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目的构建非标准化尖吻蝮(五步蛇,Agkistr-odonacutus)毒腺cDNA文库,随机挑取克隆测序,分析金属蛋白酶基因。方法以Trizol试剂提取新鲜尖吻蝮毒腺总RNA,以superscriptⅡ反转录酶合成cDNA第一链并以DNA聚合酶Ⅰ连续合成第二链。双链DNA经过含EcoRⅠ酶切位点接头加接,末端磷酸化并以XhoⅠ内切酶酶切,按照<0.25kb,0.25~0.5kb,0.5~1kb,1~2kb和>2kb5个片段大小分别回收,随后与pBluescriptⅡSK(+)载体相连转化E.coliDH10B,构建成尖吻蝮毒腺cDNA文库。随机挑取克隆5′端测序,共获得8696条高质量表达序列标签,经过序列拼接和聚类,这些序列在经过功能注释后最终被聚类成2855个基因聚类。其中,发现一个由74个克隆组成的基因聚类(Agkihagin)为新的金属蛋白酶基因。经反转录和巢式PCR扩增该基因并对其进行结构分析。结果构建好的文库含有2.048×106个重组子,新的金属蛋白酶开框读码序列全长1827个核苷酸,编码608个氨基酸,属于PⅢ型金属蛋白酶。其Zn2+结合模序HEMGHNLGIDH和去整合素模序DECD在进化上高度保守。结论该文库符合建库标准库容要求,为构建尖吻蝮毒腺基因表达谱和筛选新的目的基因提供了有效平台;克隆的金属蛋白酶基因与GenBank中其他蛇毒金属蛋白酶氨基酸序列同源性最高达87%,为研究蛇毒金属蛋白酶结构与功能的关系奠定了良好基础。
Objective To construct a non-standard cDNA library of venom gland of Agkistr-odonacutus, randomly select clones and analyze the metalloproteinase gene. Methods Trizol reagent was used to extract the total RNA from the fresh venom of Apodemus agrarius. The first strand of cDNA was synthesized by superscript Ⅱ reverse transcriptase and the second strand was synthesized by DNA polymerase Ⅰ. Double-stranded DNA was digested with EcoR I restriction site and then phosphorylated and digested with XhoI endonuclease. The size of the double-stranded DNA was <0.25kb, 0.25-0.5kb, 0.5-1kb, 1-2kb and> 2kb respectively And then transformed into E. coliDH10B by ligating with pBluescriptⅡSK (+) vector to construct a venom gland cDNA library. A total of 8696 high-quality expressed sequence tags were obtained by randomly picking the 5 ’end of the clones. After sequence splicing and clustering, these sequences were clustered into 2,855 gene clusters after functional annotation. Among them, a 74-clone gene cluster (Agkihagin) was found to be a new metalloprotease gene. The gene was amplified by reverse transcription and nested PCR and its structure was analyzed. Results The constructed library contained 2.048 × 106 recombinants. The new open reading frame of the metalloprotease was 1827 nucleotides in length and encoded 608 amino acids, belonging to the type Ⅲ metalloproteinase. The Zn2 + binding motif HEMGHNLGIDH and the disintegrin motif DECD are evolutionarily highly conserved. CONCLUSION: This library conforms to the standard of reservoir capacity, and provides an effective platform for constructing the gene expression profile of Apodemus agrarius and screening new target genes. The homology between the cloned metalloprotease gene and other snake venom metalloproteinase in GenBank is as high as 87 %, To lay a good foundation for the study of the relationship between structure and function of metalloproteinase in snake venom.