重叠PCR法构建嗜酸乳杆菌分选酶A基因外源打靶片段
PDF下载 (17765)何嘉怡1,王文文1,吴 京1,潘道东1,2,吴 振1*,曾小群1.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),2017,30(6):21-27.DOI:
HE Jia-yi1,WANG Wen-wen1,WU Jing1,PAN Dao-dong1,2,WU Zhen1*,ZENG Xiao-qun1.Construction of exogenous targeting fragments of sortase A from Lactobacillus acidophilus by overlap extension PCR[J].Journal of Ningbo University(Natural Science & Engineering Edition),2017,30(6):21-27.DOI:
| Title: | Construction of exogenous targeting fragments of sortase A from Lactobacillus acidophilus by overlap extension PCR |
| 作者: | 何嘉怡1, 王文文1, 吴 京1, 潘道东1, 2, 吴 振1*, 曾小群1 |
| Author(s): | HE Jia-yi1, WANG Wen-wen1, WU Jing1, PAN Dao-dong1, 2, WU Zhen1*, ZENG Xiao-qun1 |
| 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 |
| Keywords: | Lactobacillus acidophilus; exogenous targeting fragment; one-step overlap extension PCR; two-step overlap extension PCR |
| 分类号: | Q784 |
| 文献标识码: | A |
| 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. |
| 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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| 备注/Memo: | 收稿日期: 2017?04?10. 宁波大学学报（理工版）网址: http://journallg.nbu.edu.cn/ 基金项目: 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）. 第一作者: 何嘉怡（1992－）, 女, 浙江宁波人, 在读硕士研究生, 主要研究方向: 乳品科学. E-mail: 478809548@qq.com *通信作者: 吴振（1985－）, 男, 河南新乡人, 讲师, 主要研究方向: 乳品科学. E-mail: woodsen@163.com 宁波大学学报（理工版）网址：http://journallg.nbu.edu.cn/ |
