EB病毒胸苷激酶基因的定向克隆

采用异硫氰酸胍（ＧｕＳＣＮ）和硅藻从Ｂ９５－８细胞中快速抽摸板ＤＮＡ。根据ＥＢ病毒（ＥＢＶ）Ｂ９５－８株ＤＮＡ全序列及编码ＥＢＶ胸苷激酶（ＴＫ）的开放读框ＢＸＬＦ１的结构，设计合成一对引物，并在引物的５′一端分别引入ＥｃｏＲＩ和ＰｓｔＩ切点，用ＰＣＲ技术扩增出一含完整的ＥＢＶＴＫ基因的１．８４３ＫｂＤＮＡ片段，ＮｃｏＩ酶切分析鉴定，ＥｃｏＲＩ／ＰｓｔＩ双酶切ＰＣＲ产物和载体，使目的基因定向克隆至选

Directional Cloning of Gene Coding Epstein-Barr Virus-specific Thymidine Kinase

The study has successfully inserted the PCR product containing intact EBV TK gene in-to the expression plasmid pBV221 by directional cloning. The template DNA was quickly ex-tracted from B95-8 cells with Guanidinium Isothiocyanate (GuSCN) and silica coarse (SC).Based on the sequence of EBV DNA (B95-8 strain) and the structure of open reading frameBXLF1 coding EBV TK, a pair of primers with EcoR I and Pst I recognition sites respec-tively on their 5' -ends was designed and synthesized. A l. 843Kb DNA fragment containing, intact EBV TK gene was amplified using PCR technique and identified by cleaving with NcoI. PCR-amplified fragment and plasmid pBV221 were restricted with EcoR I and Pst lsimultaneously. The target gene and vector plasmid were directionally ligated by T4 DNAligase, yielding recombinant plasmid pBVTK- Fresh competent E- coli HB101 was trans-formed with recombinant plasmid and the transformd bacteria were grown on LB agar withAmpicillin. A little of plasmid DNA was rapidly prepared using alkaline lysis method andscreened by agarose electropheresis. The positlve recombinant plasmids were confirmed bycleaving with EcoR I or / and Pst I.