of Alashan Camelus bactrianus immunized 5 times with PCV2 Cap protein was 1∶1 024 000 using the prepared antiserum. The prepared antiserum can react with nanobodies against different viral proteins, but did not react with monoclonal antibody(1E7) derived from ascites against PCV2 Cap protein. The results indicate that the prepared antiserum can be used to detect antibody titer of immunized camel and expressed nanobodies. |
| keywords:Camelus bactrianus Affinity chromatography IgG Antiserum Nanobody |
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