猪RegⅢγ蛋白的重组表达及功能研究

doi:10.16431/j.cnki.1671-7236.2023.06.026

Abstract

Abstract: 【Objective】 This experiment was aimed to explore the biological function and molecular mechanism of recombinant porcine RegⅢγ protein.【Method】 RegⅢγ gene fragment was amplified by PCR and connected to pDCP vector to construct RegⅢγ recombinant expression system.The recombinant protein was expressed efficiently at low temperature induced by IPTG and purified by Ni-NTA and dialysis.The inhibitory effects on different bacteria, as well as its functions of promoting proliferation and wound healing, anti-oxidation, and anti-apoptosis of porcine intestinal epithelial cells (IPEC-J2 cells) were analyzed to further explore its molecular mechanism.【Result】 The purified recombinant protein was identified by Western blotting, and the characteristic band was found at 17 ku, which confirmed the successful expression of recombinant porcine RegⅢγ protein.The recombinant porcine RegⅢγ protein showed obvious bactericidal activities against two Gram-positive bacteria, Staphylococcus aureus and Streptococcus, with MIC₉₀ of 39.1 μg/mL, but no bactericidal zone appeared against two Gram-negative bacteria, Escherichia coli and Salmonella Enteritidis.The cell proliferation and wound healing of IPEC-J2 cells were observed evidently when the protein concentration was 10 μg/mL.mRNA expression levels of epidermal growth factor receptor (EGFR), extracellular regulated protein kinase 1 (ERK1), ERK2, cyclin dependent kinase 2 (CDK2), CDK4, cyclin E (Cyclin E) and Cyclin D genes were increased (P<0.05), while the expression of serine/threonine protein kinase 1 (AKT1) gene was decreased(P<0.05).When the recombinant protein was applied to the oxidation-damaged IPEC-J2 cells, it was observed that the activities of SOD and GSH-Px were extremely significantly increased (P<0.01), while MDA content (P<0.05) and LDH activity (P<0.01) were decreased.It was further found that by reducing the mRNA expression levels of proapoptotic genes Caspase-3, Caspase-8, Caspase-9 and Bax, and increasing the mRNA expression level of anti-apoptotic gene Bcl-2, the recombinant protein played the anti-oxidation and anti-apoptosis effects on the oxygen-damaged IPEC-J2 cells.【Conclusion】 Recombinant porcine RegⅢγ protein could selectively inhibit Gram-positive bacteria, promote proliferation and would healing of IPEC-J2 cells, and had certain antioxidant and anti-apoptotic effects.

Key words: RegⅢγ; prokaryotic expression; antibiosis; cell proliferation; wound healing; anti-oxidation and anti-apoptosis

CLC Number:

S828

LI Can, LI Chuchu, ZENG Wei, ZHANG Ning, JI Chunxiao, CHEN Tao. Recombinant Expression and Function Study of RegⅢγ Protein in Porcine[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2414-2426.

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Recombinant Expression and Function Study of RegⅢγ Protein in Porcine

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