摘要
利用PCR技术从质粒pSHIV89.6P中扩增猴免疫缺陷病毒(SIV)衣壳蛋白p27基因,并将其插入表达载体pET32a(+)构建重组质粒pET32a-p27,转化大肠杆菌BL21,经IPTG诱导,对其产物进行SDS-PAGE和Western blot分析,并将纯化定量后的表达产物免疫小鼠,制备单克隆抗体。结果显示,gag基因的p27相应片段为763bp,其表达产物相对分子量为46ku。经镍柱纯化,获得目的蛋白p27纯度可达90%以上。利用淋巴细胞杂交瘤技术获得5株稳定分泌抗p27的单克隆抗体细胞株,分别命名为1A5、1C7、3C2、2D12、3D12。5株杂交瘤细胞诱生小鼠产生的腹水抗体效价分别为1∶12800、1∶25600、1∶819200、1∶51200和1∶409600。免疫球蛋白类型均为IgG1型,轻链均为λ链。Western blot和IFA试验结果表明所获得的单克隆抗体均对病原检测具有很高的特异性,为进一步开发SIV早期诊断试剂盒奠定了基础。
The capsid p27 gag gene of SIVmac239 was cloned into the pET32a （＋） and expressed in E.coli BL21. The fusion protein was purified by Ni^2＋ affinity chromatography and used as antigen for monolconal antibody development. Five hybridoma cell lines that secreted monoclonal antibody （mAbs） against p27 were established and were designated as 1A5, 1C7, 3C2, 2D12 and 3D12. All these five mAbs were IgG1 subtype and consisted of λ light chain. The titers of mAbs in ascites were 1：12 800, 1：25 600, 1：819 200, 1：51 200 and 1：409 600, respectively. Furthermore, all these five mAbs reacted specifically with the capsid p27 protein of SHIV89.6P in Western blot and IFA.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2008年第11期901-905,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
哈尔滨市科技攻关项目(2006AA3AS040)
关键词
猴免疫缺陷病毒
P27蛋白
单克隆抗体
simian immunodeficiency virus
p27 protein
monoclonal antibody
