Study On Tumor-targeting Therapy With Apoptin Recombination Protein

Cancers have been threatening the health of human beings. The chemicals used inclinical now lack selectivity. Toxical effects are common seen in clinical.Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects ofanti-tumor medicines. Apoptin is a protein, which can specifically induce apoptosis oftumor cells but does not affect normal cells or diploid cells. Experiments showed thatapoptin did not induce apoptosis of normal cells. When the plasmids expressingapoptin were transfected into certain types of human normal cells, apoptosis occurredsimilar to the natural apoptotic level of normal cells. Apoptin can be used as atherapeutic agent. It can selectively inhibit oncocytes provided it could be transferredinto tumor cells in great amount. It has been confirmed that there are many receptorsof Somatostatin (SS) existing on the surface of tumor cells. Bacillus pyocyaneusexotoxin II domain encodes a transmembrane protein. Therefore if the genes encodingSS and P II could be integrated into the gene encoding apoptin, the ability of apoptinto promote the apoptosis of tumor cells would be enhanced greatly.The apoptin gene amplified by LA PCR and was ligated with an expressionvector pET-28a to construct the prokaryotic expression plasmid pET-apoptin. Thepositive recombinant plasmids were transformed into host strain E.coli BL21(DE3)and induced to express l apoptin by IPTG. The specific protein expressed (about14.0KDa) was detected by SDS-PAGE. The protein was expressed at high level,amounting to 11.4% of the total bacterial protein as confirmed by thin-layer scanning.mainly in the form of inclusion bodies. After purified by electroelution in dialysis bags,the apoptin was used to produce polyclonal antiserum against rabbits and ELISAdetection showed the titer of apoptin antiserum was 12 800. Western blot showed thatthe antiserum raised against the apoptin in rabbits could react to the protein expressedspecifically.In order to study on Tumor-targeting therapy with apoptin recombination protein,the apoptin fusion gene was amplified by LA PCR, the product of PCR were clonedinto a clone vector pMD18-T, and then the gene was sequenced and analyzed. It wasconfirmed that the gene sequence was consistent with what we had predicted.Recombinant baculovirus DNA was produced using Bac-to-Bac baculovirusexpression systems according to the manufacturer's protocol. Sf9 cells were grown in75-cm2 cell culture flasks, and infected with recombinant baculovirus (MOI of 5). Thespecific protein expressed (about 30.0KDa) was detected by SDS-PAGE. Western blotand IFA showed that the antiserum raised against the apoptin in rabbits could react tothe protein expressed specifically. Since the expressed recombinant protein contained6×histidine tag at N-terminal, it was purified using Ni-NTA resin convenientlyaccording to the manufacturer's instructions. SDS-PAGE was performed to analyzethe purified protein from the infected cells,result showed that the recombinant proteinwith 30.0KDa.The effects of apoptin recombination protein in vitro to cancer cell line wasdeterminates by light microscopy, MTT assay, DNA ladder and fluorescence activatedcell sorter (FACS) analysis. The results showed that remarkable apoptoticcharacteristics such as nuclear shrinkage appeared in tumor cells, but few apoptoticcharacteristics were observed in control groups. The apoptosis present were higherthan control, the cell cycle were exchanged also detected by FACS. It was suggestedthat the apoptin recombination protein could induce apoptosis in different tumor cellseffectively.The expression of apoptin recombination gene in Pichia expression system. Theexpression plasmids pGAPZαA-SPA in yeast were constructed. After linealization ofthe plasmids apoptin recombination gene gene was integrated into the genome of hostyeast Pichia Pastoris SMD1168 by electroporation. After the optimization ofexpression conditions apoptin recombination protein was expressed successfully inPichia yeast. Then apoptin recombination protein was purified using Ni-NTA resinconveniently.