Construction of prokaryotic expression vector carrying HIV Tat gene and the expression, purification and identification of recombinant Tat in E.coli
QIU Hui-ping1, CHENG Xiao-dong2, LU Chun2
(1.The Laboratory Center for Basic Medical Sciences, 2.Department of Microbiology and Immunology, Nanjing Medical University， Nanjing Jiangsu 210029, China)
Abstract：
[Abstract] Objective: To construct the prokaryotic expression vector carrying HIV-1 Tat gene and to purify the fusion protein in E.coli. Methods: The DNA fragment of the Tat gene from pcDNA3-1（+）／Tat101 was cloned into the prokaryotic expression vector pET-28a(+),named pET-28a(+)Tat. Then the recombinant vector was transformed into E.coli BL21 (DE3). The expression of the histidinetagged (His-Tag) fusion protein was induced with isopropyl-β-D-thiogalactopyranoside (IPTG). The fusion protein was detected and purified by Western blot and immobilized Ni2+absorption chromatographic column, respectively. Results: The prokaryotic expression vector carrying Tat gene was successfully constructed. The expression of the His-Tag fusion protein in E.coli BL21 (DE3) was detectable. Finally, the fusion protein with the relative molecular weight of 18×103 was gained after purified using the affinity chromatographic column. Conclusion: Recombinant Tat gene can be expressed in E. coli BL21 (DE3) and the fusion protein obtained can be functionally active.
收稿日期: 2013-05-16
通讯作者:
卢春,教授,博士生导师,E-mail:clu@njmu.edu.cn
作者简介: 邱会平(1982—),女,硕士研究生;
引用本文:
邱会平1, 程晓东2, 卢春2. HIV-1 Tat基因重组原核表达载体的构建及融合蛋白的表达纯化与功能鉴定[J]. 江苏大学学报:医学版, 2013, 23(4): 308-312.
QIU Hui-ping1, CHENG Xiao-dong2, LU Chun2. Construction of prokaryotic expression vector carrying HIV Tat gene and the expression, purification and identification of recombinant Tat in E.coli. Journal of Jiangsu University(Medicine Edition), 2013, 23(4): 308-312.
[1]Li L, Li HS, Pauza CD, et a1.Roles of HIV-1 auxiliary proteins in viral pathogenesis and host-pathogen interactions ［J］. Cell Res, 2005, 15(11／12): 923-934.［2］Jeang KT, Xiao H, Rich EA. Multifaceted activities of the HIV-1 transcription, Tat［J］.J Biol Chem, 1999, 274(41): 28837-28840.［3］Raha T, Cheng SW, Green MR. HIV-1 Tat stimulates transcription complex assembly through recruitment of TBP in the absence of TAFs［J／OL］. PLoS Biol,2005, 3(2): e44.［4］Aoki Y, Tosato G. HIV-1 Tat enhances Kaposi sarcoma associated herpesvirus (KSHV) infectivity ［J］. Blood, 2004, 104(3): 810-814.［5］Buttò S, Fiorelli V, Tripiciano A,et al.Sequence conservation and antibody cross-recognition of clade B human immunodeficiency virus (HIV) type 1 Tat protein in HIV-1-infected Italians, Ugandans, and South Africans ［J］. J Infect Dis, 2003, 188(8): 1171-1180.［6］Chaloin O, Peter JC, Briand JP, et al. The N-terminus of HIV-1 Tat protein is essential for Tat-TAR RNA interaction［J］. Cell Mol Life Sci, 2005, 62:355-361.［7］Zeng Y, Zhang X, Huang Z, et al. Intracellular Tat of human immunodeficiency virus type 1 activates lytic cycle replication of Kaposi′s sarcoma-associated herpesvirus: role of JAK／STAT signaling［J］. J Virol, 2007,81(5):2401-2417.［8］Chen X, Cheng L, Jia X, et al. Human immunodeficiency virus type 1 Tat accelerates Kaposi sarcomaassociated herpesvirus Kaposin A-mediated tumorigenesis of transformed fibroblasts in vitro as well as in nude and immunocompetent mice［J］.Neoplasia, 2009,11(12):1272-1284.［9］Zhou F, Xue M, Qin D,et al.HIV-1 Tat promotes Kaposi′s sarcoma-associated herpesvirus (KSHV) vIL-6-induced angiogenesis and tumorigenesis by regulating PI3K／PTEN／AKT／GSK3β signaling pathway［J］.PLoS One, 2013,8(1):e53145.［10］Midde NM, Gomez AM, Zhu J.HIV-1 Tat protein decreases dopamine transporter cell surface expression and vesicular monoamine transporter-2 function in rat striatal synaptosomes［J］.J Neuroimmune Pharmacol,2012,7(3):629-639.［11］Debaisieux S, Rayne F, Yezid H, et al.The ins and outs of HIV-1 Tat［J］.Traffic,2012,13(3):355-363.［12］Chang HC, Samaniego F, Nair BC, et al. HIV-1 Tat protein exits from cells via a leaderless secretory pathway and binds to extracellular matrix-associated heparan sulfate proteoglycans through its basic region［J］. AIDS, 1997, 11(12): 1421-1431.［13］Imamura J, Suzuki Y, Gonda K,et al.Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan crosslinkage activates Rac1 for internalization［J］.J Biol Chem,2011,286(12):10581-10592.［14］Barillari G, Gendelman R, Gallo RC, et al. The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokineactivated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence［J］. Proc Natl Acad Sci U S A, 1993, 90(17): 7941-7945.
2013, Vol. 23 
