桂北五步蛇纤溶酶金属蛋白酶基因的克隆和序列分析

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基因，利用平端连接的方法将ＰＣＲ扩增产物克隆至ｐＧＥＭ－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扩增得到一６００ｂｐ产物，并被克隆

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

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