获得重构基因的简捷方法——重叠延伸PCR

Splicing overlap extension by PCR:a convenient method to obtain recombinant gene

目的 :构建 IFN- β信号肽序列与白细胞介素 18(IL- 18)成熟肽的重组嵌合分子。方法 :从 BAL B/ c鼠基因组 DNA扩增 IFN- β信号肽序列 ;由小鼠 Pro- IL- 18真核表达质粒获得 IL- 18成熟肽序列 ;以非对称 PCR技术得到IFN- β信号肽编码区的反义链和 IL- 18成熟肽正链 ;采用重叠延伸 PCR(SOE- PCR)产生具有 IFN- β信号肽与 IL- 18成熟肽的嵌合编码序列 ;定向连方式构建嵌合 IL- 18的表达质粒并测序。结果 :连续胶 PAGE表明非对称 PCR可分别扩增出两个靶序列的特异性单链 ,大小符合预期。IFN- β信号肽对称 PCR产物与 IL- 18非对称 PCR产物混合作为模板 ,进行重叠延伸 ;电泳分析可见清晰的嵌合片段 ;测序显示重组质粒的表达框由两个设计的靶序列构成 ,且无移码。结论 :重叠延伸 PCR是一种获得重构基因的简捷方法。

Objective To construct a hybrid molecule composed of IFN-β signal sequence and IL-18 mature sequence. Methods Firstly, through routine PCR, IFN-β signal sequence was amplified from BALB/c mouse genome DNA while IL-18 mature sequence from a recombinant plasmid containing mouse Pro-IL-18 sequence. Secondly, the sense strand of IL-18 mature sequence and the anti-sense strand of IFN-β signal sequence were obtained by asymmetric PCR. Thirdly, a hybrid IL-18 containing IFN-β signal sequence and IL-18 mature sequence was obtained through splicing overlap extension by PCR. At last, the hybrid IL-18 was bonded to pCDNA3 expression vector by the way of orientation linkage and sequenced. Results Continuing PAGE showed the specific single sequence of the two target products could be amplified by asymmetric PCR, and the size was accord with expectation. The best way to obtain hybrid molecule was the mixing of IFN-β signal sequence and IL-18 mature sequence to overlap extention PCR. Squencing proved that expression box of recombinant plasmid was composed of the expected target sequence. And there was no base malposition. Conclusion Splicing overlap extension by PCR is a convenient method to obtain recombinant gene.