The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9...The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.展开更多
旨在制备猫PD-L1抗原并筛选单抗,探讨其在犬、猫肿瘤检测和诊断中的应用,同时制备猫源化嵌合单链抗体,为猫肿瘤的治疗提供基础。本研究通过PCR扩增猫PD-L1基因,构建原核表达重组质粒PET-MPD-L1,通过在宿主BL21(DE3)中表达制备PD-L1抗原...旨在制备猫PD-L1抗原并筛选单抗,探讨其在犬、猫肿瘤检测和诊断中的应用,同时制备猫源化嵌合单链抗体,为猫肿瘤的治疗提供基础。本研究通过PCR扩增猫PD-L1基因,构建原核表达重组质粒PET-MPD-L1,通过在宿主BL21(DE3)中表达制备PD-L1抗原,免疫小鼠后细胞融合,ELISA方法进行杂交瘤细胞筛选,3次亚克隆后建立MPD-L1杂交瘤细胞系。测定杂交瘤细胞上清、腹水效价、抗体亚类、Western blot和免疫组织化学(IHC)特性。扩增轻、重链可变区和猫IgG1 Fc基因,降落PCR将重链可变区(Variable region of the Heavy chain,VH)和轻链可变区(Variable region of the Light chain,VL)拼接成scFv,进一步拼接出scFv-fFc,构建pFast-MPD-L1-scFv和pFast-MPD-L1-scFv-fFc重组质粒,在昆虫杆状病毒表达系统中表达。结果表明,猫PD-L1在上清中大量表达且纯度较好,杂交瘤细胞2A5A2F4D7分泌的抗体属于IgG 2a,轻链为κ亚型,细胞上清、腹水抗体效价分别为1∶3200和1∶102400,Western blot结果表明,该单抗能与表达的猫PD-L1反应,IHC结果显示,该单抗可以识别犬猫肿瘤组织细胞表达的PD-L1,可以反映PD-L1在肿瘤组织中的表达情况。VH和VL与IMGT数据库中相应基因序列相符,具有4个框架区和3个高变区,猫源化嵌合单链抗体在昆虫杆状病毒表达系统中成功表达。综上,本研究制备的猫PD-L1单克隆抗体在犬猫肿瘤检测和预后中具有一定的价值,猫源化单链抗体的成功制备为其在犬猫肿瘤病治疗中的应用打下了基础。展开更多
文摘The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.
文摘旨在制备猫PD-L1抗原并筛选单抗,探讨其在犬、猫肿瘤检测和诊断中的应用,同时制备猫源化嵌合单链抗体,为猫肿瘤的治疗提供基础。本研究通过PCR扩增猫PD-L1基因,构建原核表达重组质粒PET-MPD-L1,通过在宿主BL21(DE3)中表达制备PD-L1抗原,免疫小鼠后细胞融合,ELISA方法进行杂交瘤细胞筛选,3次亚克隆后建立MPD-L1杂交瘤细胞系。测定杂交瘤细胞上清、腹水效价、抗体亚类、Western blot和免疫组织化学(IHC)特性。扩增轻、重链可变区和猫IgG1 Fc基因,降落PCR将重链可变区(Variable region of the Heavy chain,VH)和轻链可变区(Variable region of the Light chain,VL)拼接成scFv,进一步拼接出scFv-fFc,构建pFast-MPD-L1-scFv和pFast-MPD-L1-scFv-fFc重组质粒,在昆虫杆状病毒表达系统中表达。结果表明,猫PD-L1在上清中大量表达且纯度较好,杂交瘤细胞2A5A2F4D7分泌的抗体属于IgG 2a,轻链为κ亚型,细胞上清、腹水抗体效价分别为1∶3200和1∶102400,Western blot结果表明,该单抗能与表达的猫PD-L1反应,IHC结果显示,该单抗可以识别犬猫肿瘤组织细胞表达的PD-L1,可以反映PD-L1在肿瘤组织中的表达情况。VH和VL与IMGT数据库中相应基因序列相符,具有4个框架区和3个高变区,猫源化嵌合单链抗体在昆虫杆状病毒表达系统中成功表达。综上,本研究制备的猫PD-L1单克隆抗体在犬猫肿瘤检测和预后中具有一定的价值,猫源化单链抗体的成功制备为其在犬猫肿瘤病治疗中的应用打下了基础。
