摘要
利用噬菌体展示技术筛选人源肌红蛋白(Myoglobin,Mb)的单链抗体(single chain antibody fragment,scFv),以制备应用于临床免疫组化的Mb单克隆抗体。利用重组Mb抗原免疫新西兰白兔,提取脾脏组织的总RNA,RT-PCR扩增出轻、重链可变区基因序列,经重叠延伸PCR(overlap-PCR)拼接成scFv并克隆至pComb3XSS噬菌粒中,成功构建库容量为2×10^(7) cfu的Mb-scFv抗体库。利用噬菌体展示技术对该文库进行3轮固相筛选,经基因测序获得Mb-scFv的阳性克隆序列。将此序列的重链、轻链可变区基因与兔源IgG_(1)相应的恒定区连接后,分别克隆至pcDNA3.4表达载体中,并共转染Expi239F细胞以获得重组单克隆抗体Mb-IgG_(1)。间接ELISA显示Mb-IgG_(1)的亲和力常数为9.24×10^(12)L/mol,IHC实验显示该抗体特异性识别人骨骼肌组织中的天然Mb抗原。Mb-IgG_(1)单克隆抗体的研发,为丰富Mb抗原的临床检测以及横纹肌溶解综合症快速诊断的产品开发奠定了临床应用基础。
To generate and characterize a monoclonal antibody against myoglobin(Mb)for clinical immunohistochemistry,a singlechain antibody fragment(scFv)specific to human Mb was screened from an immunized antibody library using phage display technology.New Zealand white rabbits were immunized three times with the recombinant human Mb antigen.The high titer of rabbit serum antibodies was detected using the indirect ELISA method,indicating successful immunization.The total RNA extracted from spleen tissue was reverse transcribed into complementary DNA(cDNA)libraries.The variable region gene sequences of the light chains(VL)and heavy chains(VH)of antibodies were amplified separately using polymerase chain reaction(PCR).The VL and VH gene sequences were ligated to generate scFv using overlap extension PCR(overlap-PCR),and then subcloned into the pComb3XSS phagemid.Consequently,the Mb-scFv library with a capacity of 2×10^(7) cfu was successfully constructed.Three rounds of solid-phase screening were conducted on this library using phage display technology,and the positive clone sequences of Mb-scFv were obtained through gene sequencing.Upon sequence alignment,only one anti-myoglobin scFv was identified.The VL and VH gene sequences of this scFv were ligated to the corresponding IgG_(1) constant gene derived from rabbit and then subcloned separately into the pcDNA3.4 expression vector.These plasmids were co-transfected into Expi239F cells to express the Mb-IgG_(1) antibody,which was subsequently purified using protein G.The indirect ELISA indicated that the affinity constant of Mb-IgG_(1) was 9.24×10^(12) L/mol.Immunohistochemistry(IHC)experiments confirmed that this antibody specifically recognized the natural Mb antigen in human skeletal muscle tissue.The development of the Mb-IgG_(1) monoclonal antibody established a clinical foundation for enhancing the detection of Mb antigen and facilitating the rapid diagnosis of rhabdomyolysis syndrome.
作者
姜孟伶
曹金丹
张美珍
樊竑冶
JIANG Mengling;CHAO Jindan;ZHANG Meizheng;FAN Hongye(School of Life Science and Technology,China Pharmaceutical University,Nanjing 211198,China)
出处
《药物生物技术》
2025年第3期253-258,共6页
Pharmaceutical Biotechnology
关键词
肌红蛋白
单链抗体
噬菌体展示
Mb-scFv抗体库
固相筛选
重组单克隆抗体
Myoglobin
Single-chain antibody
Phage display technology
Mb-scFv library
Solid phase screening
Recombinant fulllength antibody
