Cloning And Expression Of The Huamn Alpha-fetoprotein Gene

Alpha-Fetoprotein (AFP) is a Mr 70,000 glycoprotein, produced at high levels by the fetal liver, transcriptionally repressed after birth. The majority of human HCCs overexpress this pncofetal antigen , so serum AFP measurements play an important role in the diagnosis and monitoring of hepatocellular carcinoma (HCC). As literatures reported, AFP may have an immunosuppressive role,which make the tumor cells escape from the immune surveillance,promote the growth of tumor indirectly.AFP may also stimulate the growth of tumor directly.Meanwhile, Peptide fragments of AFP presented in the context of major histocompatibility molecules could serve as potential recognition targets by CD8 T cells.In conclusion, AFP represent potential targets for HCC immunotherapy.But the protein extracted from serum is trace and expensive. Thus, it is the best strategy to acquire recombinant protein with bio-engineering methods. In this research,AFP gene was obtained by PCR and recombinant plasmids were constructed. And these recombinant plasmids were expressed in BL21(DE3) at last, which lay a good foundation for the research of AFP. l.Amplification of AFP genes:Fetus liver mRNA was extracted by Trizol,then AFP1, AFP2, AFP3 was amplified by RT-PCR method. Additional features of the gene were added including the terminal restriction endonuclease sites matched to the vector pGEMEX-1.2.Constructions of recombinant plasmids pGEMEX-AFPl, pGEMEX-AFP2, pGEMEX-AFP3:AFP1, AFP2, AFP3 gene was digested with BamH I and Hind III;after purification, digested products were ligated with the same digested and purified pGEMEX-1 by T4 DNA ligase, and recombinant plasmids pGEMEX-AFPl, pGEMEX-AFP2, pGEMEX-AFP3 were constructed. The recombinant plasmids were identified with PCR and enzyme digestion, and were confirmed by DNA sequencing at last. 3.Expression fusion proteins in E.coli BL21(DE3):After the E.coli BL21(DE3) transformed recombinant plasmids pGEMEX-AFPl,pGEMEX-AFP2, pGEMEX-AFP3 separately,they were detected at the gene and protein level. The E.coli BL21(DE3) transformed recombinant plasmids pGEMEX-AFPl,pGEMEX-AFP2, pGEMEX-AFP3 could be found a 588bp, 678bp , 734bp DNA band with PCR each. Induced with IPTG, an about 551kDa 59kDa, 57kDa additional protein band-the fusion protein each was expressed in E.coli BL21(DE3) transformed recombinant plasmids pGEMEX-AFPl , pGEMEX-AFP2 , pGEMEX-AFP3, which was demonstrated by SDS-PAGE. The good reactinogenicity of fusion AFP1,AFP2, AFP3 was identified with Western blot. Conclusions:1.The high fidelity recombinant plas.nids pGEMEX-AFF1, pGEMEX-AFP2,pGEMEX-AFP3 were successfully constructed, and were confirmed bysequencing.2.The recombinant plasmids pGEMEX-AFPl , pGEMEX-AFP2 ,pGEMEX-AFP3 each expressed fusion protein AFP1, AFP2, AFP3 in E.coliBL21(DE3).3.Fusion proteins AFP1. AFP2, AFP3 expressed in E.coli BL21(DE3) all hadgood reactinogenicity.