【作者】 郝军元；

【导师】 张玉静；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 凋亡素是鸡贫血病毒(CAV)vp3基因编码一个功能性蛋白,主要引起鸡单核细胞凋亡。多年来对凋亡素研究证明它能够特异诱导肿瘤细胞发生凋亡而对正常体细胞无损伤作用。凋亡素在肿瘤的治疗中是一个独立的因子,凋亡素诱导的细胞凋亡不依赖于p53基因,在肿瘤细胞内高水平表达的Bcl-2能够促进凋亡素诱导的凋亡。凋亡素的这些特征在肿瘤治疗中体现了巨大的潜力,已成为肿瘤治疗研究领域的热点。为探索凋亡素蛋白导向治疗肿瘤的方法,本研究将凋亡素基因与生长抑素基因和绿脓杆菌外毒素转膜系统基因重组,重组蛋白能以生长抑素为导向富集于肿瘤组织,并通过绿脓杆菌外毒素转膜系统将凋亡素进入肿瘤细胞,以促进肿瘤细胞的凋亡。本研究首先采用原核表达系统表达了凋亡素蛋白并制备其抗体为下一步凋亡素重组蛋白的检测奠定基础,随后通过PCR重叠延伸技术将凋亡素基因与生长抑素基因和绿脓杆菌外毒素转膜系统基因重组,获得凋亡素重组蛋白基因,并在杆状病毒和酵母表达系统实现了凋亡素重组蛋白的表达。凋亡素重组蛋白在体外对肿瘤细胞的凋亡作用试验表明,凋亡素重组蛋白对肿瘤细胞有凋亡作用,能够抑制肿瘤细胞增殖,但不同肿瘤细胞凋亡率存在差异。通过上述实验,证实凋亡素重组蛋白能够诱导肿瘤细胞凋亡,为进一步利用凋亡素治疗肿瘤奠定基础。更多还原

【Abstract】 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.更多还原