鸡血源抗菌肽分离鉴定及其生物活性的研究
发布时间：2019-05-30 03:21
【摘要】：抗菌肽作为天然免疫的重要组成部分,因其独特的抗菌机制而日益受到关注。作为禽类屠宰加工业副产品甚至是屠宰废水中难降解成分之一,鸡血来源丰富、价格低廉,是提取抗菌肽的优良原材料,但是目前从鸡血中提取抗菌肽的研究还罕见报导。本研究利用蛋白酶解法成功提取了具有广谱、高效、稳定性较强、毒性较低的鸡血源抗菌肽(antimicrobial peptides from chicken hemoglobin,CHAP),并对其抗菌活性、杀菌活性、抗热应激活性及免疫调节活性进行了系统的研究,主要研究内容及结果如下:1.CHAP的提取纯化及鉴定新鲜鸡血经木瓜蛋白酶酶解和乙酸浸提后获得鸡血源抗菌肽粗提物,并从酶解时间、酶解温度、酶用量、浸提时间和浸提温度等方面进一步优化其提取工艺。凝胶层析法获得纯化的CHAP,经Tricine-SDS-PAGE和质谱检测得出该抗菌肽为鸡血红蛋白α链片段,分子量为3.3KDa。2.CHAP抗菌活性及抗菌机制通过微量琼脂糖弥散试验,最低抑菌浓度试验,最低杀菌浓度试验,抑菌和杀菌动力学试验测定CHAP对19株细菌(包括8种细菌,9株多重耐药菌)的抗菌活性,试验结果表明CHAP对19株细菌均有较强抗菌作用:CHAP对多重耐药(MR)-大肠杆菌(马)的抑菌圈直径为33.5 cm,对MR-绿脓杆菌(M140)的最低抑菌浓度(MIC)为1.56μg mL~(-1),最低杀菌浓度(MBC)为5μg mL~(-1),在90min对6株代表细菌的抑菌率均达到100%,与对照组相比CHAP对对数期早期和晚期细菌在10～240 min均有极显著的杀菌作用(P0.01),对静止期细菌有较好的溶菌作用。细菌生物膜试验和NaCl渗透试验结果表明,与对照组相比CHAP可极显著干扰细菌生物膜形成(P0.01),增加细菌对NaCl的渗透性,电镜观察发现CHAP处理后细菌大小不一,表面聚集大量粘稠物质、粗糙、有球状的突起、局部形成空洞并有内容物释放,表明CHAP主要通过破坏细菌细胞膜结构来实现其抑制和杀灭细菌作用的。3.CHAP稳定性和安全性研究热稳定检测试验和血清稳定检测试验结果表明经121℃处理30 min后CHAP的抗菌活性没有显著降低(P0.05),经兔或鸡的血清处理6 h后其抗菌活性也无显著降低(P0.05),说明CHAP具有较好的热稳定性和血清稳定性。安全性试验结果表明CHAP浓度为360 μg mL~(-1)时其溶血率为38.94%,鸡胚接种CHAP后无死亡和明显毒性作用,提示CHAP对原核细胞和真核细胞有较好的选择性,因而具有较低的溶血性和鸡胚毒性。4.CHAP抗热应激研究采用鸡慢性热应激(10 d)和急性热应激(8 h)试验模型研究CHAP的抗热应激效果。试验结果表明在慢性热应激试验中,与单纯热应激组相比较,CHAP添加热应激组的平均体重和平均日增重显著升高(P0.05),饲料转化率显著降低(P0.01);血清甲状腺激素含量和MDA含量显著降低(P0.05),SOD和ATP酶显著升高(P0.05);肠组织结构完整,损伤程度轻,绒毛长度、绒毛宽度和绒毛长度/隐窝深度值显著升高,十二指肠AKP酶含量显著升高(P0.01),各段小肠中IEL和SlgA显著增多,GC数量明显减少,HSP70和PCNA表达量显著降低;脾脏、胸腺和法氏囊脏器指数显著升高,免疫器官充血、水肿和坏死均不明显,TUNEL染色阳性细胞数量显著减少,Bax和Caspase-3表达量显著降低,Bcl-2表达量显著升高,说明CHAP能有效拮抗热应激造成的鸡生产性能降低、氧化应激损伤和肠道与免疫器官组织结构变化及免疫功能的损伤。5.CHAP免疫调节活性初步研究CHAP对SPF雏鸡免疫机能的影响,试验结果表明,添加CHAP组鸡的体重显著高于对照组(P0.05),NDV和AIV两种疫苗的血清抗体水平明显高于对照组(P0.05),免疫器官指数和T、B淋巴细胞增殖率显著高于对照组。此结果说明CHAP能够提高SPF鸡的特异性免疫功能。综上所述,本研究采用酶解法成功分离提取到鸡血源抗菌肽,其具有广谱高效的抗菌作用,热稳定性和血清稳定性良好,溶血毒性和鸡胚毒性低,CHAP可以有效抵抗热应激造成的生产性能降低、氧化应激损伤、小肠和免疫器官的损伤与免疫功能低下,并对动物的免疫机能具有增强作用。鸡血源抗菌肽的研发为治疗耐药性细菌感染疾病、抵抗热应激以及开发机体免疫增强剂提供了新思路,对动物医学、公共卫生等都具有重要实践意义。
[Abstract]:As an important part of the natural immunity, the antibacterial peptide is increasingly concerned by its unique antibacterial mechanism. As a by-product of the poultry slaughtering and processing industry, even one of the difficult degradation components in the slaughter wastewater, the chicken blood source is rich, the prices are low and factory-direct.We even have the best raw material for extracting antibacterial peptide, but the research on extracting antibacterial peptide from chicken blood is rare. In this study, the antibacterial activity, the bactericidal activity, the heat stress activity and the immunoregulation activity of the chicken blood source antibacterial peptide (CHAP) with broad spectrum, high efficiency, high stability and low toxicity were successfully extracted by the proteolysis method. The main contents and results are as follows:1. The extraction and purification of CHAP and the identification of fresh chicken blood are carried out by enzymolysis with papain and acetic acid, and the crude extract of the antibacterial peptide of the chicken blood is obtained, and the extraction process is further optimized from the aspects of the enzymolysis time, the enzymolysis temperature, the enzyme dosage, the extraction time and the extraction temperature, and the like. The purified CHAP was obtained by gel chromatography, and it was detected by Tricine-SDS-PAGE and mass spectrometry. The antibacterial activity and the anti-bacterial mechanism of the antibacterial peptide were tested by the micro-agarose diffusion test, the minimum inhibitory concentration test and the minimum bactericidal concentration. The antibacterial activity of CHAP on 19 strains of bacteria (including 8 kinds of bacteria and 9 strains of multiple drug-resistant bacteria) was determined by the inhibition and sterilization dynamics test. The results showed that CHAP has a strong antibacterial effect on the 19 strains of bacteria: the diameter of the antibacterial ring of CHAP to multiple drug-resistant (MR)-E. coli (horse) is 33.5 cm, The minimal inhibitory concentration (MIC) of MR-Pseudomonas aeruginosa (M140) was 1.56 & mu; g/ mL ~ (-1), and the minimum bactericidal concentration (MBC) was 5. m Compared with the control group, CHAP has a very significant bactericidal effect on the early and late-stage bacteria in the logarithmic phase at 10-240 min (P0.01), and has a good colytic effect on the bacteria in the stationary period. The results of the bacterial biofilm test and the NaCl penetration test show that, compared with the control group, the CHAP can significantly interfere with the formation of the bacterial biofilm (P0.01), increase the permeability of the bacteria to the NaCl, there is a spherical projection that partially forms a cavity and has a release of the contents, 3. CHAP stability and safety study have no significant reduction in the antibacterial activity of CHAP after 30 min at 121 鈩，
本文编号：2488474
[Abstract]:As an important part of the natural immunity, the antibacterial peptide is increasingly concerned by its unique antibacterial mechanism. As a by-product of the poultry slaughtering and processing industry, even one of the difficult degradation components in the slaughter wastewater, the chicken blood source is rich, the prices are low and factory-direct.We even have the best raw material for extracting antibacterial peptide, but the research on extracting antibacterial peptide from chicken blood is rare. In this study, the antibacterial activity, the bactericidal activity, the heat stress activity and the immunoregulation activity of the chicken blood source antibacterial peptide (CHAP) with broad spectrum, high efficiency, high stability and low toxicity were successfully extracted by the proteolysis method. The main contents and results are as follows:1. The extraction and purification of CHAP and the identification of fresh chicken blood are carried out by enzymolysis with papain and acetic acid, and the crude extract of the antibacterial peptide of the chicken blood is obtained, and the extraction process is further optimized from the aspects of the enzymolysis time, the enzymolysis temperature, the enzyme dosage, the extraction time and the extraction temperature, and the like. The purified CHAP was obtained by gel chromatography, and it was detected by Tricine-SDS-PAGE and mass spectrometry. The antibacterial activity and the anti-bacterial mechanism of the antibacterial peptide were tested by the micro-agarose diffusion test, the minimum inhibitory concentration test and the minimum bactericidal concentration. The antibacterial activity of CHAP on 19 strains of bacteria (including 8 kinds of bacteria and 9 strains of multiple drug-resistant bacteria) was determined by the inhibition and sterilization dynamics test. The results showed that CHAP has a strong antibacterial effect on the 19 strains of bacteria: the diameter of the antibacterial ring of CHAP to multiple drug-resistant (MR)-E. coli (horse) is 33.5 cm, The minimal inhibitory concentration (MIC) of MR-Pseudomonas aeruginosa (M140) was 1.56 & mu; g/ mL ~ (-1), and the minimum bactericidal concentration (MBC) was 5. m Compared with the control group, CHAP has a very significant bactericidal effect on the early and late-stage bacteria in the logarithmic phase at 10-240 min (P0.01), and has a good colytic effect on the bacteria in the stationary period. The results of the bacterial biofilm test and the NaCl penetration test show that, compared with the control group, the CHAP can significantly interfere with the formation of the bacterial biofilm (P0.01), increase the permeability of the bacteria to the NaCl, there is a spherical projection that partially forms a cavity and has a release of the contents, 3. CHAP stability and safety study have no significant reduction in the antibacterial activity of CHAP after 30 min at 121 鈩，
本文编号：2488474
本文链接：https://www.wllwen.com/shoufeilunwen/nykjbs/2488474.html
最近更新
教材专著
