Cry2A毒素抗独特型单链抗体库的构建

Construction of the anti-idiotypic single-chain variable fragment(scFv)library of Cry2A toxin

为了降低苏云金杆菌Cry2A毒素的免疫检测成本,开发廉价、无毒试剂盒,采用整体抗体免疫和噬菌体展示技术的路线,设计和构建了Cry2A抗独特型单链抗体库。以Protein A纯化的Cry2A兔多克隆抗体为免疫原,对雌性Balb/c系小鼠进行了免疫。在优化的包被原浓度下,用ELISA法测定小鼠血清效价及抗独特型抗体组分。选取效价最高的小鼠,取脾脏提取总RNA,RT-PCR法合成c DNA第一链。设计简并引物,利用PCR法扩增小鼠VH、Vκ基因,再用SOE-PCR法进行单链抗体基因的拼接。SOE-PCR产物和p IT2载体经双酶切后连接,电转入感受态E.Coli. TG1完成建库,并用多克隆噬菌体ELISA对抗体库与免疫原的结合能力进行了鉴定。结果表明,免疫鼠血清的效价在1∶1.6×105到1∶6.4×105倍之间。免疫鼠血清可对Cry2A与其兔多克隆抗体的结合最高可产生17. 6%的抑制,证明Ab2β和Ab2γ型抗独特型抗体的存在。最终构建了库容为1.2×107的Cry2A抗独特型单链抗体库,该抗体库与免疫原的结合信号/背景比值为6. 04。

In order to reduce the cost of immunoassay of Bacillus thuringiensis Cry2A toxin and develop a cheap and toxin-free ELISA kit,an anti-idiotypic single-chain variable fragment(scFv)library of Cry2A toxin was designed and constructed by immunization with the whole molecule of rabbit polyclonal antibody and phage display.Rabbit polyclonal antibody against Cry2A toxin was purified by protein A and used as immunogen for the female Balb/c mice.The titer and the component of anti-idiotypic antibodies in mice antiserum were determined by ELISA under the optimized concentration of coating antigens.Then the best mouse was chosen to extract total RNA,and the first strand of cDNA was synthesized by RT-PCR.The VH,Vκgenes of mice and linker fragments were amplified by PCR with designed primers,and the single chain antibody gene was spliced by SOE-PCR.The SOE-PCR product and the pIT2 vector were digested by restriction enzymes and linked together,then were electroporated into competent E.coli.TG1.The binding ability of library with the immunogen was determined by polyclonal phage ELISA.The results showed that the titers of immunized mouse antiserum were between 1∶1.6×10 5 and 1∶6.4×10 5.The antiserum could also produce 17.6%inhibition on the binding of Cry2A with its rabbit polyclonal antibodies,and that proved the existence of Ab2βand Ab2γtype anti-idiotypic antibodies.And eventually an anti-idiotypic scFv library was built with the capacity of 1.2×10 7.The ratio of signal to background of library with immunogen was 6.04.