Acta Veterinaria et Zootechnica Sinica ›› 2025, Vol. 56 ›› Issue (11): 5864-5874.doi: 10.11843/j.issn.0366-6964.2025.11.042
• Basic Veterinary Medicine • Previous Articles Next Articles
CAO Qiuxia1,2(
), YAN Kexin2, CHENG Zhenkong2, BIAN Xianyu2, WANG Chuanhong2, LI Sufen2, ZHANG Xuehan2, FAN Baochao2, GUO Rongli2, YANG Shanshan2,*(), WANG Xiaodu1,*(), LI Bin1,2,*()
Received:2025-01-21
Online:2025-11-23
Published:2025-11-27
Contact:
YANG Shanshan, WANG Xiaodu, LI Bin
E-mail:1161824097@qq.com;yangshanshan@caas.cn;xdwang@zafu.edu.cn;libinana@126.com
CLC Number:
CAO Qiuxia, YAN Kexin, CHENG Zhenkong, BIAN Xianyu, WANG Chuanhong, LI Sufen, ZHANG Xuehan, FAN Baochao, GUO Rongli, YANG Shanshan, WANG Xiaodu, LI Bin. Expression and Biological Activity Analysis of Porcine CCL25 Recombinant Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(11): 5864-5874.
Table 1
Primers for RT-qPCR"
| 基因 Gene | 序列(5′→3′) Sequence |
| caspase-3 | F: TGGGATTGAGACGGACAGTG；R: CGCTGCACAAAGTGACTGGA |
| IL-8 | F: TGCACTTACTCTTGCCAGAACTG；R: CAAACTGGCTGTTGCCTTCTT |
| ACTIN | F: GGACTTCGAGCAGGAGATGG；R: AGGAAGGAGGGCTGGAAGAG |
Fig. 1
Construction and identification of the recombinant plasmid vector pCold-SUMO-CCL25 A. Secondary structure prediction of CCL25; B. Prediction of the transmembrane region of CCL25; C. Schematic diagram of the construction of pCold-SUMO-CCL25 recombinant plasmid; D. Verification of the recombinant plasmid by double digestion (M. KB Ladder; 1. pCold-SUMO-CCL25 plasmid; 2. pCold-SUMO-CCL25 plasmid digested by EcoRI and EcoRV)"
Fig. 2
Optimization of the induction expression conditions of porcine-derived recombinant CCL25 A. Screening at different sampling times (M. Marker; 1.pCold-SUMO supernatant; 2.pCold-SUMO pellet; 3, 5, 7, 9, 11. pCold-SUMO-CCL25 supernatant 8, 12, 16, 20, 24 h sampling; 4, 6, 8, 10, 12. pCold-SUMO-CCL25 pellet at 8, 12, 16, 20, 24 h sampling); B. Screening at different OD600 nm (1. pCold-SUMO supernatant; 2 pCold-SUMO pellet; 3, 5, 7, 9, 11.pCold-SUMO-CCL25 supernat at OD600 nm=0.2, 0.4, 0.6, 0.8, 1.0; 4, 6, 8, 10, 12. pCold-SUMO-CCL25 pellet at OD600 nm=0.2, 0.4, 0.6, 0.8, 1.0, 1.2); C. Screening at different IPTG (M. Marker; 1, 3, 5, 7, 9, 11.pCold-SUMO-CCL25 supernatant at IPTG=0, 0.2, 0.4, 0.6, 0.8, 1.0 mmol ·L-1; 2, 4, 6, 8, 10, 12.pCold-SUMO-CCL25 pellet at IPTG=0, 0.2, 0.4, 0.6, 0.8, 1.0 mmol ·L-1)"
Fig. 3
Purification and identification of porcine CCL25 A.Identification of purified pCold-SUMO-CCL25 by SDS-PAGE (M. Marker; 1. pCold-SUM supernatant; 2. pCold-SUMO pellet; 3. pCold-SUMO-CCL25 supernatant; 4. pCold-SUMO-CCL25 pellet; 5. Purified pCold-SUMO-CCL25 protein); B. Identification of purified pCold-SUMO-CCL25 by Western blot (1. pCold-SUMO; 2. Purified porcine CCL25 protein); C. Purified porcine CCL25 by ELISA"
Fig. 4
Functional identification of porcine-derived recombinant CCL25 A.CCK8 assay; B.Western blot (1.5.10 μg ·L-1 SUMO; 2.6.10 μg ·L-1 CCL25; 3.7.100 μg ·L-1 SUMO; 4.8.100 μg ·L-1 CCL25); C.Bar analysis of the gray value of B by ImageJ. D.Scratch assay (Scale bar: 500 μm); E.Bar analysis of the scratch distance of D by ImageJ (ns. No significant difference, *.P < 0.05 **.P < 0.01, ***.P < 0.001)"
Fig. 5
The effects of CCL25 on cell inflammation, apoptosis and death by RT-qPCR and FCM A.RT-qPCR detection of IL-8 and caspase-3 expression at the transcriptional level in PBMC cells; B.RT-qPCR detection of IL-8 and caspase-3 expression at the transcriptional level in IPEC-J2 cells; C.PBMC flow cytometry 7-AAD expression"
Fig. 6
Detection of CCL25 chemotaxis of PBMC cells using Transwell A. Counting of cells in the lower chamber of the Transwell (scale bar: 500 μm); B. Schematic diagram the operation of the Transwell experiment; C. Histogram analysis of the cell number in A by ImageJ (ns. No significant difference, *. P < 005, **. P<0.01, ***. P < 0.001)"
Fig. 7
Preparation and identification of rabbit polyclonal antibody A.Schematic diagram of the immunization process for the rabbit polyclonal antibody; B.ELISA detection of antibody titer; C.Construction and double enzyme digestion identification of pcDNA3.1-CCL25 (1.2 000 DNA marker; 2.PCR amplification product CCL25 gene; 3.Double enzyme digestion of pcDNA3.1 plasmid; 4.6.1 kd marker; 5.pcDNA3.1-CCL25 plasmid digested by EcoRI and XhoⅠ); D.Western blot (1.3.10 μg pcDNA3.1; 2.4.1.0 μg pcDNA3.1-CCL25); E.Conf detection of the co-localization of CCL25 rabbit polyclonal antibody and tag (Scale bar: 10 μm)"
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