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。
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.
出处
《动物医学进展》
CSCD
北大核心
2012年第2期51-54,共4页
Progress In Veterinary Medicine
