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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 the plasmin metalloproteinase gene from Wubei snake in northern Guangxi and analyze its sequence. METHODS: Total RNA was extracted from the venom gland of the five-step snake venom from northern Guangxi and the plasmin metalloproteinase gene was amplified by one-step method (RT-PCR and PCR in the same tube). The PCR amplification product was cloned by blunt end ligation. To p GEM-T Easy vector, white colonies were picked and identified by enzyme digestion and PCR. The purified PCR products were used for sequencing or positive colonies were extracted for sequencing. Results : RT-PCR and PCR amplification yielded a 600 bp product, which was cloned and sequenced. Conclusion : This experimental product has 90.6 % homology with the reported amino acid sequence of the five-step snake broth plasmin metalloproteinase, which provides the conditions for further study of the expression of the product and the activity of the expression product.