[The fusion construction of HIV-1 Tat gene and efficient expression in E.coli].
OBJECTIVE
To express high-level the Tat protein in E.coli.


METHODS
Full-length HIV-1 Tat gene was amplified artificially by PCR and Tat gene was mutated site-specifically (substitution the codons AAG encoding the lysine at the 28th and the 50th site by the CAG encoding glutamine) in order to eliminate the transcriptional activity of Tat protein. The site-mutated Tat gene was fused with chaperone10 gene, and then was subcloned into vector pET28a. The recombinant plasmid was expressed in E.coli BL21(DE3). The expressed products were identified by Western blot.


RESULTS
Full-length HIV-1 Tat gene was amplified successfully by three rounds of PCR. The recombinant plasmid pET28a-chaperone 10-Tat was expressed efficiently in E.coli BL21(DE3). Western blot analysis showed the expressed Tat fusion protein with relative molecular mass (M(r)) 24 000 could bind to anti-His-tag monoclonal antibody.


CONCLUSIONS
Full-length HIV-1 Tat gene was cloned and chaperone 10-Tat fusion protein was expressed efficiently in E.coli BL21(DE3), which will lay the foundation for researching the pathogenic effect of HIV-1 Tat on AIDS.
Lin
Jian-Ping
JP
Department of Immunology, Basic Medical College of Jilin University, Changchun 130021, China.
Wang
Hua
H
Wang
Ling
L
Wei
Hong-Fei
HF
Hu
Xiao-Ping
XP
Wang
Li-Ying
LY
Yu
Yong-Li
YL
D000818:Animals; D019677:Artificial Gene Fusion / Q000379:methods*; D015153:Blotting, Western; D018835:Chaperonin 10 / Q000235:genetics / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D015497:HIV-1 / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
D018835:Chaperonin 10; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D004262:DNA Restriction Enzymes