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的论文.docVIP

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的论文 【摘要】 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-gcfu-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age i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survivin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。 【关键词】 基因；克隆细胞；基因，病毒 value of in-fusion cloning techhique on routine vector construction　lin chaogui　fan lin　chen lianglongdepartment of cardiology，union hospital，fujian medical university，fuzhou，fujian，350001，　　abstract：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. 　　key words：genes;clone cells;genes,viral 　　传统的聚合酶链式反应（pcr）产物克隆技术包括补平末端克隆、ta克隆以及连接酶依赖性克隆等。WWW.11665.coM这些方法的共同特点是在实际操作过程中需要用多种不同的酶（如t4连接