【摘要】 在计算机辅助下,设计并合成西维因半抗原,与载体蛋白KLH连接后免疫Balb/C小鼠,经过5次免疫后,最高效价和特异性抑制率分别达到254 000和26%;提取免疫后小鼠脾脏总RNA,纯化mRNA后反转录获得cDNA,利用设计的兼并引物获得长度约为400 bp的轻链可变区和重链可变区基因片段,重叠延伸后获得长度约为780 bp的单链抗体基因。经过EcoR I和HindⅢ酶切后,连入T7噬菌体臂,构建西维因特异性的抗体库,抗体库原始滴度为1.74×107pfu。以西维因-OVA为配体对抗体库进行淘选,经过4轮淘选后,利用直接ELISA对获得噬菌体克隆进行特异性检测,西维因对克隆A4-7和A4-16的抑制率分别达到54.1%和66.4%。经过序列测定,2个克隆的轻链可变区分别由108和112个氨基酸构成,重链可变区分别由117和112个氨基酸构成。更多还原

【Abstract】 Carbaryl hapten,which was designed using computer softwares and synthesized by chemical synthesized methods,was connected with carrier protein KLH.Then Balb/C mice were immunized using the connected product as the immunogen.After 5 rounds of immunization,the highest titer and the specific inhibition rate of the immune serum reached 254 000 and 26 % respectively.Total RNA was extracted from the immunized mice spleen and mRNA was purified.The cDNA was obtained by reverse transcription and used as template for PCR amplification of variable regions of antibodies.The variable regions of light chain(VL) and heavy chain(VH) were about 400 bp in length.Then the single-chain antibody gene(scFv) about 780 bp was obtained with overlap PCR method.The scFv fragments were digested by restriction enzyme EcoR I and Hind III.The digestion production was ligated into T7 phage arms and the carbaryl-specific phage-displaying antibody library was constructed.After examination original titer of the primary phage library reached 1.74×107 pfu.The antibody library was panning using carbaryl-OVA as the coating ligand.After 4 rounds of panning,affinities and specificities of the individual colonies were detected with direct ELISA and the inhibition rates of free carbaryl against clone A4-7 and A4-16 reached 54.1 % and 66.4 % respectively.Sequencing results indicated that the light chain variable regions of the 2 clones comprised 108 and 112 amino acids respectively.The heavy chain variable regions of the 2 clones comprised 117 and 112 amino acids respectively.更多还原