一步法快速构建多片段连接的同源臂载体

摘要

目的：建立一种简便、高效，可一步完成多个片段连接，从而构建含同源臂的载体的方法。方法：按照酶切后可产生前后片段相匹配的粘性末端接头的原则设计PCR引物，在目的片段两端均引入BsaⅠ酶切位点。以G160基因为例，PCR扩增打靶用左右同源臂片段、示踪基因CMV-EGFP片段、载体骨架pMD19-T等4个片段，纯化后一起加入一个反应管中，并加入BsaⅠ限制性内切酶和T7 DNA连接酶及相应缓冲液，进行酶切、酶连接共10~50个循环反应，一步构建含同源臂载体的质粒；产物经高温处理后，直接转化感受态细胞，并进行重组子PCR鉴定；对pMD19-T载体进行优化，突变载体上的BsaⅠ酶切位点，把示踪基因CMV-EGFP片段引入pHSG298-T载体，再选择不同的G160基因同源臂片段组合对构建系统进行验证。结果：重组质粒酶切和PCR结果表明，应用一步法可成功连接多个片段来构建含同源臂及示踪基因的克隆载体；用优化后的pMD19-T-O载体体系，在2 d内即完成了6种各含4个片段的载体的构建。结论：多个基因片段一步无缝连接的方法简便、易行、可靠，不仅可快速构建某类载体系统，还可对基因进行精确的点突变，该系统可用于快速构建基因打靶载体。%　　Objective: To set up a simple and high efficiency method to construct the homologous arm vector composed of multi-fragments within one step. Methods: According to the principle that the matched cohesive ends between the tandem fragments can be generated after restriction digestion, BsaⅠ sites were added at the two ends of PCR primers to amplify target fragment. The G160 gene was chosen as the target gene, and the four frag⁃ments including left and right homologous arms, tracer gene fragment CMV-EGFP and vector frame pMD19-T were amplified by PCR using the designed primers. Then all the DNA fragments, BsaⅠ, T7 ligase and the corre⁃sponding buffer were added into the reaction solution with 10~50 cycles of digestion and ligation to construct the homologous arm vector composed of multi-DNA fragments. The reaction was stopped at 72℃, and the ligated prod⁃ucts were transformed into the competent cells for the subsequent identification by enzyme digestion and PCR. pMD19-T was then optimized to eliminate the BsaⅠ site on it, and the racer gene fragment CMV-EGFP was in⁃serted into pHSG298-T vector containing resistant marker. The different combination of homologous arms were used to validated this methodology. Results: The results of enzyme digestion and PCR demonstrated that one step approach could successfully construct the homologous arm vector composed of multi-DNA fragments. Six different four-fragments composing vectors can be constructed using the optimized pMD19-T-O vector system within two days. Conclusion: The one step method for vector construction was an simple, feasible and reliable approach to connect multiple fragments seamlessly into one vector. This methodology can not only be used to construct some kinds of vector systems but also make precise point mutations. This approach was suitable for quick construction of gene targeting vector.