ScFv(3G11)-mms13融合基因克隆及表达载体构建

Cloning of ScFv(3G11)-mms13 Fusion Gene and Construction of Its Expression Vector

目的通过重叠延伸拼接PCR(SOE-PCR)技术克隆融合基因ScFv(3G11)-mms13并构建融合蛋白表达载体pET30a(+)-ScFv(3G11)-mms13,为重组制备肿瘤靶向细菌磁小体提供转基因载体。方法根据mms13基因和ScFv(3G11)基因的序列,设计以PCR引物,以克隆载体pMD18-mms13和pMD18-ScFv为模板,采用SOE-PCR技术构建融合基因ScFv-Linker-mms13。进而将融合基因ScFv-mms13与pET30a(+)连接构建重组表达载体pET30a(+)-ScFv-mms13,测序后应用软件DNAMAN进行分析。结果应用重叠延伸拼接PCR方法构建融合基因ScFv-linker-mms13(1158bp),然后通过克隆技术得到重组质粒pMD18-ScFvmms13。将融合基因ScFv-Linker-mms13插入到pET30a(+)构建了重组表达载体pET30a(+)-ScFv-mms13;菌落PCR、酶切、DNA测序鉴定结果均表明目的片段准确地插入到表达载体中。结论通过SOE-PCR技术扩增了预先设计的长达1158bp的融合基因片段ScFv-Linker-mms13,并成功构建了重组质粒pMD18-ScFv-mms13及重组表达载体pET30a(+)-ScFv-mms13,为磁小体用于肿瘤靶向治疗研究提供了实验材料。

Objective The purpose of the study was to generate the DNA of ScFv（ 3G11） -mmsl3 by SOE-PCR and construct fu- sion protein expression vector pET30a（ ＋ ） -SeFv（3GI 1 ） -mmsl 3. To prepare gene transfer vector fur obtaining tumor-targeting bacterial mag- netosornes. Methods In this study,c.onstruction of the fusion gene ScFv-Linker-mmsl3 connected by two primer sels were designed according to the coding region DNA sequences of mmsl3 and ScFv （3Gll）. ScFv-Linker-mmsl3 was generated by SOE-PCR amplification, pMD18-mmsl3 and pMD18-ScFv were used as the templates. And the fusion gene SeFv-mmsl3 was inserted into expression vector pET30a（＋） to construct the recombinant plasmid pET30a（ ＋ ）-ScFv-mmsl3. The sequencing result of the recombinant expression plasmid was analyzed by software DNAMAN. Results The fusion gene of ScFv-linker-mmsl3（1158bp） was generated by SOE-PCR. The fusion gene of SeFv-linker- mmsl 3 was i nserled into the recombinant expression vector pET30a （＋） -mmsl 3-ScFv, and the results of colony PCR test, restriction enzyme digestion and DNA sequencing showed the target fragment was inserted into the expression vector pET30a（ ＋ ） correctly. Conclusion By SOE-PCR technology,we successfnlly amplified fusion gene fragment（1158bp） as the same as ScFv-linker-mmsl3 pre-designed. And we suc- cessfully constructed the recombinant plasmids pMDI 8-ScFv-mmsl3 and pET30a（ ＋ ）-SeFv-mmsl3. This study provided experimental materi- al fnr targeted cancer therapy researeh using the magnetosome.