桂北五步蛇金属蛋白酶原基因800bp片段的克隆和序列分析

STUDY OF THE METALLOPROTEINASE GENES OF AGKISTRODON ACUTUS VENOM FROM NORTHER MOUNTAIN AREA OF GUANGXI

目的 :扩增桂北五步蛇金属蛋白酶原基因 ,并对其进行序列分析。方法 :从广西桂北山区五步蛇毒腺中抽提蛇毒总RNA,采用一步法 (RT- PCR和 PCR在同一试管内进行 )扩增金属蛋白酶原基因 ,对其进行电泳检测 ,得到 3条特异的 DNA条带 ,大小分别为 1.5 kb、 1.3kb和 80 0 bp,利用平端连接方法将 80 0 bp克隆至 p GEM- T载体 ,挑选白色菌落 ,用酶切和 PCR法对其进行鉴定。提取阳性菌落进行测序并推导编码的氨基酸序列。结果 :信号肽和前肽和已报道的皖南五步蛇金属蛋白酶很相似 ,同源性为 97.9% ,但仅含有部分金属蛋白酶成熟肽的氨基酸序列。结论 :推测此 80 0 bp片段为广西五步蛇金属蛋白酶原基因的一部分。

Objective:To clone and sequence the metalloproteinase genes of Agkistrodon acutus venom from northern mountain area of Guangxi Methods:One step method was taken to extract total RNA from the venom of Agkistrodon acutus which was found in northern mountain area of Guangxi The DNAs encoding metalloproteinase were amplified by one step method (RT PCR and PCR reactions occurred in the same tube) Results:The PCR products were 1 5 kb,1 3 kb and 800 bp The 800 bp PCR product was cloned into the pGEM T vector Plasmid DNA was obtained from positive clones identified by means of digestion with restriction enzyme and PCR Its cDNA sequence was determined with positive clones The cDNA sequence with 786 bp encodes an uncompleted open reading frame (ORF) of 263 amino acids, which included a signal peptide of 18 amino acids,a proposed propeptide of 171 amino acids, a partial mature protein of 74 amino acids Conclusion:The sequences of signal peptide and propeptide are very similar to ones from Wannan Agkistrodon acutus reported by Fan Chunyan, but it only contains partial amino acids of mature protein The 800 bp is likely to be a part of gene encoding metalloproteinase Agkistrodon acutus from Guangxi