Molecular Cloning, Induced Expression And Functional Prediction Of Apoptin

Chicken anemia virus (CAV) is the pathogen of chicken infectious anemia (CIA). CAV belongs to circoviridae. CAV genome encodes three kinds of protein: VP1 (51.6KD), VP2 (24. OKD) and VP3 (13.6KD). Apoptin (VP,) can selectively induce apoptosis in human transformed and malignant cells but not in normal diploid cells. Apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Apoptin is becoming a potential candidate as a therapeutic and diagnostic tool in cancer treatment.A pair of primers were designed and synthesized according to the conservative sequence of vp3 gene. The fragment amplified by PCR technique was enriched by electrophoresis and jointed into plasmid pUC18. The recombined plasmid pUC18-vp3 was got. The sequence of the fragment amplified by PCR was analyzed with F primer. It was found that it had well homology to the reported apoptin genes by comparing the gene with all genes in Genbank and Shandong strain SJ1 of China.The vp3 gene was cloned into pET30(a) and pcDNA(3. 1) vectors separately. The recombined plasmids pET30-vp3 and pcDNA-vp3 were got. The pET30-vp3 was induced to express in E. coli DE3 by IPTG, then the expression was purified by G-75 filtration and affinity chromatography. It was proved that the protein was got by SDS-PAGE. The successful cloning and expressing of vp3 gene become the basis for the study and exploiture of apoptin.The physical and chemical characters and secondary structure of apoptin were predicted with softwares Antheprot and DNAstar. According to the result from BLAST, apoptin maybe take part in the signal pathway of programmed cell death with activity of enzyme. Especially, apoptin is partially similar with ribonucleotide reductase, uridylate kinase and phosphotriesterase in the amino acid sequence. The similarity between them indicates that apoptin maybe takes part in the signal pathway of apoptosis with phosphotriesterase activity or operates on hnRNA with ribonucleotide reductase or uridylate kinase activity.