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猪瘟病毒E2蛋白主要抗原区编码基因的原核表达
及其单克隆抗体的制备
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侯强，彭伍平，孙元，仇华吉*
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（中国农业科学院 哈尔滨兽医研究所 兽医生物技术国家重点实验室，黑龙江 哈尔滨150001）
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摘要：
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利用RT-PCR扩增了猪瘟病毒（CSFV) 兔化弱毒疫苗株（C株）的E2蛋白主要抗原区（tE2）编码区，并定向克隆到表达载体pPROEX-HTb中，获得重组表达载体pPROEX-tE2，将其转化大肠杆菌DH5α菌株，经IPTG诱导，tE2蛋白获得高效表达；经检测，该重组蛋白能被CSFV C株抗血清识别。用纯化的重组蛋白免疫BALB/c小鼠，取其脾淋巴细胞与SP2/0骨髓瘤细胞融合，获得了1株稳定分泌抗tE2蛋白单克隆抗体的杂交瘤细胞株。经检测，该单克隆抗体能够与CSFV C株和石门株以及杆状病毒表达的重组E2蛋白发生特异性反应。推测，该单克隆抗体是针对CSFV E2蛋白的一个保守线性表位。 |
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关键词：
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猪瘟病毒；E2蛋白；原核表达；单克隆抗体 |
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中图分类号：S 852.659.6 文献标识码：
A 文章编号：1673-4696(2008)01-0001-05 |
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Expression of the truncated E2 protein-encoding gene of classical swine
fever virus in Escherichia coli and preparation of a monoclonal
antibody against E2 protein |
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HOU Qiang,PENG Wu-ping,SUN Yuan,QIU Hua-ji |
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(National Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences,Harbin 150001,China)
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Abstract: A truncated gene encoding the major antigenic domains of E2 protein of classical swine fever virus(CSFV) was amplified by RT-PCR from the genomic RNA of CSFV C-strain and cloned into pPROEX-HTb expression vector to obtain recombinant pPROEX-tE2.The truncated E2 protein(tE2) was expressed with high level in pPROEX-tE2-transformed Escherichia coli cells after induction with IPTG.The recombinant protein could be recognized by CSFV C-strain antisera in either Western-blotting or ELISA.The tE2 protein purified using a Ni-chelating HisTrap affinity column was used to immunize BALB/c mice,of which splenocytes were fused with SP2/0 cells with PEG3250 after the 3rd immunization.A hybridoma cell line stably secreting a monoclonal antibody(McAb) directed against the tE2 protein was screened by ELISA.The McAb was able to react specifically with C-strain and Shimen strain,and recognized a linear conserved epitope on the E2 protein.The McAb was identified to be IgG1 subtype. |
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Key words: classical swine fever virus(CSFV);E2 protein;prokaryotic expression;monoclonal antibody |
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