Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig

Bao, Yuanyuan; Li, L; Zhang, H; Gao, Chunyu; Xiao, Chang; Li, C L

doi:10.4238/2015.december.28.36

Cited by 12 publications

(6 citation statements)

References 29 publications

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“…Porcine β-defensin 2 (PBD-2) was first determined by sequence alignment with the existing sequence of porcine β-defensin 1 [6]. It was further identified that PBD-2 was distributed in different pig tissues using a polyclonal antibody against PBD-2 [7]. The antimicrobial activity of PBD-2 against gram-positive and gram-negative bacteria has been well described [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

Huang

Wang

et al. 2020

Virol J

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Background: Porcine β-defensin 2 (PBD-2), produced by host cells, is an antimicrobial cysteine-rich cationic peptide with multi-functions. Previous studies have demonstrated that PBD-2 can kill various bacteria, regulate host immune responses and promote growth of piglets. However, the antiviral role of PBD-2 is rarely investigated. This study aimed to reveal the antiviral ability of PBD-2 against pseudorabies virus (PRV), the causative pathogen of Aujeszky's disease, in PK-15 cells and in a PBD-2 expressing transgenic (TG) mouse model. Methods: In this study, the cytotoxicity of PBD-2 on PK-15 cells was measured by CCK-8 assay. PK-15 cells were incubated with PRV pre-treated with different concentrations of PBD-2 and PRV titers in cell culture supernatants were determined by real-time quantitative PCR (RT-qPCR). TG mice and wild-type (WT) mice were intraperitoneally injected with PRV and the survival rate was recorded for 10 days. Meanwhile, tissue lesions in brain, spleen and liver of infected mice were observed and the viral loads of PRV in brain, liver and lung were analyzed by RT-qPCR.Results: PBD-2 at a maximum concentration of 80 μg/mL displayed no significant cytotoxicity on PK-15 cells. A threshold concentration of PBD-2 at 40 μg/mL was required to inhibit PRV proliferation in PK-15 cells. The survival rate in PBD-2 TG mice was 50% higher than that of WT mice. In addition, TG mice showed alleviated tissue lesions in brain, spleen and liver compared with their WT littermates after PRV challenge, while viral loads of PRV in brain, liver and lung of TG mice were significantly lower than that of WT mice.Conclusions: PBD-2 could inhibit PRV proliferation in PK-15 cells and protect mice from PRV infection, which confirmed the antiviral ability of PBD-2 both in vitro and in vivo. The application of PBD-2 in developing anti-viral drugs or disease-resistant animals can be further investigated.

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Section: Introductionmentioning

confidence: 99%

Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

Huang

Wang

et al. 2020

Virol J

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“…The use of pigs as an animal model for translational research focusing on β‐defensins and infectious diseases is growing (Alizadeh et al, ; Bao et al, ; Liu et al, ; Ren et al, ; Tang et al, ; X. Yang et al, ). The messenger RNA (mRNA) expression of thirteen different porcine β‐defensins has been described in several tissues (G. Zhang et al, ; Sang, Patil, Zhang, Ross, & Blecha, ) and, with regard to the transcriptomics of the swine epididymis, seven β‐defensins showed marked regionalized expression, including the porcine β‐defensins 3 (pBD3), 4 (pBD4), 125 (pBD125) and epididymal protein splicing variant 2C (pEP2C; Guyonnet, Dacheux, Dacheux, & Gatti, ).…”

Section: Introductionmentioning

confidence: 99%

Structural modeling and mRNA expression of epididymal β‐defensins in GnRH immunized boars: A model for secondary hypogonadism in man

Weber

Alves

Abujamra

et al. 2018

Molecular Reproduction Devel

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Human secondary hypogonadism is associated with impaired testicular function, however, little is known about its impact on sperm epididymal maturation. Endocrine disruption in the epididymis could impair the secretion of key proteins, such as β‐defensins, responsible for spermatozoa maturation during epididymal transit. This study evaluated the sequence and structural similarities between porcine epididymal β‐defensins porcine β‐defensins (pBD3), pBD4, pBD125, and pEP2C and their human homologs using bioinformatics integrated with information derived from protein databanks. We then verified whether the expression of pBD3, pBD4, pBD125, and pEP2C genes in the testis and epididymis are influenced by disruption of the hypothalamic‐pituitary‐testicular (HPT) axis in a pig model for male human secondary hypogonadism. Upon modeling porcine β‐defensins, structural and functional analysis confirmed the presence of motifs associated with β‐defensin function, validating the models generated in silico. pBD3 and pBD4 showed acceptable structural alignments with human β‐defensins BDEF103 and BDEF110, respectively. In addition, evaluation of hormonal regulation of β‐defensins was assessed by analyzing the expression of these four β‐defensins in adult boars immunized against gonadotropin‐releasing hormone (GnRH). Our results indicate that HPT axis disruption modifies the expression of pBD3, pBD4, pBD125, and pEP2C in boar testis and epididymis, suggesting an endocrine‐dependent regulation of β‐defensins in swine epididymis. In conclusion, sequence and structural homology between pBD3 and pBD4 and their human homologs provide a basis for using the pig as a model for the study of human secondary hypogonadism. Further investigation of the human homologs in hypogonadal men could elucidate the connections between fertility and epididymal expression of β‐defensins.

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“…Samples were then washed with 10 mM PBS buffer for 3 × 5 min and were incubated with 10% normal goat serum in PBS for 30 min at room temperate in a wet box. After drying with absorbent paper, bacterial samples were incubated with 50 µl polyclonal pBD2 (prepared by us from rabbit 58 , diluted to 1:500) at 4 °C overnight. Afterwards, cells were incubated with fluorescently (FITC) labeled secondary antibodies for 1 h at 37 °C in the box, washed with PBS for 3 × 5 min and then stained by DAPI solution for 5 min in the dark.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the antimicrobial mechanism of porcine beta defensin 2 against E. coli by electron microscopy and differentially expressed genes

Chen

Zhang

et al. 2018

Sci Rep

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Porcine beta defensin 2 (pBD2) is a cationic antimicrobial peptide with broad spectrum antibacterial activity, which makes it a potential alternative to antibiotics to prevent and cure diseases of pigs. However, development of pBD2 as an effective antibiotic agent requires molecular understanding of its functional mechanism against pathogens. In this study, we investigated the functional mechanism of pBD2 antibacterial activity. Escherichia coli was incubated with different pBD2 concentrations for different times. Electron microscopy was used to analyze the locations of pBD2 and its induced morphological changes in E. coli. Gene expression analysis was also performed to further understand the molecular changes of E. coli in response to pBD2 incubation. The results demonstrated that E. coli membranes were broken, holed, and wrinkled after treatment with pBD2, and pBD2 was located on the cell membranes and manly in the cytoplasm. Furthermore, 38 differentially expressed genes (DEGs) were detected, successfully sequenced and confirmed by quantitative real-time PCR (qRT-PCR). Most of the known functional DEGs were associated with DNA transcription and translation and located in the cytoplasm. Collectively, the results suggest that pBD2 could have multiple modes of action and the main mechanism for killing E. coli might be influence on DNA transcription and translation by targeting intracellular molecules after membrane damage, although transport and metabolism proteins were also affected.

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Preparation of polyclonal antibody against porcine beta defensin 2 and identification of its distribution in tissues of pig

Cited by 12 publications

References 29 publications

Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

Porcine β-defensin 2 inhibits proliferation of pseudorabies virus in vitro and in transgenic mice

Structural modeling and mRNA expression of epididymal β‐defensins in GnRH immunized boars: A model for secondary hypogonadism in man

Analysis of the antimicrobial mechanism of porcine beta defensin 2 against E. coli by electron microscopy and differentially expressed genes

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