[Construction of prokaryotic expression vector and expression of human beta defensin 4 gene].
Human beta defensin 4 is a small cationic peptide with a broad range of antimicrobial activity. It plays an important role in innate immunity of human body, especially in mucosal and epithelial defense. In this study, the full-length encoding gene of HBD4 was synthesized by overlap extension polymerase chain reaction and inserted into cloning vector pMD18-T. The gene encoding mature peptide of HBD4 was amplified by PCR and cloned into prokaryotic expression vector pGEX-4T-2. Then pGEX-4T-2/mHBD4 was transformed into E. coli DH5 alpha, which was induced by isopropy-beta-D-thiogalactoside (IPTG). The identification was made by means of endonuclease digestion, DNA sequencing, sodium dodecyl sulphate-polyacrylamine gel electrophoresis (SDS-PAGE). The results showed that the synthesized gene and cloned gene were identical to the HBD4 gene sequence registered in GenBank and were successfully cloned into cloning vector pMD18-T and prokaryotic expression vector pGEX-4T-2. After IPTG induction, the GST-HBD4 fusion protein was successfully expressed in E. coli.
Cao
Yuhong
Y
Xijing Hospital, the Fourth Military Medical University, Xi'an 710032, China.
Zhang
Guocheng
G
Zhang
Guangyun
G
Xu
Yanming
Y
Wang
Lifeng
L
D000890:Anti-Infective Agents; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D023083:beta-Defensins / Q000096:biosynthesis* / Q000235:genetics
D000890:Anti-Infective Agents; C106641:DEFB4A protein, human; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins