潍坊医学院学报
濰坊醫學院學報
유방의학원학보
JOURNAL OF WEIFANG MEDICAL COLLEGE
2013年
3期
168-170
,共3页
纪国强*%邢秋翠%孔登%付新华%王小柯
紀國彊*%邢鞦翠%孔登%付新華%王小柯
기국강*%형추취%공등%부신화%왕소가
AMB-1%mms13%单链抗体%重叠延伸拼接PCR%融合基因
AMB-1%mms13%單鏈抗體%重疊延伸拼接PCR%融閤基因
AMB-1%mms13%단련항체%중첩연신병접PCR%융합기인
AMB-1%mms13%ScFv%SOE-PCR%Fusion gene
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-ScFv-mms13.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.
目的 通過重疊延伸拼接PCR(SOE-PCR)技術剋隆融閤基因ScFv(3G11)-mms13併構建融閤蛋白錶達載體pET30a(+)-ScFv(3G11)-mms13,為重組製備腫瘤靶嚮細菌磁小體提供轉基因載體.方法 根據mms13基因和ScFv(3G11)基因的序列,設計以PCR引物,以剋隆載體pMD18-mms13和pMD18-ScFv為模闆,採用SOE-PCR技術構建融閤基因ScFv-Linker-mms13.進而將融閤基因ScFv-mms13與pET30a(+)連接構建重組錶達載體pET30a(+)-ScFv-mms13,測序後應用軟件DNAMAN進行分析.結果 應用重疊延伸拼接PCR方法構建融閤基因ScFv-linker-mms13(1158bp),然後通過剋隆技術得到重組質粒pMD18-ScFv-mms13.將融閤基因ScFv-Linker-mms13插入到pET30a(+)構建瞭重組錶達載體pET30a(+)-ScFv-mms13;菌落PCR、酶切、DNA測序鑒定結果均錶明目的片段準確地插入到錶達載體中.結論 通過SOE-PCR技術擴增瞭預先設計的長達1158bp的融閤基因片段ScFv-Linker-mms13,併成功構建瞭重組質粒pMD18-ScFv-mms13及重組錶達載體pET30a(+)-ScFv-mms13,為磁小體用于腫瘤靶嚮治療研究提供瞭實驗材料.
목적 통과중첩연신병접PCR(SOE-PCR)기술극륭융합기인ScFv(3G11)-mms13병구건융합단백표체재체pET30a(+)-ScFv(3G11)-mms13,위중조제비종류파향세균자소체제공전기인재체.방법 근거mms13기인화ScFv(3G11)기인적서렬,설계이PCR인물,이극륭재체pMD18-mms13화pMD18-ScFv위모판,채용SOE-PCR기술구건융합기인ScFv-Linker-mms13.진이장융합기인ScFv-mms13여pET30a(+)련접구건중조표체재체pET30a(+)-ScFv-mms13,측서후응용연건DNAMAN진행분석.결과 응용중첩연신병접PCR방법구건융합기인ScFv-linker-mms13(1158bp),연후통과극륭기술득도중조질립pMD18-ScFv-mms13.장융합기인ScFv-Linker-mms13삽입도pET30a(+)구건료중조표체재체pET30a(+)-ScFv-mms13;균락PCR、매절、DNA측서감정결과균표명목적편단준학지삽입도표체재체중.결론 통과SOE-PCR기술확증료예선설계적장체1158bp적융합기인편단ScFv-Linker-mms13,병성공구건료중조질립pMD18-ScFv-mms13급중조표체재체pET30a(+)-ScFv-mms13,위자소체용우종류파향치료연구제공료실험재료.
@@@@ Objective The purpose of the study was to generate the DNA of ScFv (3G11)-mms13 by SOE-PCR and construct fu-sion protein expression vector pET30a(+)-ScFv(3G11)-mms13.To prepare gene transfer vector for obtaining tumor-targeting bacterial mag-netosomes.Methods In this study,construction of the fusion gene ScFv-Linker-mms13 connected by two primer sets were designed according to the coding region DNA sequences of mms13 and ScFv(3G11).ScFv-Linker-mms13 was generated by SOE-PCR amplification,pMD18-mms13 and pMD18-ScFv were used as the templates .And the fusion gene ScFv-mms13 was inserted into expression vector pET30a( +) to construct the recombinant plasmid pET30a(+)-ScFv-mms13.The sequencing result of the recombinant expression plasmid was analyzed by software DNAMAN.Results The fusion gene of ScFv-linker-mms13(1158bp) was generated by SOE-PCR.The fusion gene of ScFv-linker-mms13 was inserted into the recombinant expression vector pET 30a(+)-mms13-ScFv,and the results of colony PCR test,restriction enzyme digestion and DNA sequencing showed the target fragment was inserted into the expression vector pET 30a( +) correctly.Conclusion By SOE-PCR technology,we successfully amplified fusion gene fragment (1158bp) as the same as ScFv-linker-mms13 pre-designed.And we suc-cessfully constructed the recombinant plasmids pMD18-ScFv-mms13 and pET30a(+)-ScFv-mms13.This study provided experimental materi-al for targeted cancer therapy research using the magnetosome .
