中国蛇岛蝮蛇毒腺cDNA文库的构建

Construction of a cDNA Library from the Venom Gland of Chinese Gloydius shedaoensis shedaoensis

为研究中国蛇岛蝮蛇(Gloydius shedaoensis shedaoensis)基因工程产品,采用SMART(switching mechanism at 5′end of RNA transcript)技术构建其毒腺的cDNA表达文库。采用RNAiso试剂提取毒腺总RNA,再采用Poly(A)PuristTM试剂盒分离纯化mRNA,第一链cDNA经反转录合成,双链cDNA经LD-PCR合成并扩增;分级分离去除小片段,收集大于400 bp的cDNA片段,与质粒载体pDNR-LIB连接,采用电转化法将重组质粒转入大肠杆菌感受态细胞DH10B。构建的蛇岛蝮蛇毒腺cDNA文库库容量为8×107cfu/ml,大多数插入片段大于1 000 bp,重组质粒表达率高(100%)。本研究成功构建了高质量的蛇岛蝮蛇毒腺cDNA文库,表达文库质量高,可用于进一步克隆和表达中国蛇岛蝮蛇蛋白活性组分基因,并对其功能进行研究。

A high quality cDNA library from the venom gland of Chinese Gloydius shedaoensis shedaoensis was constructed using SMART（switching mechanism at 5′ end of RNA transcript） technique.Total RNA was extracted using RNAiso reagent kit and mRNA was purified using Poly（A） PuristTM kit,respectively.Singlestrand cDNA was synthesized using reverse transcription PCR and double-strand cDNA was synthesized and amplified using LD（long-distance） PCR.The cDNA fragments with the size over 400 bp were fractionated and ligated to pDNR-LIB vector.Recombined plasmids were then elelctrotransformed in Escherichia coli DH10B strains.The capacity of the constructed library was 8 × 107 cfu /ml.The size of most inserts was larger than 1 000 bp.The percentage of the selected recombinant clones was very high（100%）.Thus,a cDNA library from the venom gland of G.s.shedaoensis has been successfully constructed.It has a high recombinant expression level,and can be used for cloning and expressing bioactive protein genes from G.s.shedaoensis and for studying their biological functions.