重叠区扩增基因拼接法在构建膜锚定金黄色葡萄球菌肠毒素A融合基因上的应用  

Construction of a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension

作　　者：易平勇[1] 余海[1] 马文学[1] 孙文均[1] 姜槐[1] 

机构地区：[1]浙江大学医学院,浙江杭州310006

出　　处：《浙江大学学报（医学版）》2003年第5期412-414,共3页Journal of Zhejiang University(Medical Sciences)

基　　金：浙江省自然科学基金资助 (399131)

摘　　要：目的 :将超抗原金黄色葡萄球菌肠毒素 A(SEA)基因与人胎盘碱性磷酸酶 (h PL AP- 1) C末端信号肽序列进行拼接 ,形成融合基因。方法 :利用重叠区扩增基因拼接法 ,分别扩增 h PLAP- 1C末端信号肽序列和 SEA基因序列。然后 ,将二段基因拼接并与 p GEM- T克隆载体连接 ,酶切和测序鉴定。结果 :成功扩增出 h PLAP- 1C末端信号肽序列 131bp和 SEA基因序列 796 bp;经测序证实 ,二段基因序列成功拼接 (90 1bp)。结论 :重叠区扩增基因拼接法能将 SEA基因与 h PL AP- 1C末端信号肽序列拼接 ,形成融合基因。Objective: To construct a chimeric SEA-hPLAP-1 cDNA with gene splicing by overlap extension. Methods: The SEA gene and a DNA fragment encoding the signal for GPI-anchor attachment of hPLAP-1 were amplified by PCR.The two amplified gene sequence was annealed to form a chimeric GPI-anchored SEA molecule with gene splicing by overlap extension. The resulting chimera was cloned in pGEM-T vector and verified by sequencing analysis. Results: A chimeric SEA-hPLAP-1 cDNA was successfully constructed with gene splicing by overlap extension. Conclusion: Gene splicing by overlap extension is a successful specific PCR technique for gene recombination.

关 键 词：金黄色葡萄球菌肠毒素A 碱性磷酸酶 重叠区扩增基因拼接法 

分 类 号：R346[医药卫生—基础医学]