【摘要】 【目的】制备抗PthA-NLS多肽单克隆抗体并构建其单链抗体(single chain variable fragment,ScFv)基因,为从PthA角度进一步研究柑橘溃疡病致病机理创造条件,并为构建单链抗体基因植物表达载体,尝试利用转基因和植物抗体技术创制抗溃疡病柑橘新种质奠定基础。【方法】利用PthA-NLS重组多肽免疫Balb/c小鼠,制备抗PthA-NLS多肽单克隆抗体,从分泌抗PthA-NLS多肽单克隆抗体的杂交瘤细胞中提取总RNA,采用RT-PCR和重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)法构建其单链抗体基因,并克隆到pGEM-T载体和原核表达载体pET32a(+)中,重组原核表达质粒转化大肠杆菌BL21(DE3)后诱导重组蛋白的表达。【结果】获得了3株能稳定分泌抗PthA-NLS多肽的单克隆抗体杂交瘤细胞株1C8H1、2D12B6、3D8A10。成功构建了单链抗体基因ScFv,获得的ScFv基因大小约750bp,其中重链可变区(VH)基因有360bp,轻链可变区(VL)基因有342bp,中间连接肽基因45bp,经过IPTG诱导ScFv原核表达,获得了大小为44.5kD的ScFv重组蛋白。【结论】试验获得了3株能稳定分泌抗PthA-NLS多肽的单克隆抗体杂交瘤细胞株,成功构建了ScFv基因,并实现了原核表达,为进一步探索PthA的致病机理和下一步利用抗体技术获得抗溃疡病柑橘种质[0]的研究打下了基础。更多还原

【Abstract】 【Objective】 The present study aimed at preparation of monoclonal antibody against the recombinant PthA-NLS and isolaton of the relative ScFv(Single Chain Variable Fragment) genes,providing a possibility to better understand the pathogenesis mechanism via PthA,and to develop proper construct for future experimentation to obtain citrus plants resistant to citrus canker disease by transformation and plant antibody techniques.【Method】 The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody.Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti-PthA-NLS McAb with Trizol reagent,and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR(splicing by overlap extension),and then the ScFv gene was constructed.The recombinant ScFv gene was cloned into pGEM-T and pET32a(+) vector.The later plasmid was transferred into E.coli BL21(DE3) and the expression of the recombinant protein was induced.【Result】 Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1,2D12B6 and 3D8A10.The recombinant ScFv gene of about 750 bp was constructed.The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain,a 342 bp light chain and a 45 bp linker region.The recombinant fusion ScFv protein was expressed by IPTG induction,and a 44.5 KDa of recombinant fusion protein was obtained.【Conclusion】 Three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS were obtained,and the relative ScFv gene was constructed and successfully expressed in E.coli.These results may play an important role in further understanding of the pathogenesis mechanismand in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.更多还原