利用In-Fusion技术构建存活素-增强型绿色荧光蛋白融合基因重组慢病毒表达载体

Value of In-Fusion cloning techhique on routine vector construction

目的:本研究以构建存活素-增强型绿色荧光蛋白(Survivin&eGFP)融合基因慢病毒表达载体(p-GC-FU-Survivin)为例来探讨In-Fusion克隆技术在常规载体构建中的应用价值。方法:根据In-Fusion技术原理,克隆引物设计时,在Survivin同源序列的两侧分别引入经AgeI线性化的载体p-GCFU两端各15个碱基,将以此引物扩增的聚合酶链式反应(PCR)产物与线性化p-GCFU用In-Fusion交换酶在室温下作用30min,使Sur-vivin特异性扩增产物两端的序列与线性化载体两端的序列发生同源交换,取2μl交换液进行转化,挑取阳性克隆,进行酶切和测序鉴定。将鉴定正确的阳性克隆瞬时转染293T细胞,观察Survivin&eGFP融合蛋白在293T细胞中的表达。结果:每2μl克隆交换液获得大约103个克隆数,阳性率达90%以上,瞬时转染p-GCFU-Survivin可获得Survivin&eGFP融合蛋白在293T细胞中的表达。结论:该技术是一种非连接酶依赖性克隆技术,使基因克隆步骤简化并大大节省了实验时间和经费。

Objective：To introduce a simple method for the cloning of PCR products.Methods：The In-Fusion cloning technique was described by constructing a recombinant lentivirus vector （pGCFU） with surviving ＆ eGFP fusion gene as a sample.The survivin cDNA was amplified with survivin gene-specific primers with 15 bp extensions homologous to the pGCFU ends.By the action of the In-Fusion enzyme at room temperature for 30 minutes,the single-stranded PCR fragment and vector ends were fused due to the 15 bp homology.Finally,clones derived from transformation were chosen randomly and identified.Results：About 103 positive clones for inserts were obtained after transformation with 2 μl of exchanging products,and the ratio of the positive colonies was more than 90%.After 24 h the p-GCFU-survivin was transfected into 293T eukaryotic cells,the expression of the survivin ＆ eGFP fusion gene can be confirmed with fluorescence microscope.Conclusion：The ligation-independent property makes the In-Fusion PCR cloning technique rapid,reliable and higher cost-effective,avoiding the need for multiple sub-cloning steps.