凋亡素基因序列重组及其真核表达载体的构建

目的:提高凋亡素基因在肿瘤细胞中的表达效率,增强其诱导肿瘤细胞凋亡的生物学活性.方法:通过两次PCR,以pcDNA-VP3质粒为模板,扩增出带Kozak序列(真核核糖体结合位点)的凋亡素基因VP3.将其克隆到真核表达载体pRevTRE的多克隆位点,构建成凋亡素的真核表达载体pRevTRE-VP3,将该质粒分别以限制性内切酶BamHI和XhohI进行酶切鉴定,将初步鉴定显示可能克隆有目的基因的质粒进行测序鉴定.结果:测序结果表明,本研究合成的凋亡素基因与Noteborn报告的凋亡素基因序列一致,同源性为l 00%.在其起始密码子前添加了Ko zak序列的凋亡素基因已成功克隆到真核表达载体pRevTRE.结论:采用两次PCR法可提高表达效率的真核核糖体结合位点添加到凋亡素基因序列起始密码子前端,并构建成凋亡素真核表达载体.

Study on recombination of sequence of apoptin gene and construction of a eukaryn vector for expression of apopt

Objective: To increase efficiency in expression of apoptin gene(vp3) in tumor cells and promote its bioactivity to induce apoptosis of tumor cells. Methods: The plasmid pcDNA-VP3 was used as a template and the apoptin gene with Kozak sequence,i,e.eukaryn ribosome locus was amplified through twice PCR method. The recombined apoptin gene was subcloned into the multiple clone site of plasmid pRevTRE to obtain a plasmid pRevTRE-VP3. The plasmid pRevTRE-VP3 was cut and identified by means of restriction endonuclease BamHI and XhoI. The sequence of selected plasmid pRevTRE-VP3 was analysed. Results: The sequence of recombined apoptin gene was identical with that reported by Noteborn et al. The apoptin gene in which Kozak sequence was added to the front of ATG code was subcloned into the eukaryn vector pRevTRE. Conclussion: The eukaryn ribosome locus which promotes expression efficiency may be added to the front of ATG code of sequence of apoptin gene by twice PCR and the eukaryn vector for expression of apoptin can be constructed.