In order to study PBD-2 and PoIFNgamma, the chimeric gene PBD-2-PoIFNgamma was synthesized by overlap extension PCR, and amplified PoIFNgamma on the basis of this sequence, then cloned into yeast expression vector pPICZalphaA separately to get the recombinant plasmid pPICZalphaA-PBD-2-PoINFgamma and pPICZalphaA-PoINFgamma. The recombinant plasmid was digested by Sac I and introduced into Pichia pastoris X33 cells by electroporation. Positive clones were screened and cultivated in BMMY medium containing 0.5% methanol for 72 h. SDS-PAGE and Western blotting analysis showed that the screened recombinant could secrete PBD-2-PoINFgamma and PoINFgamma separately. The activity of fusion protein was not detected by cytopathic effect inhibition assay and agar diffusion assay, but detected obvious antiviral activity of PoINFgamma. The helix and random coil contents was showed vary greatly between PoIFNgamma and PBD-2-PoLNFgamma by circular dichroism analysis. It was speculated that the fusion protein was not correctly folded and may affect the activity of PBD-2-PoINFgamma.
Animals ; Genetic Vectors ; genetics ; Interferon-gamma ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; Swine ; beta-Defensins ; biosynthesis ; genetics

The inductive conditions for the flask-shaking of E.coli BL21-pET-hBD3-Bra had been optimized, at the same time, the expressed protein had been purified and analyzed. The effect of three factors which were IPTG concentration, induction time and temperature on growth of strain and on the yield of hBD3-Bra was analyzed in detail. The result indicated that the concentration of IPTG had little effect on the growth and the expression of target protein between 0.2-1 mmol/L, Biomass would be improved as time passed, but the target protein didn't increase obviously as the same time, temperature was the most important factor, the expressed level of hBD3-Bra, as high as about 35% of total cell protein, could be gained when strain was induced by IPTG under 30 degrees C. Further analysis showed the best temperature for growth was 30 degrees C-32 degrees C and for expression protein was 30 degrees C.The purified hBD3-Bra has a weak antimicrobial activity, but is 200 times sweeter than that of sucrose. After digested by thrombin and purified by affinity column, the natural des-pGlul-Brazzein also has 600-time sweetness of sucrose, and the recombinant hBD3 has a high antimicrobial activity again E. coli and S. aureus.
Anti-Infective Agents ; Escherichia coli ; genetics ; metabolism ; Humans ; Plant Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Sweetening Agents ; Temperature ; beta-Defensins ; biosynthesis ; genetics

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.
Anti-Infective Agents ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics

To investigate whether LL-37 and human beta defensin-2 (hBD-2) is related to the patients with psoriasis seldom having skin infections and explore the role of the two peptides and CCR6 (the receptor of hBD-2) in the pathogenesis of psoriasis, the expression levels of mRNA of LL-37, hBD-2, and CCR6 in skin lesions of patients with psoriasis vulgaris were detected by using RT-PCR. The results showed that the mRNA expression levels of the two peptides and CCR6 in psoriatic lesions all increased compared with the normal skin (P<0.001). It was suggested that up-regulated expression of LL-37 and hBD-2 might be the main reason that result in the the skin of patients with psoriasis being seldom infected, and the two peptides and CCR6 might play crucial roles in the pathogenesis of psoriasis.
Adult ; Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; Female ; Humans ; Male ; Middle Aged ; Psoriasis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, CCR6 ; Receptors, Chemokine ; biosynthesis ; genetics ; Up-Regulation ; beta-Defensins ; biosynthesis ; genetics

The objective of the study was to clone avian beta-defensin (AvBD) 5 gene from pigeon bone marrow tissues and liver tissues, to express the recombinant AvBD5 protein in E. coli, and to determine its antimicrobial activity. The mRNA of duck AvBD5 was cloned from pigeon bone marrow tissues and liver tissues by RT-PCR. In addition, phylogenetic relationships between amino acid sequence of the pigeon AvBD5, AvBDs from other avian species, and some mammalian beta-defensin-5 were analyzed. The cDNA of pigeon AvBD5 was sub-cloned into pGEX-6p-1 vector to construct recombinant plasmid pGEX-pigeon AvBD5. The recombinant protein was expressed into E. coli and purified. Antimicrobial activity and physical-chemical stability of the recombinant fusion protein were measured in vitro. The complete nucleotide sequence of both cDNAs contained 201 bp nucleotides, encoding a polypeptide of 66 amino acids. Both beta-defensins have six conserved cysteines. Phylogenetic relationships were analyzed. Both pigeon AvBDs shared the highest amino acid homology (87.9% and 78.8%) with duck AvBD5. So it was named as pigeon AvBD5alpha (bone marrow) and AvBD5beta (liver). Both recombinant plasmids were transformed into E. coli BL21 and the bacteria were induced with Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). After purification, antibacterial activity of the purified was investigated. In addition, effect of ionic strength on the antibacterial activity, and hemolytic recombinant protein activity of the purified recombinant protein were investigated. A 32 kDa protein was highly expressed. Both purified recombinant pigeon AvBD5alpha and AvBD5beta exhibited extensive antimicrobial activities against 12 bacteria, including Gram-positive and Gram-negative. In high salt ions concentrations, antibacterial activity of both recombinant proteins was decreased. In addition, the hemolysis activity of recombinant protein was extremely low.
Amino Acid Sequence ; Animals ; Anti-Infective Agents ; metabolism ; pharmacology ; Avian Proteins ; biosynthesis ; genetics ; pharmacology ; Cloning, Molecular ; Columbidae ; genetics ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; beta-Defensins ; biosynthesis ; genetics ; pharmacology

OBJECTIVETo investigate the effect of IL-10 on the inducibility of human beta-defensin 2 (hBD-2) in human peripheral blood cells.

METHODSPeripheral blood samples were collected from 22 healthy individuals and co-cultured with 0 ng/ml lipopolysaccharide (LPS), 100 ng/ml LPS, 10 ng/ml IL-10, or 100 ng/ml LPS plus 10 ng/ml IL-10 at 37 degree for 6 h. Total RNA was extracted from peripheral blood cells and the mRNA level of hBD-2 was detected by relative quantitative real-time PCR..

RESULTNo detectable level of hBD-2 mRNA was found in normal peripheral blood cells with stimulation of 0 ng/ml LPS or 10 ng/ml IL-10. The mRNA level of hBD-2 was increased to 166.9 +/- 35.14 after 100 ng/ml LPS challenge, while the mRNA level of hBD-2 was decreased to 30.40 +/- 9.18 after the co-stimulation of 100 ng/ml LPS plus 10 ng/ml IL-10; which was significantly lower than that with LPS alone (P<0.05).

CONCLUSIONThe expression of hBD-2 gene can be induced by LPS.IL-10 inhibits the inducible expression of hBD-2.

Blood Cells ; cytology ; drug effects ; metabolism ; Cells, Cultured ; Humans ; Interleukin-10 ; pharmacology ; Lipopolysaccharides ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; beta-Defensins ; genetics

To establish human beta-defensin-3 gene transgenic cell lines as competent donor cells for the production of transgenic animals using somatic cell nuclear transfer (SCNT). Firstly, we obtained human beta-defensin-3 by RT-PCR from human placenta, and subsequently inserted the fragment hBD into the corresponding site of the plasmid pBCP. Then we moved the combined fragment BCD (including 5' and 3' regulating region of beta-casein and hBD) into the corresponding site of the plasmid pEGFP-C1. Finally we successfully constructed mammary-specific expression vector pEBCD. We transected pEBCD into Holstein Fetal fibroblast cells by Lipofectamine TM-2000 and selected in medium with G418 for three to four weeks. We identified G418 resistant transfectants by PCR, RT-PCR and EGFP detection. Our results indicated that human beta-defensin-3 gene stably was integrated into the open region of the chromatin in G418 resistant fibroblast cells. Meanwhile we identified the expression of human beta-defensin-3 in the supernatant of stable transfected mammary epithelial cells by Western blotting. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.
Animals ; Animals, Genetically Modified ; Caseins ; genetics ; Cattle ; Epithelial Cells ; metabolism ; Genes, erbB-1 ; genetics ; Genetic Vectors ; genetics ; Humans ; Mammary Glands, Animal ; cytology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; beta-Defensins ; biosynthesis ; genetics

The objective of the study was to clone avian beta-defensin (AvBD) 3 gene from goose tissues, express the recombinant AvBD3 protein in Escherichia coli, and determine its antimicrobial activity. The mRNA of goose AvBD3 was cloned from spleen and bursa of Fabricius of the gooses by RT-PCR. The sequence analysis showed that the genefragment of AvBD3 contained 182 bp, and encoded 60 amino acids. Homology analysis showed that goose AvBD3 shared the highest percentage of amino acid homology (100%) with chicken AvBD3. The cDNA of goose AvBD3 was sub-cloned into BamH I and Sal I sites of pGEX-6p-1 vector to construct recombinant plasmid pGEX-goose AvBD3. The recombinant plasmid was transformed into E. coli BL21 and the bacteria was induced with IPTG It was demonstrated by SDS-PAGE that a 31 kDa protein which was equal to goose AvBD3 protein in molecular weight was highly expressed. The purified recombinant goose AvBD3 exhibited extensive antimicrobial activity against twelve bacteria strains, including Gram-positive and Gram-negative investigated. At high salt ions conditions, antimicrobial activity of recombinant goose AvBD3 protein against both Staphylococcus aureus and Pasteurella multocida decreased significantly. In addition, hemolysis activity of the recombinant protein was extremely low, and the recombinant protein remained antimicrobial activity under different pH values.
Amino Acid Sequence ; Animals ; Anti-Bacterial Agents ; chemistry ; metabolism ; pharmacology ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Geese ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; beta-Defensins ; genetics ; isolation & purification ; metabolism

Mammalian epithelia produce the various antimicrobial peptides against the bacterial or viral infection, thereby acting as the active immune modulators in the innate immunity. In this study, we examined the effects of the various proinflammatory cytokines or LPS on cell viability and antimicrobial beta-defensin gene expressions in human corneal epithelial cells. Results showed that the cytokines or LPS did not exert severe cytotoxic effects on the cells, and that beta-defensin 1 was constitutively expressed, while beta-defensin 2 was specifically induced by IL-1beta, supporting the idea that these cytokines or LPS involve the defense mechanism in the cornea. Furthermore, the reporter and gel shift assay to define the induction mechanism of beta-defensin 2 by IL-1beta demonstrated that the most proximal NF-kB site on the promoter region of beta-defensin 2 was not critical for the process. Data obtained from the normal or patients with the varying ocular diseases showed that our in vitro results were relevant in the clinical settings. Our results clearly demonstrated that beta-defensin 1 and 2 are important antimicrobial peptides in the corneal tissues, and that the mechanistic induction process of beta-defensin 2 by IL-1beta is not solely dependent on proximal NF-kB site activation, thus suggesting that the long distal portion of the promoter is needed for the full responsiveness toward IL-1beta.
Binding, Competitive ; Cell Survival ; Cells, Cultured ; Corneal Diseases/metabolism ; Electrophoretic Mobility Shift Assay ; Epithelium, Corneal/drug effects/*immunology/metabolism ; Gene Expression ; Humans ; Interferon Type II/metabolism/pharmacology ; Interleukin-1/*pharmacology ; Lipopolysaccharides/metabolism/pharmacology ; NF-kappa B/metabolism ; Promoter Regions (Genetics)/drug effects/genetics ; Research Support, Non-U.S. Gov't ; Tumor Necrosis Factor-alpha/metabolism/pharmacology ; beta-Defensins/*biosynthesis/genetics/metabolism

OBJECTIVETo investigate the expression of human beta-defensin-2 (HBD-2) mRNA in skin lesions of patients with recurrent genital herpes (RGH) and the effect of Huangbai Liquid (HL) on it.

METHODSTwenty-seven patients were randomly assigned to 2 groups, the HL group (n = 14) treated with HL and the famciclovir group (n = 13) with famciclovir. HBD-2 expression of patients were detected before and after the treatment and compared with that of 10 healthy subjects.

RESULTSHBD-2 expression was found in the skin lesions of both the healthy persons and the RGH patients before treatment. It is higher significantly in the HL group than in the famciclovir group after treatment.

CONCLUSIONHL was suitable for treatment of RGH since it could improve immune function of RGH patients and keep a rather higher concentration of HBD-2 expression in local skin lesions.

Adult ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression ; drug effects ; Herpes Genitalis ; drug therapy ; genetics ; virology ; Herpesvirus 2, Human ; drug effects ; Humans ; Male ; Phytotherapy ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Secondary Prevention ; Skin ; drug effects ; metabolism ; pathology ; beta-Defensins ; genetics