猪RegⅢγ蛋白的重组表达及功能研究

doi:10.16431/j.cnki.1671-7236.2023.06.026

摘要/Abstract

摘要： 【目的】探究重组猪RegⅢγ蛋白的生物功能及相应分子机理，为该蛋白的开发应用提供参考。【方法】利用PCR扩增RegⅢγ基因片段，并连接至pDCP载体，以构建RegⅢγ蛋白重组表达系统。加入IPTG低温诱导促使重组蛋白高效表达，利用镍柱亲和层析纯化蛋白并进行透析后研究其对不同细菌的抑制作用，以及对猪小肠上皮细胞（IPEC-J2细胞）的促增殖与损伤修复及抗氧化与凋亡的功能，进一步探究其分子机理。【结果】纯化后重组蛋白Western blotting鉴定结果显示，17 ku处存在特征条带，确定重组猪RegⅢγ蛋白成功表达。重组猪RegⅢγ蛋白对金黄色葡萄球菌和化脓性链球菌2种革兰阳性菌有明显的抑菌效果，MIC₉₀均为39.1 μg/mL，对大肠杆菌和肠炎沙门氏菌2种革兰阴性菌未产生抑菌圈；可促进IPEC-J2细胞的增殖及损伤修复，且浓度为10 μg/mL时增殖与修复效果最佳。与对照组相比，表皮生长因子受体（EGFR）、细胞外调节蛋白激酶1（ERK1）、ERK2、细胞周期蛋白依赖激酶2（CDK2）、CDK4、细胞周期蛋白E （Cyclin E）和Cyclin D基因mRNA表达水平显著升高（P<0.05），而丝氨酸/苏氨酸蛋白激酶1（AKT1）基因mRNA表达水平显著降低（P<0.05）。将重组蛋白作用于氧化损伤IPEC-J2细胞时，与对照组相比，超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性极显著增强（P<0.01），丙二醛（MDA）含量（P<0.05）、乳酸脱氢酶（LDH）（P<0.01）活性均降低，并发现其通过降低促凋亡基因Caspase-3、Caspase-8、Caspase-9及Bax基因的mRNA表达水平，提高抗凋亡基因Bcl-2的mRNA表达水平，发挥对氧化损伤IPEC-J2细胞的抗氧化与凋亡作用。【结论】重组猪RegⅢγ蛋白对革兰阳性菌有选择性抑制作用，对IPEC-J2细胞增殖与损伤修复均有促进作用，且具有一定抗氧化与凋亡作用。

关键词: RegⅢγ; 原核表达; 抗菌; 细胞增殖; 损伤修复; 抗氧化与凋亡

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

中图分类号:

S828

李灿, 李楚楚, 曾维, 张宁, 纪春晓, 陈韬. 猪RegⅢγ蛋白的重组表达及功能研究[J]. 中国畜牧兽医, 2023, 50(6): 2414-2426.

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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