人源性促甲状腺激素受体单链抗体Fc融合蛋白的制备及鉴定

Preparation and determination of human thyrotrophin receptor single-chain antibody Fc fusion protein

目的:构建含有促甲状腺激素受体抗体(TRAb)单链抗体的重组载体pFastBacTM1-ScFv-IgG1Fc,通过Bac-to-Bac表达系统实现单链抗体融合蛋白的高效表达,并检测目的蛋白表达和免疫学活性。方法:TRAb抗体的重链可变区(HV)和轻链可变区(LV)的序列以linker(G4S)连接,构建ScFv;Genbank检索人IgG1Fc序列,SnapGene软件分析上述序列和载体pFastBacTM1,以基因合成方式构建重组质粒pFastBacTM1-ScFv-IgG1Fc;pFastBacTM1-ScFv-IgG1Fc转化感受态大肠杆菌DH10Bac生成重组杆粒,pUC/M13正向和反向引物进行PCR分析确认目的基因的序列;重组杆粒采用Cellfectin®Ⅱ试剂转染昆虫细胞Sf9生成P1病毒储液,继续感染Sf9生成高滴度的P2和P3病毒储液;P3病毒储液感染High FiveTM细胞,收集上清通过SPA-Sepharose CL-4B亲和层析纯化获得较纯的单链抗体融合蛋白,SDS-PAGE鉴定目的蛋白;化学发光和免疫组化检测目的蛋白抗原结合力。结果:PCR扩增得到约3747 bp的基因序列,重组杆粒中目的基因正确插入;目的蛋白位于55 kDa附近且无杂带,纯度较高;目的蛋白浓度为248 U/L,单链抗体融合蛋白的结合力较好。结论:Bac-to-Bac表达系统成功构建并表达了TRAb单链抗体融合蛋白,抗原亲和力良好。

Objective:To construct a recombinant vector pFastBacTM1-ScFv-IgG1Fc with single-chain antibody against human thyrotrophin receptor antibody.To express fusion protein efficiently through Bac-to-Bac expression system,and to detect expression level and immunological activity of target protein.Methods:ScFv was composed of sequences of heavy chain variable region(HV)and light chain variable region(LV)of TRAb,which were linked by linker(G4S).Human IgG1Fc sequence was obtained from GenBank.SnapGene software was used to analyze above sequences and pFastBacTM1 vectors to construct recombinant plasmid pFastBacTM1-ScFv-IgG1Fc in form of gene synthesis.Recombinant plasmid was transformed to DH10Bac to generate recombinant rod.PUC/M13 forward and reverse primers were used to confirm sequence of target gene by PCR.Recombinant rod was transfected to insect cell Sf9 to generate P1 virus reservoir using Cellfectin®Ⅱ,and higher titer P2 and P3 virus reservoir with generated with iterative transfection.High FiveTM cells were infected with P3 virvus reservoir.Cell supernatant was collected and purified by SPA-Sepharose CL-4B affinity chromatography to obtain relatively pure single-chain antibody fusion protein,and target protein was identified by SDS-PAGE.Binding activity of target protein was detected by chemiluminescence and immunohistochemistry assay.Results:PCR amplification obtained gene sequence,of 3747 bp,and recombinant rod contained target gene.Target protein was located near 55 kDa without other bands,which confirmed that high purity target protein was obtained.Concentration of target protein was 248 U/L,and had high binding activity of single-chain antibody fusion proteins.Conclusion:TRAb single-chain antibody fusion protein was successfully expressed by Bac-to-Bac expression system,which had good antigenic affinity.