• 1.第四軍醫(yī)大學唐都醫(yī)院普通外科（西安710038）;;
  • 2.第四軍醫(yī)大學西京醫(yī)院肝膽外科（西安710033）;;
  • 3.第四軍醫(yī)大學西京醫(yī)院病理科;;
  • 4.新加坡約翰霍普金斯大學生物醫(yī)學部（新加坡138669）;

目的構(gòu)建人CD59真核表達載體pSecTag2/HygroB-CD59，并利用殼聚糖（chitosan）組裝成納米微粒復合體，轉(zhuǎn)染小鼠NIH3T3細胞。
方法應用PCR方法，從CD59-pGEM-T Easy Vector克隆相關(guān)的DNA序列，插入到具有相應酶切位點的真核表達載體pSecTag2/HygroB中，構(gòu)建pSecTag2/HygroB-CD59真核表達質(zhì)粒，并進行酶切鑒定及序列測定。利用殼聚糖包裹質(zhì)粒，轉(zhuǎn)染小鼠NIH3T3細胞，并用抗CD59抗體對轉(zhuǎn)染細胞進行免疫組織化學染色。
結(jié)果CD59基因大小為312 bp，與Genbank中記載的人CD59 cDNA序列結(jié)果完全一致。利用殼聚糖-CD59納米微粒轉(zhuǎn)染NIH3T3細胞24 h后，免疫組織化學染色顯示轉(zhuǎn)染細胞CD59呈彌漫性胞漿陽性。
結(jié)論成功構(gòu)建了人CD59真核表達載體并通過殼聚糖介導轉(zhuǎn)染NIH3T3細胞，為進一步探討及研究轉(zhuǎn)基因肝臟奠定了基礎(chǔ)。

引用本文： 喬慶,竇科峰,陳勇,張靜,李劍平,王映梅,毛海泉. 構(gòu)建人CD59真核表達載體并利用殼聚糖介導轉(zhuǎn)染NIH3T3細胞. 中國普外基礎(chǔ)與臨床雜志, 2006, 13(4): 424-426. doi: 復制

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5. Tada T, Okada H, Okada N, et al. Membrane attack complex of complement and 20 kDa homologous restriction factor(CD59) in myocardial infarction [J]. Virchows Arch， 1997; 430(4)∶327.
6. Byrne GW, McCurry KR, Martin MJ, et al. Transgenic pigs expressing human CD59 and decayaccelerating factor produce an intrinsic barrier to complementmediated damage [J]. Transplantation， 1997; 63(1)∶149.
7. Longhi MP, Sivasankar B, Omidvar N, et al. Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complementindependent manner [J]. J Immunol, 2005; 175(11)∶7098.
8. Hanna SM, Spiller OB, Linton SM, et al. Rat T cells express neither CD55 nor CD59 and are dependent on Crry for protection from homologous complement [J]. Eur J Immunol, 2002; 32(2)∶502.
9. Hill A, Ridley SH, Esser D, et al. Protection of erythrocytes from human complementmediated lysis by membranetargeted recombinant soluble CD59: a new approach to PNH therapy [J]. Blood, 2006; 107(5)∶2131.
10. Harada Shiba M, Yamauchi K, Harada A, et al. Polyion complex micelles as vectors in gene therapypharmacokinetics and in vivo gene transfer [J]. Gene Ther, 2002; 9(6)∶407.
11. Ishii T, Okahata Y, Sato T. Mechanism of cell transfection with plasmid/chitosan complexes [J]. Biochim Biophys Acta, 2001; 1514(1)∶51.
  1. 1. 阿永俊, 李立, 李曉延, 等. 人工肝支持系統(tǒng)在肝衰竭和肝臟移植中的臨床應用研究 [J]. 中國普外基礎(chǔ)與臨床雜志, 2005; 12(5)∶496.
  2. 2. 夏穗生. 肝移植進展 [J]. 中國普外基礎(chǔ)與臨床雜志, 2005; 12(2)∶97.
  3. 3. Ramirez P, Montoya MJ, Rios A, et al. Prevention of hyperacute rejection in a model of orthotopic liver xenotransplantation from pig to baboon using polytransgenic pig livers (CD55, CD59, and Htransferase) [J]. Transplant Proc， 2005; 37(9)∶4103.
  4. 4. Huang Y, Smith CA, Song H, et al. Insights into the human CD59 complement binding interface toward engineering new therapeutics [J]. J Biol Chem， 2005; 280(40)∶34073.
  5. 5. Tada T, Okada H, Okada N, et al. Membrane attack complex of complement and 20 kDa homologous restriction factor(CD59) in myocardial infarction [J]. Virchows Arch， 1997; 430(4)∶327.
  6. 6. Byrne GW, McCurry KR, Martin MJ, et al. Transgenic pigs expressing human CD59 and decayaccelerating factor produce an intrinsic barrier to complementmediated damage [J]. Transplantation， 1997; 63(1)∶149.
  7. 7. Longhi MP, Sivasankar B, Omidvar N, et al. Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complementindependent manner [J]. J Immunol, 2005; 175(11)∶7098.
  8. 8. Hanna SM, Spiller OB, Linton SM, et al. Rat T cells express neither CD55 nor CD59 and are dependent on Crry for protection from homologous complement [J]. Eur J Immunol, 2002; 32(2)∶502.
  9. 9. Hill A, Ridley SH, Esser D, et al. Protection of erythrocytes from human complementmediated lysis by membranetargeted recombinant soluble CD59: a new approach to PNH therapy [J]. Blood, 2006; 107(5)∶2131.
  10. 10. Harada Shiba M, Yamauchi K, Harada A, et al. Polyion complex micelles as vectors in gene therapypharmacokinetics and in vivo gene transfer [J]. Gene Ther, 2002; 9(6)∶407.
  11. 11. Ishii T, Okahata Y, Sato T. Mechanism of cell transfection with plasmid/chitosan complexes [J]. Biochim Biophys Acta, 2001; 1514(1)∶51.