T7噬菌体DNA的提取及其反向遗传拯救方法的建立

DNA extraction of T7 phage and development of its reverse genetics rescue system

从T7噬菌体培养液中粗提噬菌体颗粒,经热裂解后用苯酚、氯仿抽提进而获得纯净的T7噬菌体DNA。用PCR、酶切法鉴定T7噬菌体DNA的完整性。通过对不同感受态细菌浓度、T7噬菌体DNA用量、电转化电压条件的优化,建立了T7噬菌体反向遗传拯救方法。结果显示,提取的DNA结构完整,能够被特异性酶切割,多克隆位点序列正确。T7噬菌体的反向遗传拯救方法最优化条件为200 ng T7噬菌体DNA、1 ml 5×109感受态细菌、1.5 kV电转化电压,在此条件下获得的拯救效率为3.5×105 PFU/ng(DNA)。

The T7 phage DNA was purified with benzene polyphenol and chloroform from T7 phage particles after heat cracking.The integrity of T7 phage DNA was identified by PCR and enzemy digestion,and a reverse genetic rescue system for the purified T7 DNA was established through the optimization of the conditions,such as T7 phage DNA input dosage,density of complete cells,and electrotransformation voltage.The results showed that the purified T7 DNA could be digested with EcoRⅠor Hind Ⅲ,and the multiclon site was correct.The reverse genetics rescue system was successfully established.The highest rescue efficiency was obtained under the conditions of 200 ng T7 phage DNA,5×109 complete cell per milliliter and 1.5 kV electrotransformation voltage,with a output of 3.5×105 PFU/ng（DNA）.