摘要
在已知基因序列但缺乏DNA模版的情况下,人工设计合成多条引物,采用重叠PCR技术体外人工合成899bp的MluⅠ基因和1 312bp的M.MluⅠ基因.并将合成的MluⅠ基因连接到表达载体pet30a上,重组表达载体构成pet30a-MluⅠ,将M.MluⅠ基因连接到表达载体PUC19上,重组表达载体构成PUC19-M.MluⅠ.测序结果显示,载体构建成功后利用体外重组转化技术,将pet30a-MluⅠ和PUC19-M.MluⅠ转入T7expression E.coli表达菌株中.重组工程菌经IPTG诱导,用SDS-PAGE鉴定,发现限制性内切酶MluⅠ成功表达.该酶的成功表达不仅为后续研究提供了材料,而且为实验室制备限制性内切酶在方法上开辟了新的途径,为更多新发现的内切酶基因的成功克隆提供了参考.
With a known gene sequence but in the absence of DNA template,we have artificially designed and synthesized a number of primers to synthesize MluⅠ gene of 899 bp and M.MluⅠ gene of 1 312 bp in vitro using overlapping PCR technique.After that we successfully constituted a recombinant expression vector Pet30a-MluⅠ with linking MluⅠ gene to expression vector pet30 a,as PUC19-M.MluⅠ do equally.Pet30a-MluⅠ and PUC19-M.MluⅠ were transformed into T7 expression E.coli expression strains after sequencing.Expression strains were induced by IPTG,identified by SDS-PAGE and found successful expression of MluⅠ.This successful case not only provide for follow-up study of the material,but also for the preparation of restriction endonuclease enzymes in new ways in lab,as more within providing a reference of the newly discovered and successfully cloned restriction endonuclease enzyme gene.
出处
《西南师范大学学报（自然科学版）》
CAS
北大核心
2016年第10期46-53,共8页
Journal of Southwest China Normal University(Natural Science Edition)
基金
苏州工业园区服务外包职业学院教研教改研究课题(JG-201601)
