| 重叠PCR法构建嗜酸乳杆菌分选酶A基因外源打靶片段 |
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| 引用本文： | 何嘉怡,王文文,吴,京,潘道东,吴,振,曾小群.重叠PCR法构建嗜酸乳杆菌分选酶A基因外源打靶片段[J].宁波大学学报(理工版),2017,0(6):21-27. |
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| 作者姓名： | 何嘉怡 王文文 吴 京 潘道东 吴 振 曾小群 |
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| 作者单位： | (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宁波 315211; 2.南京师范大学 食品科学与营养系 南京 210097) |
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| 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20~25bp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.
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| 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 |
Construction of exogenous targeting fragments of sortase A from Lactobacillus acidophilus by overlap extension PCR |
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| HE Jia-yi,WANG Wen-wen,WU Jing,PAN Dao-dong,,WU Zhen,ZENG Xiao-qun.Construction of exogenous targeting fragments of sortase A from Lactobacillus acidophilus by overlap extension PCR[J].Journal of Ningbo University(Natural Science and Engineering Edition),2017,0(6):21-27. |
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| Authors: | HE Jia-yi WANG Wen-wen WU Jing PAN Dao-dong WU Zhen ZENG Xiao-qun |
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| Affiliation: | ( 1.Key Laboratory of Animal Protein Food Deep Processing Technology of Zhejiang Province, Ningbo University, Ningbo 315211, China; 2.Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, China ) |
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| Abstract: | 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 ATCC 4356, 5 couples of primers with 20-25bp 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. |
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| Keywords: | Lactobacillus acidophilus exogenous targeting fragment one-step overlap extension PCR two-step overlap extension PCR |
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