| 摘要: |
| 【目的】制备鼠抗猪繁殖与呼吸综合征病毒（PRRSV）细胞受体CD163 SRCR5-SRCR6多克隆抗体。【方法】提取PAM细胞总RNA，通过RT-PCR方法获得CD163 SRCR5-SRCR6(第1 417～2 136 bp，共720个bp)基因，将其克隆到pET-28a(+)载体上，构建重组表达载体pET-28a(+)-CD163，经酶切、PCR鉴定和测序验证后转化大肠杆菌，在BL21(DE3)中诱导重组目的蛋白表达，然后用Ni-NTA方法纯化目的蛋白，免疫Balb/c小鼠制备多克隆抗体，通过间接ELISA和IFA方法检测多克隆抗体效价，并初步鉴定多克隆抗体血清结合细胞及阻断PRRSV感染细胞的活性。【结果】克隆了720 bp的CD163 SRCR5-SRCR6基因，成功构建了pET-28a(+)-CD163重组表达载体；SDS-PAGE结果表明，CD163 SRCR5-SRCR6重组蛋白被成功诱导并以包涵体形式表达；Western blot结果表明，重组蛋白具有良好的免疫原性，间接ELISA方法测得免疫Balb/c小鼠制备的抗CD163 SRCR5-SRCR6重组蛋白的多克隆抗体效价可达105；IFA试验结果显示，制备的抗CD163 SRCR5-SRCR6重组蛋白的多克隆抗体可与Marc-145细胞结合，且在1∶20、1∶40倍稀释时，能部分阻断高致病性PRRSV SD16毒株感染Marc145细胞。【结论】用大肠杆菌原核表达系统成功诱导表达了CD163 SRCR5-SRCR 6重组蛋白，用重组蛋白免疫小鼠获得了高效价多克隆抗体，其可以结合细胞并阻断PRRSV感染细胞。 |
| 关键词: PRRSV CD163 CD163 SRCR5-SRCR6重组蛋白 多克隆抗体 |
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| 基金项目:国家自然科学基金项目（U0931003/L01）；国家“863”高技术研究发展计划项目（2011AA10A208） |
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| Preparation of polyclonal antibodies against PRRSV cellular receptor CD163 SRCR5-SRCR6 |
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MA Yu-ping,KONG Ning,WANG Xiang-peng,et al
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| Abstract: |
| 【Objective】The objective of the study was to prepare the murine polyclonal antibodies against the recombinant protein of cellular receptor CD163 SRCR5-SRCR6 of PRRSV.【Method】Total RNA was extracted from PAM cells and CD163 SRCR5-SRCR6 gene was amplified by RT-PCR and cloned into pET-28a (+) vector.Then,the positive recombinant plasmid was transformed into BL21 (DE3) competent cells to express the recombinant protein.The recombinant protein was identified by SDS-PAGE and Western blot,and purified by Ni-NTA method.To produce polyclonal antibodies,Balb/c mice were immunized with the purified protein.The titres of antibodies were detected by ELISA and the blocking functions of antibodies to PRRSV infected permissive cells were characterized by IFA.【Result】The recombinant plasmid containing CD163 SRCR5-SRCR6 gene named pET-28a(+)-CD163 with 720 bp was successfully constructed.SDS-PAGE analysis results showed that the recombinant protein was expressed in the form of inclusion body and Western blot results showed that the protein had immunogenicity.The titer of anti-CD163 SRCR5-SRCR6 polyclonal antibodies was 105 as determined by indirect-ELISA.IFA results showed that the polyclonal antibodies could combine Marc-145 cells and block Marc-145 cells infected with high pathogenic PRRSV SD16 strain when diluted by 1∶20 and 1∶40.【Conclusion】CD163 SRCR5-SRCR6 recombinant protein was expressed.Polyclonal antibodies,which could combine Marc-145 cells and block PRRSV infection,were obtained by immunizing mice with the recombinant protein. |
| Key words: PRRSV CD163 gene protein encoded by CD163 SRCR5-SRCR6 gene polyclonal antibody |
