摘要
试验旨在对转座酶Tn3的酶活力提高和进化进行初步探索。采用PCR扩增、限制性酶切、DNA连接、重组质粒的转化、易错PCR的优化及构建、D值的测定、酶切鉴定方法对转座酶Tn3进行体外定向进化研究。结果表明,用SacⅠ和XbaⅠ对重组质粒进行酶切,得到了450 bp的酶切产物;在含有卡那霉素的LB培养基培养菌体24 h,测量其D值,经过3轮重复性的研究,其D值和增殖能力从开始的0上升至0.18、0.42、0.60,说明Tn3活性有了明显的提高;将筛选得到的进化型重组质粒进行质粒抽提,用内切酶XhoⅠ和XbaⅠ进行双酶切鉴定,发现进化型重组质粒酶切产物条带相对较小;之后对进化型重组质粒进行基因测序分析,发现Tn 3基因序列的多个位点发生了突变以及Tn3-Gal4靶向序列中间的CCR5-delta32基因被切除。说明成功完成了转座酶Tn 3基因的克隆;确立了易错PCR的最优反应体系;选择卡那霉素作为筛选标记对进化型Tn3进行筛选,初步验证利用含有卡那霉素的LB培养基和对菌液D值的测定来进行筛选是可行的;Tn 3基因序列突变和基因敲除,说明不论是在功能上还是在基因序列上Tn3都发生了改变,这种变化正是向着需要的方向进行的。
The aim was to explore the improvement and evolution of the enzyme activity of the transposase Tn3.The in vitro directional evolution of transposase Tn3 was studied by primers design and synthesis,PCR amplification,restriction enzyme digestion,error-prone PCR optimization and construction,DNA rearrangement construction and optimization,D-value determination and enzyme digestion identification.The results showed that the recombinant plasmids were digested with SacⅠand XbaⅠand 450 bp enzyme digestion products were obtained.After 24 h culture in LB medium containing kanamycin,D-value was measured.After three rounds of repeatability studies,D-value and proliferation ability increased from 0 to 0.18,0.42 and 0.60,indicating that Tn3 activity was significantly improved.The selected evolution-type recombinant plasmid was extracted by plasmid extraction,and the endonuclease XhoⅠand XbaⅠwere used for double digestion identification.It was found that the bands of the cleavage products of the evolutionary recombinant plasmids were relatively small.After that,gene sequencing analysis of the recombinant plasmid of evolutionary type showed that mutations occurred in multiple sites of Tn 3 gene sequence and CCR5-delta32 gene in the middle of Tn3-Gal4 targeted sequence was removed.It showed that the cloning of the transposase Tn 3 gene was successfully completed in this study.The optimal reaction system for error-prone PCR was established,it was feasible to use the LB medium containing kanamycin and D-value of the bacterial solution for screening.Mutations in the gene sequence of Tn3 and excision of the gene indicate that Tn3 had changed both in function and in gene sequence,which was exactly in the direction of need.
作者
宋尚桥
马围围
李晓剑
曾素先
李鑫
严瑾
孙翠翠
黎宗强
SONG Shangqiao;MA Weiwei;LI Xiaojian;ZENG Suxian;LI Xin;YAN Jin;SUN Cuicui;LI Zongqiang(College of Animal Science and Technology of Guangxi University,Nanning 530004,China;Shajing Subdistrict Office of Jiangnan District of Nanning City Aquatic Animal Husbandry and Veterinary Station,Nanning 530045,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第4期984-991,共8页
China Animal Husbandry & Veterinary Medicine
基金
广西壮族自治区科技计划项目-重点研发计划(桂科AB16380072)。