| 小鼠抗HCMV-gB多克隆抗体的制备及初步应用 |
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| 引用本文： | 韩慧平,于三科.小鼠抗HCMV-gB多克隆抗体的制备及初步应用[J].动物医学进展,2012(2):51-54. |
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| 作者姓名： | 韩慧平 于三科 |
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| 作者单位： | 西北农林科技大学动物医学院 |
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| 摘 要： | 为了快速制备抗HCMV-gB小鼠多克隆抗体,通过PCR扩增HCMV-gB(57aa～146aa)编码序列并克隆到原核表达载体pET21b(+)多克隆位点,用重组质粒转化大肠埃希菌Rosetta(DE3)并通过IPTG诱导表达,经镍胶亲和层析纯化和透析复性获得重组蛋白。以CpG-ODN和Al(OH)3复合佐剂作为重组蛋白的免疫佐剂,通过0周和3周2次肌肉注射免疫Balb/c小鼠,并在第5周采血分离免疫血清。用ELISA检测免疫血清效价,并通过Western blot检测免疫血清对哺乳动物细胞表达的HCMV-gB1和gB2的反应性。结果显示,本研究制备的免疫血清ELISA滴度达到1∶51 200～1∶102 400,进行Western blot能够与哺乳动物细胞表达的HCMV-gB1和gB2发生特异性反应。此试验获得了能够用于后续研究工作的抗HCMV-gB小鼠多克隆抗体,本方法具有制备速度快、抗体滴度高和抗原用量少等优点。
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| 关 键 词： | CpG-ODN Al(OH)3 免疫佐剂 多克隆抗体 |
Preparation of Polyclonal Antibodies Against Glycoprotein B of Human Cytomegalovirus in Mouse |
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| HAN Hui-ping,YU San-ke.Preparation of Polyclonal Antibodies Against Glycoprotein B of Human Cytomegalovirus in Mouse[J].Progress In Veterinary Medicine,2012(2):51-54. |
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| Authors: | HAN Hui-ping YU San-ke |
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| Institution: | (College of Veterinary Medicine,Northwest A & F University,Yangling,Shaanxi,712100,China) |
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| Abstract: | The present study was designed for rapid preparation of polyclonal antibodies against glycoprotein B(gB) of human cytomegalovirus(HCMV).The coding sequence of HCMV-gB(57 aa-146 aa) was amplified by PCR and cloned into prokaryotic expression vector pET21b(+).The expression of the recombinant protein was induced with IPTG after transformation of the recombinant plasmid into E.coli Rosetta(DE3).The recombinant protein was obtained through purification by Ni metal-chelating chromatography and refolding by dialysis.Balb/c mice were immunized intramuscularly on day 0 and boosted 3 weeks later with CpG-ODN and Al(OH)3 as adjuvants,and the immune serum was collected 2 weeks after boosting.The ELISA titers of anti-serum could reach as high as 1∶51 200-1∶102 400.This polyclonal antibody was HCMV-gB specific as showed in Western blot analysis.The method described in the present study has the advantages of time-saving,high antibody titers and antigen-sparing. |
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| Keywords: | CpG-ODN Al(OH)3 adjuvant polyclonal antibody |
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