桂北五步蛇纤溶酶金属蛋白酶基因的克隆和序列分析

MOLECULAR CLONING AND SEQUENCE OF A cDNA ENCODING FIBRINOLYSIN METALLOPROTEINASE FROM THE VENOM OF AGKISTRODON ACUTUS FROM NORTH GUANGXI

目的 :克隆桂北五步蛇纤溶酶金属蛋白酶基因 ,并对其进行序列分析。方法 :从桂北五步蛇毒腺中抽提总 RNA,采用一步法 (RT- PCR和 PCR在同一管内进行 )扩增出纤溶酶金属蛋白酶基因 ,利用平端连接的方法将 PCR扩增产物克隆至p GEM- T Easy载体 ,挑选白色菌落 ,用酶切和 PCR法对其进行鉴定 ,直接利用纯化 PCR产物进行测序或提取阳性菌落进行测序。结果 :RT- PCR和 PCR扩增得到一 6 0 0 bp产物 ,并被克隆及测序。结论 :该实验产物和已报道的皖南五步蛇纤溶酶金属蛋白酶氨基酸序列相比较 ,有 90 .6 %的同源性 ,这为进一步研究该产物的表达 ,以及表达产物的活性提供条件。

Objective:To clone and sequence a cDNA Encoding fibrinolysin metalloproteinase from the Venom of Agkistrodon acutus from north guangxi Methods:One step method was used to extract total RNA from the venom of Agkistrodon acutus found in northern mountain area of Guangxi The DNAs encoding fibrinolysin metalloproteinase were amplified by one step method (RT PCR and PCR reactions occurred in the same tube)The 600bp PCR product was cloned into the pGEM T easy vector Plasmids obtained from positive clones were identified by means of digestion with EcoR I and PCR reaction The 600bp PCR product was sequenced Results:We got 600bp of amplified product that was cloned into E coli JM109 Its sequence was determined Conclusion:Compared with the amino acids sequence for the fibrinolysin metalloproteinase from the Venom of Agkistrodon acutus from the southern of Anhui Province,their homology is 90 6% This result provides the condition for its research into expression and analyses of its activity