摘要
利用RNA 5'末端移动反转录(switching mechanism at 5'end RNA transcription,SMART)技术构建及鉴定疣缟芋螺毒管cDNA噬菌体文库,克隆新型芋螺毒素基因.提取疣缟芋螺毒管总RNA,用RT-PC反转录成单链cDNA(single-stranded cDNA,ss-cDNA),长距离PCR(long-distance PCR,LD-PCR)扩增合成双链cDNA(double-stranded cDNA,ds-cDNA),依次用蛋白酶K和SfiⅠ酶进行酶切孵育,用CHROMA SPIN-400柱进行分级分离.得到的ds-cDNA与λTripIE×2噬菌体载体体外连接并体外包装,感染大肠杆菌E.coli XL1-blue,测定原始文库滴度和重组率.进行文库扩增,测定扩增文库滴度.用PCR鉴定插入cDNA片段的大小,构建的疣缟芋螺毒管cDNA噬菌体原始文库滴度为1.47×106pfu/mL,重组率为90.72%,扩增文库的滴度为4.07×108pfu/mL,插入cDNA片段为200～1 200 bp.利用SMART技术成功构建了疣缟芋螺毒管cDNA噬菌体文库并克隆出新型芋螺毒素基因,为芋螺毒素基因的筛选和鉴定奠定了基础.
To construct and identify a cDNA phage library of the venom duct of Conus lividus,and clone novel conotoxin genes,total RNA was extracted from the venom duct of C.lividus.Single-stranded cDNA（ss-cDNA） and double-stranded cDNA（ds-cDNA） were synthesized by reverse transcription PCR（RT-PCR） and long distance PCR（LD-PCR）.Double-stranded cDNA fragments were digested by proteinase K and SfiⅠenzyme and fractionated by CHROMA SPIN-400.The isolated fragments were recombined to λTripIE×2 phage vector.The recombined cDNA was packaged with GigapackⅢ Gold packing extract in vitro.A small portion of packed phages was used to infect E.coli XL1-blue for titration and determination of the percentage of recombinant clones.The remainder of packed phages was used to infect E.coli XL1-blue for amplification.The titer of the amplified library was detected.The size of the inserted cDNA was identified by PCR.The constructed cDNA phage library of the venom duct of C.lividus consisted of 1.47×106 pfu/mL independent clones with a recombination rate of 90.72%.After amplification,the titer of the amplified library was 4.07×108 pfu/mL.The lengths of the inserted cDNA fragments ranged from 200 to 1200 bp.A high-quality cDNA phage library of the venom duct of C.lividus was successfully constructed and a novel conotoxin gene was cloned.The results would be the basis for screening and identifying more new conotoxin genes of C.lividus.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2012年第5期484-488,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金(No.81160503)
长江学者和创新团队发展计划(No.IRT1123)
海南省重点科技计划项目(No.ZDXM20110040)~~
