【摘要】：抗菌肽作為天然免疫的重要組成部分,因其獨(dú)特的抗菌機(jī)制而日益受到關(guān)注。作為禽類(lèi)屠宰加工業(yè)副產(chǎn)品甚至是屠宰廢水中難降解成分之一,雞血來(lái)源豐富、價(jià)格低廉,是提取抗菌肽的優(yōu)良原材料,但是目前從雞血中提取抗菌肽的研究還罕見(jiàn)報(bào)導(dǎo)。本研究利用蛋白酶解法成功提取了具有廣譜、高效、穩(wěn)定性較強(qiáng)、毒性較低的雞血源抗菌肽(antimicrobial peptides from chicken hemoglobin,CHAP),并對(duì)其抗菌活性、殺菌活性、抗熱應(yīng)激活性及免疫調(diào)節(jié)活性進(jìn)行了系統(tǒng)的研究,主要研究?jī)?nèi)容及結(jié)果如下:1.CHAP的提取純化及鑒定新鮮雞血經(jīng)木瓜蛋白酶酶解和乙酸浸提后獲得雞血源抗菌肽粗提物,并從酶解時(shí)間、酶解溫度、酶用量、浸提時(shí)間和浸提溫度等方面進(jìn)一步優(yōu)化其提取工藝。凝膠層析法獲得純化的CHAP,經(jīng)Tricine-SDS-PAGE和質(zhì)譜檢測(cè)得出該抗菌肽為雞血紅蛋白α鏈片段,分子量為3.3KDa。2.CHAP抗菌活性及抗菌機(jī)制通過(guò)微量瓊脂糖彌散試驗(yàn),最低抑菌濃度試驗(yàn),最低殺菌濃度試驗(yàn),抑菌和殺菌動(dòng)力學(xué)試驗(yàn)測(cè)定CHAP對(duì)19株細(xì)菌(包括8種細(xì)菌,9株多重耐藥菌)的抗菌活性,試驗(yàn)結(jié)果表明CHAP對(duì)19株細(xì)菌均有較強(qiáng)抗菌作用:CHAP對(duì)多重耐藥(MR)-大腸桿菌(馬)的抑菌圈直徑為33.5 cm,對(duì)MR-綠膿桿菌(M140)的最低抑菌濃度(MIC)為1.56μg mL~(-1),最低殺菌濃度(MBC)為5μg mL~(-1),在90min對(duì)6株代表細(xì)菌的抑菌率均達(dá)到100%,與對(duì)照組相比CHAP對(duì)對(duì)數(shù)期早期和晚期細(xì)菌在10～240 min均有極顯著的殺菌作用(P0.01),對(duì)靜止期細(xì)菌有較好的溶菌作用。細(xì)菌生物膜試驗(yàn)和NaCl滲透試驗(yàn)結(jié)果表明,與對(duì)照組相比CHAP可極顯著干擾細(xì)菌生物膜形成(P0.01),增加細(xì)菌對(duì)NaCl的滲透性,電鏡觀察發(fā)現(xiàn)CHAP處理后細(xì)菌大小不一,表面聚集大量粘稠物質(zhì)、粗糙、有球狀的突起、局部形成空洞并有內(nèi)容物釋放,表明CHAP主要通過(guò)破壞細(xì)菌細(xì)胞膜結(jié)構(gòu)來(lái)實(shí)現(xiàn)其抑制和殺滅細(xì)菌作用的。3.CHAP穩(wěn)定性和安全性研究熱穩(wěn)定檢測(cè)試驗(yàn)和血清穩(wěn)定檢測(cè)試驗(yàn)結(jié)果表明經(jīng)121℃處理30 min后CHAP的抗菌活性沒(méi)有顯著降低(P0.05),經(jīng)兔或雞的血清處理6 h后其抗菌活性也無(wú)顯著降低(P0.05),說(shuō)明CHAP具有較好的熱穩(wěn)定性和血清穩(wěn)定性。安全性試驗(yàn)結(jié)果表明CHAP濃度為360 μg mL~(-1)時(shí)其溶血率為38.94%,雞胚接種CHAP后無(wú)死亡和明顯毒性作用,提示CHAP對(duì)原核細(xì)胞和真核細(xì)胞有較好的選擇性,因而具有較低的溶血性和雞胚毒性。4.CHAP抗熱應(yīng)激研究采用雞慢性熱應(yīng)激(10 d)和急性熱應(yīng)激(8 h)試驗(yàn)?zāi)Ｐ脱芯緾HAP的抗熱應(yīng)激效果。試驗(yàn)結(jié)果表明在慢性熱應(yīng)激試驗(yàn)中,與單純熱應(yīng)激組相比較,CHAP添加熱應(yīng)激組的平均體重和平均日增重顯著升高(P0.05),飼料轉(zhuǎn)化率顯著降低(P0.01);血清甲狀腺激素含量和MDA含量顯著降低(P0.05),SOD和ATP酶顯著升高(P0.05);腸組織結(jié)構(gòu)完整,損傷程度輕,絨毛長(zhǎng)度、絨毛寬度和絨毛長(zhǎng)度/隱窩深度值顯著升高,十二指腸AKP酶含量顯著升高(P0.01),各段小腸中IEL和SlgA顯著增多,GC數(shù)量明顯減少,HSP70和PCNA表達(dá)量顯著降低;脾臟、胸腺和法氏囊臟器指數(shù)顯著升高,免疫器官充血、水腫和壞死均不明顯,TUNEL染色陽(yáng)性細(xì)胞數(shù)量顯著減少,Bax和Caspase-3表達(dá)量顯著降低,Bcl-2表達(dá)量顯著升高,說(shuō)明CHAP能有效拮抗熱應(yīng)激造成的雞生產(chǎn)性能降低、氧化應(yīng)激損傷和腸道與免疫器官組織結(jié)構(gòu)變化及免疫功能的損傷。5.CHAP免疫調(diào)節(jié)活性初步研究CHAP對(duì)SPF雛雞免疫機(jī)能的影響,試驗(yàn)結(jié)果表明,添加CHAP組雞的體重顯著高于對(duì)照組(P0.05),NDV和AIV兩種疫苗的血清抗體水平明顯高于對(duì)照組(P0.05),免疫器官指數(shù)和T、B淋巴細(xì)胞增殖率顯著高于對(duì)照組。此結(jié)果說(shuō)明CHAP能夠提高SPF雞的特異性免疫功能。綜上所述,本研究采用酶解法成功分離提取到雞血源抗菌肽,其具有廣譜高效的抗菌作用,熱穩(wěn)定性和血清穩(wěn)定性良好,溶血毒性和雞胚毒性低,CHAP可以有效抵抗熱應(yīng)激造成的生產(chǎn)性能降低、氧化應(yīng)激損傷、小腸和免疫器官的損傷與免疫功能低下,并對(duì)動(dòng)物的免疫機(jī)能具有增強(qiáng)作用。雞血源抗菌肽的研發(fā)為治療耐藥性細(xì)菌感染疾病、抵抗熱應(yīng)激以及開(kāi)發(fā)機(jī)體免疫增強(qiáng)劑提供了新思路,對(duì)動(dòng)物醫(yī)學(xué)、公共衛(wèi)生等都具有重要實(shí)踐意義。
[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，

本文編號(hào)：2488474