中华眼镜蛇毒腺cDNA表达文库的建立和分析  

Construction of cDNA library from venom of Chinese Cobra

作　　者：万榕[1] 宋军[1] 林旭[1] 林建银[1] 

机构地区：[1]福建医科大学分子医学中心,病理学系福建福州350004

出　　处：《第四军医大学学报》2004年第13期1208-1211,共4页Journal of the Fourth Military Medical University

基　　金：福建省重大科技项目 (2 0 0 2Y0 0 3)

摘　　要：目的 :为研究开发眼镜蛇毒药用基因工程产品 ,筛选克隆及表达相关药用功能基因 ,构建中华眼镜蛇毒腺cDNA表达文库 .方法 :一步法提取总RNA ,Oligo(dT)纤维素层析柱纯化mRNA ,逆转录PCR合成双链cDNA ,分级分离除去小片段后 ,收集大于 5 0 0bp的cDNA片段 ,取 1 0ngcDNA与质粒载体pSPORT1连接、转化 .结果 :获得克隆总数为 2× 1 0 5的眼镜蛇毒腺cDNA表达文库 ,重组率 97% .利用PCR技术从该文库扩增了心脏毒素 3(CTX 3)和磷脂酶A2 (PLA2 )基因的cD NA .通过对文库克隆的序列测定和初步生物信息学分析 ,获得 2 4个中华眼镜蛇毒腺EST序列 .结论 :所建立的毒腺cDNA表达文库质量较高 ,可用于进一步筛选。AIM: To construct a high quality cDNA expression library from the venom of Chinese Cobra. METHODS: Total RNA and purified mRNA were extracted by using ”single step method” and by chromatography on oligo (dT) cellulose. The cDNA was synthesized through reverse transcription. After cDNA size fractionation was purified by chromatography column, the double cDNA was ligated to pSPORT1 plasmid vector. RESULTS: The directional library was confirmed to be about 2×10 5 independent clones in which the percentage of recombinant clones was 97% and the whole length cDNA of CTX 3 and PLA2 gene were obtained by PCR amplification. CONCLUSION: The constructed cDNA library can be used for further screening and cloning of new protease inhibitor gene in Chinese cobra.

关 键 词：中华眼镜蛇 毒腺 CDNA表达文库 PCR 

分 类 号：Q75[生物学—分子生物学]