眼镜蛇毒腺cDNA文库的构建

cDNA Library Construction of the Venom Gland in Naja naja atra

目的 蛇毒含有多种生物活性成分， 从天然蛇毒中提取分离蛇毒有效成分受到蛇毒资源和质量的限制。为开发蛇毒有效成分的基因工程产品， 本研究构建了蛇毒腺的ｃ Ｄ Ｎ Ａ 文库， 为进一步筛选、克隆和表达蛇毒有关基因做准备。 方法 从眼镜蛇（ Ｎａｊａ ｎａｊａ ａｔｒａ ）毒腺中提取ｍ Ｒ Ｎ Ａ，经反转录合成ｃ Ｄ Ｎ Ａ后， 以λｇｔ１０ 噬菌体为载体， 构建非表达的ｃ Ｄ Ｎ Ａ 文库。 结果 蛇毒腺ｃ Ｄ Ｎ Ａ 非表达文库库容量为２×１０６ｐｆｕ／μｇ 重组子， 经大肠杆菌 Ｃ６００ ｈｌｆ 菌株平皿测定， 重组率为 ７０％ 。 结论 经平板鉴定和 Ｐ Ｃ Ｒ 快速鉴定表明， 所构 ｃ Ｄ Ｎ Ａ 文库达到建库要求， 能够用于目的基因筛选和克隆表达。

Objective Non expression cDNA library of venom gland ofNaja naja atra was constructed for the development of genetically engineered products Method After mRNA extracted from the venom gland, cDNA was obtained by in vitro transcription, and then ligated to EcoR Iarm of bacteriophageλgt10 vector, followed by λ DNA packaging and infected host bacterial strain C600hlf Results According to the phage plaques bright selection, the percentage of recombinant phages are 70% and the titer is 2×10 6 pfu/μg Conclusion Ths size of the library was determined by the number of independent recombinant p;aques and PCR The result showed that the library is available for screening target gene and expression of proteins