Cloning And Expression Of Sus Scrofa Annexin A2 In Prokaryotic And Eukaryotic System And Preparation Of Its Polyclonal Antibody

Annexin A2 is a member of the multigene family of Annexin proteins, which are characterized by binding to negatively charged phospholipids in the presence of Ca2+. Recent studies highlight a very close relationship between Annexin A2 and virus infection. The studies of Annexin A2 and the other Annexin members in respect of virology contribute to elucidate the interaction of virus with host cell, reveal virus pathogenic mechanism and develop strategies to prevent and cure viral diseases.According to reported Sus Scrofa Annexin A2 sequence (Enter number: NM-001005726), Annexin A2 from pig alveolar macrophage was amplified and gained by RT-PCR and enhanced green fluorescent protein from pEGFP-1 vector was amplified by PCR. Then the recombinant gene (ANXA2-EGFP) of enhanced green fluorescent protein(EGFP) with Sus Scrofa Annexin A2 was constructed by recombinant PCR, which was cloned into pMD-18T vector(This recombinant pMD-18T vector was called pA2-EGFP for short). After identification of restriction endonuclease and sequencing, it was confirmed that ORF of ANXA2-EGFP in pMD-18T vector was correct. On the base of this, with ANXA2-EGFP amplified from pA2-EGFP and cloned into pET-30a(+)vector, p30a/AE prokaryotic expression vector was constructed and then expressed 66kD recombinant protein of ANXA2-EGFP in the form of inclusion body by IPTG induction. Likewise, with intact Annexin A2, the C-terminal (426bp)and N- terminal (156bp) Annexin A2 amplified and cloned into pGEX-6P-1 vector respectively, pGEX/A2,pGEX/CA and pGEX/NA2 prokaryotic expression vector were constructed and pGEX/A2,pGEX/CA expressed GST-ANXA2 and GST-ANXA2-C fusion protein in soluble form by IPTG induction. However, pGEX/NA2 prokaryotic expression vector still expressed GST-ANXA2-N fusion protein in the form of inclusion body by IPTG induction, after change of IPTG gradient, LB pH and host cell. Rabbit was injected with recombinant protein of ANXA2-EGFP and serum was harvested after a month. Serum was purified by two steps, one by caprylic acid -ammonium sulfate and second by Affinity purification with GST-ANXA2 and GST-ANXA2-N. Affinity purification serum titer mounted to 12800 and Western blot detection showed high specificity. ANXA2-EGFP of pA2-EGFP was subcloned into pcDNA3.1(+) vector to construct pcDNA/A2-EGFP eukaryotic expression vector. pcDNA/A2-EGFP eukaryotic expression vector was transfected PK-15 transiently and express product was used for western blot analysis. Western blot results showed that affinity purification antiserum was available. In the end, modeling structure of Sus Scrofa Annexin A2 was get by bioinformatics analysis software and suggested a ideal result by anolea and PROCHECK assessing.Antibody was recognized as a beneficial tool for study of interaction between Annexin A2 and virus. In this study, we prepared antiserum of intact ANXA2 and ANXA2-C, which were conducive to Annexin A2 investigation with respect to tissue distribution, block of corresponding ligand and upregulation detection of Annexin A2 after virus infection. Meanwhile, it was also helpful to further research interaction of annxin A2 with virus and reveal virus pathogenic mechanism.