【作者】 李显；

【导师】 齐雪峰； 陈希文；

【作者基本信息】 西北农林科技大学 ， 兽医博士（专业学位）， 2025， 博士

【摘要】 长期大量使用抗生素会导致细菌耐药性增加、抗生素残留等问题,从而危害公共卫生安全。抗菌肽(Antimicrobial peptides,AMPs)作为一类高效的天然抗菌产物,具有安全性高、抗菌活性广以及不易产生耐药性等优点,已经成为潜在抗生素替代物。然而,由于抗菌肽天然提取率低、化学合成费用高且易被蛋白酶降解等,很大程度限制了它的规模化应用。猪β-防御素2(Porcine beta-defensin 2,PBD-2)因其对致病性肠道细菌具有广谱抗菌活性以及良好的免疫调节作用而被广泛研究。白细胞介素22(Interleukin-22,IL-22)是一种抑制细菌黏附、促进肠黏膜损伤修复的细胞因子。研究表明,与单一抗菌肽相比,细胞因子与抗菌肽融合表达可提升抗菌活性并降低细胞毒性。然而,IL-22与抗菌肽融合表达的功能研究较少,特别在猪体的抗菌及促生长中的作用尚未见报道。本研究通过毕赤酵母菌GS115对猪IL-22(PIL-22)与PBD2进行重组融合表达,检测其体外抑菌活性、肠黏膜上皮细胞修复及炎性因子调节作用。为提升其稳定性,利用生物可降解材料聚乳酸羟基乙酸共聚物(Poly(lactic-co-glycolic acid),PLGA)作为载体,制备具有抗酶降解和缓释作用的PLGA-PIL22-PBD2纳米抗菌蛋白。最后,通过建立的败血症小鼠和仔猪腹泻模型,评价体内治疗效果。研究结果如下:1.重组蛋白PIL22-PBD2在毕赤酵母中的表达。构建含PIL22-PBD2基因的毕赤酵母表达载体。通过电击转化p PIC9K-PIL22-PBD2到酵母宿主菌GS115中,筛选能够高效分泌表达PIL22-PBD2的酵母工程菌。在摇瓶中进行重组蛋白的初步表达并以大肠杆菌和金黄色葡萄球菌标准菌株为指示菌进行抑菌活性检测。结果表明:成功构建了能够高效分泌表达PIL22-PBD2的毕赤酵母表达菌株;融合蛋白经镍离子亲和层析柱纯化后纯度约为89%,表达蛋白的浓度约为50.7 mg/L。牛津杯抑菌试验初步表明该菌株表达上清对大肠杆菌ATCC25922株和金黄色葡萄球菌ATCC25923株均有良好抑菌活性。2.PIL22-PBD2体外特性研究。通过牛津杯抑菌试验和透射电镜等检测PIL22-PBD2重组蛋白抑菌能力,以及其对猪小肠上皮细胞J2(Intestinal porcine epithelial cell line-J2,IPEC-J2)损伤修复、抗多重耐药产肠毒素大肠杆菌O8(Enterotoxigenic Escherichia coli,ETEC O8)引起细胞凋亡和炎性因子表达的能力。结果表明:PIL22-PBD-2对ETEC O8、大肠杆菌ATCC25922株、鼠伤寒沙门氏菌ATCC19659株和金黄色葡萄球菌ATCC25923株具有广谱抗菌活性;划痕试验显示PIL22-PBD-2对IPEC-J2划痕损伤具有显著修复能力;PIL22-PBD-2显著增强了IPEC-J2中主要细胞间连接相关蛋白ZO-1和E-cadherin的表达,显著减少了ETEC O8感染引起的IPEC-J2细胞凋亡(p<0.05)和细菌粘附(p<0.05);另外,PIL22-PBD-2通过增加内源性抗菌肽的表达,显著降低ETEC O8诱导的IL-6和TNF-α的m RNA表达水平,减轻了IPEC-J2炎性反应。3.PIL22-PBD2高密度发酵表达,以及PLGA纳米化PIL22-PBD2的制备及性质评价。使用响应面法优化了GS115-PPIC9K-PIL22-PBD2的诱导表达条件,进行小规模高密度发酵表达。结果表明:重组抗菌蛋白PIL22-PBD2在5L发酵罐的最优表达条件为:温度28℃、甲醇浓度为1.5%、p H值5.0、发酵时间84 h;扫描电镜等显示制备的PLGA-PIL22-PBD2纳米颗粒为粒径均一(230±6.2 nm)的微球,可保护负载的PIL22-PBD2蛋白抵抗胰酶降解且对ETEC O8具有抑制作用;毒性验证试验显示PLGA-PIL22-PBD2对猪红细胞无溶血作用,在生理温度下可持续释放。4.PLGA-PIL22-PBD2对败血症模型小鼠的治疗效果评价。通过建立的小鼠败血症模型,检测PLGA-PIL22-PBD2(300 mg/kg)对小鼠体重、临床症状、血清生化指标水平、脏器指数、组织病理损伤和主要器官中ETEC O8负菌量的影响。此外,通过检测肠道细胞间连接蛋白ZO-1和E-cadherin表达、内源性抗菌肽Cryptdin-1和Reg3γ表达、炎性因子IL-6、IL-1β和TNF-α表达,探究PLGA-PIL22-PBD2纳米颗粒修复肠细胞损伤的作用。结果表明:PLGA-PIL22-PBD2具有良好的安全性,可显著改善败血症小鼠体重减轻和临床症状,降低血清生化指标水平与脏器指数,减轻粪便、肝脏、脾脏和肾脏中ETEC O8的负荷与病理损伤程度;此外,PLGA-PIL22-PBD2饲喂小鼠中十二指肠绒毛高度和VH/CD值升高,十二指肠细胞间连接蛋白ZO-1和E-cadherin和内源性抗菌肽Cryptdin-1和Reg3γ表达上调,炎性因子IL-6、IL-1β和TNF-α表达下调。5.PLGA-PIL22-PBD2纳米颗粒对ETEC O8感染引起仔猪腹泻的治疗效果评价。建立致病性ETEC O8感染仔猪腹泻模型,通过H&E染色法、微生态组学分析技术等检测口服PLGA-PIL22-PBD2(300 mg/kg)对大肠杆菌感染引起仔猪腹泻的影响。结果表明:PLGA-PIL22-PBD2饲喂断奶仔猪后,可显著增加平均日增重和饲料转化率;此外,饲喂PLGA-PIL22-PBD2可显著减轻ETEC O8感染引起的仔猪临床腹泻症状、减少盲肠内容物荷菌量、改善了器官和肠道损伤的严重程度、减少了炎性因子表达;饲粮添加PLGA-PIL22-PBD2增加了小肠菌群Alpha多样性和Beta多样性,改变了菌群结构和群落组成,使断奶仔猪形成了多样性强和丰度较高的稳定肠道菌群,增强其抵抗病原菌入侵的能力。综上,本研究通过毕赤酵母表达系统表达的PIL22-PBD2重组蛋白在体外和体内均具有广谱抑菌活性和细胞修复功能。PLGA纳米化在增强PIL22-PBD2抗消化道降解以及改善败血症小鼠和仔猪细菌性腹泻病理损伤中均具有显著作用。该研究不仅为口服类融合型抗菌肽在断奶仔猪腹泻防治提供重要的研究依据,也为替代抗生素防治畜禽疫病提供了防治策略。更多还原

【Abstract】 Long-term and excessive use of antibiotics can lead to increased bacterial resistance and generate significant public health safety issues such as antibiotic residue accumulation during treatment.Antimicrobial peptides(AMPs),as a class of highly efficient natural antibacterial products,have emerged as potential alternatives to antibiotics due to their advantages of high safety profile,broad-spectrum antimicrobial activity,and low propensity to induce resistance.Nevertheless,the large-scale application of AMPs has been limited by challenges including low natural extraction yield,high costs of chemical synthesis,and susceptibility to protease degradation that causes loss of biological activity.Porcine beta-defensin 2(PBD-2)has been extensively studied due to its broad-spectrum antimicrobial activity against pathogenic intestinal bacteria and its beneficial immunomodulatory effects in pigs.Interleukin-22(IL-22),a cytokine that inhibits bacterial adhesion and promotes the repair of intestinal mucosal damage,synergistically contributes to gastrointestinal defense mechanisms.However,functional studies on the fusion expression of IL-22 with antimicrobial peptides are limited,particularly regarding their roles in antibacterial activity and growth promotion in swine,which remains unreported to date.In this study,the recombinant fusion expression of porcine IL-22(PIL-22)and PBD2 was carried out using Pichia pastoris GS115,and their antibacterial activity,intestinal repair,and inflammation regulating effects were verified in vitro.To improve their stability,the biodegradable material poly(lactic-co-glycolic acid)(PLGA)was used for encapsulation to prepare PLGA-PIL22-PBD2 nano-antibacterial proteins with anti-enzymatic hydrolysis and sustained-release effects.Finally,these proteins were applied to septic mice and weaned piglet diarrhea models to evaluate their in vivo therapeutic effects.The main research results obtained are as follows:1.Expression of recombinant protein PIL22-PBD2 in P.pastoris.In this study,molecular biology techniques were employed to link the PBD-2 and PIL-22 genes.Subsequently,the PIL-22-PBD2 gene was cloned into the Pichia pastoris expression vector p PIC9K to construct a Pichia pastoris expression vector containing the PIL-22-PBD2 gene.The constructed vector was then transformed into the yeast host strain GS115 by electroporation,and yeast engineering strains capable of highly efficient secretory expression of PIL-22-PBD2 were screened.Preliminary expression screening was carried out in shake-flasks,and the antibacterial activity was detected using Escherichia coli and Staphylococcus aureus as indicator bacteria.The experimental results demonstrated that:A Pichia pastoris expression strain capable of efficiently secreting PIL22-PBD2 fusion protein was successfully constructed;after purification via nickel ion affinity chromatography,the fusion protein achieved approximately 89%purity with a concentration of about 50.7 mg/L.Preliminary Oxford cup bacteriostatic assays indicated that the expression supernatant exhibited significant inhibitory activity against both Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923.2.Research on the bacteriostatic ability and in vitro characteristics of PIL22-PBD2.Through Oxford cup assay and transmission electron microscopy(TEM),we evaluated the antibacterial capacity of the PIL22-PBD2 recombinant protein,as well as its ability to repair damage in porcine intestinal epithelial cell line J2(IPEC-J2),resist apoptosis and inflammatory cytokine expression induced by multidrug-resistant Enterotoxigenic Escherichia coli O8(ETEC O8).The results indicated that PIL22-PBD2 had broad-spectrum antibacterial activity against multi-drug resistant ETEC O8,E.coli,Salmonella typhimurium,and S.aureus.The wound healing assay demonstrated its scratch repair ability in IPEC-J2cells.Moreover,PIL22-PBD2 significantly upregulated the expression of the main cell-junction-related proteins ZO-1 and E-cadherin in IPEC-J2.Notably,PIL22-PBD2significantly reduced ETEC O8-induced apoptosis of intestinal epithelial cells(p<0.05)and bacterial adhesion(p<0.05)in IPEC-J2.It was also found that PIL22-PBD2 significantly decreased the m RNA expression levels of IL-6 and TNF-αand alleviated ETEC O8-induced inflammatory response in IPEC-J2 by exerting antibacterial activity and increasing the expression of endogenous antibacterial peptides.3.Small-scale fermentation expression of PIL22-PBD2 and preparation and property evaluation of PLGA-nanoparticle-encapsulated PIL22-PBD2.The induction expression conditions of GS115-PPIC9K-PIL22-PBD2 were optimized using Response Surface Methodology(RSM),and small-scale high-density fermentation expression was conducted.The results indicated that the optimal expression conditions for the recombinant antimicrobial protein PIL22-PBD2 in a 5L fermenter were:temperature 28℃,methanol concentration 1.5%,p H 5.0,and fermentation duration 84 hours.Scanning electron microscopy(SEM)and other characterization methods demonstrated that the prepared PLGA-PIL22-PBD2 nanoparticles exhibited uniform particle size(230±6.2 nm)and spherical morphology.These nanoparticles effectively protected the encapsulated PIL22-PBD2 protein from trypsin degradation and displayed inhibitory activity against ETEC O8.Toxicity evaluation revealed that PLGA-PIL22-PBD2 caused no hemolytic effect on porcine erythrocytes and achieved sustained release under physiological temperature conditions.4.Evaluation of the therapeutic effect of PLGA-PIL22-PBD2 on septic mice.Mice were continuously fed with PLGA-PIL22-PBD2 nanoparticles to evaluate their in vivo safety.By establishing a mouse sepsis model,the effects of PLGA-PIL22-PBD2(300 mg/kg)on weight loss,clinical symptoms,serum biochemical indicators levels,organ index,and the load of ETEC O8 in major organs of septic mice were detected.In addition,through pathological sections of major tissue organs,the therapeutic effects of PLGA-PIL22-PBD2 on the degree of organ pathological damage and the state of duodenal villi were examined.Finally,by detecting the expression of intestinal cell junction proteins ZO-1 and E-cadherin,the expression of endogenous antimicrobial peptides Cryptdin-1 and Reg3γ,and the expression of inflammatory factors IL-6,IL-1β,and TNF-α,the role of PLGA-PIL22-PBD2 nanoparticles in repairing intestinal cell damage was further investigated.The results showed that PLGA-PIL22-PBD2 had good biocompatibility.The treatment results of mouse sepsis indicated that,compared with the ETEC O8 group,PLGA-PIL22-PBD2(300 mg/kg)could improve weight loss and clinical symptoms in mice,reduce serum biochemical indicators levels,decrease the organ index,and alleviate the load of ETEC O8 in feces,liver,spleen,and kidneys.In the PLGA-PIL22-PBD2 group,the degree of organ pathological damage decreased,the height of duodenal villi and the VH/CD value increased,the expression of duodenal cell junction proteins ZO-1 and E-cadherin and endogenous antimicrobial peptides Cryptdin-1 and Reg3γwas up-regulated,and the expression of inflammatory factors IL-6,IL-1β,and TNF-αwas down-regulated.5.Evaluation of the therapeutic effect of PLGA-PIL22-PBD2 nanoparticles on weaned piglet diarrhea caused by E.coli infection.A pathogenic ETEC O8-infected piglet diarrheal model was established.The effects of oral administration of PLGA-PIL22-PBD2nanoparticles(300 mg/kg)on ETEC-induced diarrhea were assessed through Hematoxylin and Eosin(H&E)staining,microbiomics analysis,and related detection techniques.The results demonstrated that:Feeding weaned piglets with PLGA-PIL22-PBD2 significantly increased average daily gain(ADG)and feed conversion ratio.Furthermore,PLGA-PIL22-PBD2 supplementation notably alleviated clinical diarrhea symptoms induced by ETEC O8infection,reduced bacterial load in cecal content,improved the severity of organ and intestinal damage,decreased the expression of inflammatory cytokines,and enhanced the expression of endogenous antimicrobial peptides.Dietary supplementation with PLGA-PIL22-PBD2increased both alpha and beta diversity of intestinal microbiota in weaned piglets,altered microbial community structure and composition,and promoted the formation of a stable probiotic community characterized by enhanced diversity and abundance.This intervention effectively strengthened the resistance against pathogenic bacterial invasion.In conclusion,the recombinant protein PIL22-PBD2 expressed via the P.pastoris expression system demonstrated broad-spectrum antibacterial activity and cellular repair functions both in vitro and in vivo(porcine models).PLGA nano-encapsulation significantly enhanced the resistance of PIL22-PBD2 to gastrointestinal degradation and ameliorated pathological damage in septic mice and piglets with bacterial diarrhea.This research not only provides critical experimental evidence for the application of oral fusion-type antimicrobial peptides in preventing and treating diarrhea in weaned piglets but also offers a strategic framework for substituting antibiotics in the prevention and control of livestock and poultry diseases.更多还原