重叠PCR法构建嗜酸乳杆菌分选酶A基因外源打靶片段  

Construction of exogenous targeting fragments of sortase A from Lactobacillus acidophilus by overlap extension PCR

作　　者：何嘉怡 王文文[1] 吴京 潘道东[1,2] 吴振[1] 曾小群[1] HE Jia-yi;WANG Wen-wen;WU Jing;PAN Dao-dong;WU Zhen;ZENG Xiao-qun(Key Laboratory of Animal Protein Food Deep Processing Technology of Zhejiang Province, Ningbo University, Ningbo 315211,China;Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, China)

机构地区：[1]宁波大学浙江省动物蛋白食品精深加工技术重点实验室,浙江宁波315211 [2]南京师范大学食品科学与营养系,江苏南京210097

出　　处：《宁波大学学报（理工版）》2017年第6期21-27,共7页Journal of Ningbo University：Natural Science and Engineering Edition

基　　金：国家自然科学基金(31671487;31471598);浙江省自然科学基金青年基金(LQ16C200002);宁波市富民项目(2016C10022);浙江省动物蛋白食品精深加工技术重点实验室开放基金(ZX2015000928)

摘　　要：为比较单步法、分步法重叠PCR在构建嗜酸乳杆菌Lactobacillus acidophilus ATCC4356分选酶A(SrtA)外源打靶片段的差异性,设计了5对具有20~25 bp互补末端的引物,分别扩增两对上下游同源臂和卡那霉素基因,先采用分步法、单步法重叠PCR构建不含筛选基因的外源打靶片段SrtA-up2-SrtA-down2,比较两者优劣,随后取较优者构建含抗生素筛选基因的打靶片段SrtA-up1-Kan-SrtA-down1.结果显示单步法优于分步法,非特异性扩增少,拖尾现象少,且扩增打靶片段SrtA-up1-Kan-SrtA-down1条带单一,无非特异性扩增.即在构建两种外源打靶片段时,单步法重叠PCR优于分步法PCR,为之后构建嗜酸乳杆菌基因打靶载体、构建融合基因打下了基础.To compare the efficiency of one-step and two-step overlap extension PCR in constructing exogenous targeting fragments of sortase A gene(srtA)from Lactobacillus acidophilus ATCC4356,5couples of primers with20-25bp complementary ends were designed to amplify two pairs of upstream and downstream homologous genes as well as kanamycin resistant gene.Firstly,the exogenous targeting fragment,SrtA-up2-SrtA-down2,which included no selective gene,was constructed by one-step and two-step overlap extension PCR respectively.After comparing the advantages and disadvantages of two methods,the exogenous targeting fragment including antibiotic screening gene,SrtA-up1-Kan-SrtA-down1,was constructed by the better one.It is found that one-step overlap extension PCR is more efficient than two-step,causing less nonspecsific amplification and smearing.Furthermore,the amplification result of exogenous targeting fragment,SrtA-up1-Kan-SrtA-down1,suggested no nonspecific amplification.One-step overlap extension PCR is more efficient in constructing the exogenous targeting fragment,which lays a solid foundation on constructing targeting vector of Lactobacillus acidophilus and the fusion genes.

关 键 词：嗜酸乳杆菌 外源打靶片段 单步法重叠PCR 分步法重叠PCR 

分 类 号：Q784[生物学—分子生物学]