Objective To amplificate,clone and sequence the thymidine kinase (TK) gene of herpes simplex virusⅡ(HSVⅡ); to construct and appraise the fusion gene eukaryon expression vector, pcDNA3/HSVⅡ TK/angiostatin. MethodsThe Hep2 cells were infected by HSVⅡ Sav strain. HSVⅡ genomic DNA was purified from the Hep2 cells suspension and used as template to run PCR for TK gene amplification. The amplified products were cloned into PC DNA3 vector and sequenced. The vector pcDNA/HSVⅡ TK was cut by endonuclease. The gained TK gene was cloned into eukaryon expression vector. pcDNA3/angiostation, which had been constructed. ResultsCoding region of HSVⅡTK gene consisted of 1 128 bp except stop code, it encoded 376 amino acids.After cutting the new vector by endonuclease Hind Ⅲ and BamH Ⅰ,we gained the following gene fragment: 1000 bp (TK) and 700 bp (angiostation).Conclusion The fusion gene eukaryon expression vector， pcDNA3/HSVⅡ TK/angiostatin has been constructed.
Citation：
WANG Lin,GUO Yongzhang,LI Li,et al.. Constructin and Appraisement of Fusion Gene Eukaryon Expression VectorpcDNA3/HSVⅡ TK/Angiostatin. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2002, 9(4): 246-248. doi:
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- 2. Nagy H, Panis Y, Fabre M, et al.Are hepatomas a good target for suicide gene therapy? An experimental study in rats using retroviralmediated transfer of thymidine kinase gene [J]. Surgery,1998; 123(1)∶ 19.
- 3. O’Reilly MS, Holmgren L, Shing Y,et al.Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastasis by a Lewis lung carcinoma [J]. Cell,1994; 79(2)∶ 315.
- 4. Kit S, Kit M, Qavi H, et al. Nucleotide sequence of the herpes simplex virus type 2(HSV2)thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV1 thymidine kinase gene [J]. Biochim Biophys Acta,1983; 741(2)∶158.
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- 6. Kuriyama S, Nakatani T, Masui K, et al. Bystander effect caused by suicide gene expression indicates the feasibility of gene therapy for hepatocellular carcinoma [J]. Hepatology,1995; 22(6)∶1838.
- 7. Gately S,Twardowski P,Stack MS,et al.The mechanism of cancermediated conversion of plasminogen to the angiogenesis inhibitor angiostatin [J]. Proc Natl Acad Sci USA,1997; 94(20)∶10868.
- 8. Sim BK,O’Reilly MS,Liang H,et al. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer [J]. Cancer Res, 1997; 57(7)∶1329.
