Construction Eukaryotic Expression Vector of Human CD59 and Transfection NIH3T3 Cells by Chitosan Encapsulate_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

QIAO Qing ¹ , DOU Kefeng ² , CHEN Yong ² , ZHANG Jing ³ , LI Jianping ² , WANG Yingmei ³ , MAO Haiquan. ⁴

Department of General Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, China;

Corresponding author：

Keywords：

CD59 Chitosan Nanoparticle Gene clone Gene transfection Immunohistochemistry

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【Abstract】ObjectiveTo construct eukaryotic expression vector pSecTag2/HygroB-CD59 of human CD59 and transfect NIH3T3 cells after encapsulated by chitosan.
MethodsThe human CD59 fragments were obtained by PCR form CD59-pGEM-T Easy Vector, cloned into the eukaryotic expression vector pSecTag2/HygroB, identified by restriction endonuclease’s digestion and DNA sequencing. After the particles of pSecTag2/HygroB-CD59 were encapsulated by chitosan, the NIH3T3 cells were transfected by chitosanCD59 nanoparticles and detected CD59 expression by immunohistochemistry stain.
ResultsThe CD59 fragment was 312 bp. Its sequence was as same as CD59 cDNA in Genbank. After having been transfected by chitosan-CD59 nanoparticles in 24 hours, the 3T3 cells showed diffusely positive in the cytoplasms by anti-CD59 immunohistochemistry.
ConclusionThe eukaryotic expression vector of human CD59 is constructed and transfected to NIH3T3 cells after encapsulated by chitosan. It will be very helpful for further study on transgenic livers.

Citation： QIAO Qing,DOU Kefeng,CHEN Yong,ZHANG Jing,LI Jianping,WANG Yingmei,MAO Haiquan.. Construction Eukaryotic Expression Vector of Human CD59 and Transfection NIH3T3 Cells by Chitosan Encapsulate. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2006, 13(4): 424-426. doi: Copy

1.	阿永俊, 李立, 李晓延, 等. 人工肝支持系统在肝衰竭和肝脏移植中的临床应用研究 [J]. 中国普外基础与临床杂志, 2005; 12(5)∶496.
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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.	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. 阿永俊, 李立, 李晓延, 等. 人工肝支持系统在肝衰竭和肝脏移植中的临床应用研究 [J]. 中国普外基础与临床杂志, 2005; 12(5)∶496.
2. 夏穗生. 肝移植进展 [J]. 中国普外基础与临床杂志, 2005; 12(2)∶97.
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. 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. 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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Construction Eukaryotic Expression Vector of Human CD59 and Transfection NIH3T3 Cells by Chitosan Encapsulate

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