细胞因子受体-嵌合抗体真核表达载体的构建

目的：构建细胞因子受体-抗人AFP嵌合抗体基因真核表达载体。方法：用RT-PCR方法从人胚肝组织中扩增EpoR胞外区基因及gp130跨膜区基因，并用限制性酶切连接方法拼接。用PCR方法从抗人AFP单链抗体表达载体中扩增VL、VH基因，分别与人kappa轻链和人gammal重链恒定区基因重组，获得人鼠嵌合轻链和重链，再将EpoR-gp130与嵌合重链连接，最后将嵌合轻链和H—EpoR-gp130分别连接到质粒pVITRO上，构建pVITRO-E-g-Ig载体。结果：获得EpoR胞外区基因750bp，gp130跨膜区基因900bp。重组真核表达载体经限制性酶切鉴定示EpoR、gp130基因、嵌合重链基因和嵌合轻链基因正确连接到载体pVITRO。结论：成功构建细胞因子受体-抗人AFP嵌合抗体基因表达载体。

Construction of the eukaryotic expression plasmid for the cytokine receptor-chimeric antibody

Objective： To construct cytokine receptor-anti human AFP chimeric antibody gene. Methods.. Extracellular region of EpoR gene and transmembrane domain of gp130 gene were cloned by RT-PCR method from hepatic tissue of fetus, and the two genes were spliced by endonuclease digestion. VL gene was cloned from anti-human AFP ScFv gene, and was spliced with Human CK gene to construct chimeric light chain. VH gene was cloned from anti-human AFP ScFv gene, and ligated into pAH4604 containing human 71 gene to construct VH-CH gene. EpoR-gp130 gene and VH-CH gene were spliced by endonuclease digestion, and cloned into pVITRO-L to construct pVITRO-E-g-Ig. Results, Extracellular region of EpoR gene is 700hp, transmemhrane domain of gp130 gene is 900hp. The recombinant gene was confirmed to he correct by restriction enzyme digestion and DNA sequencing. Conclusion： The cytokine receptor-anti human AFP chimeric antibody fusion protein was constructed and expressed successfully.