【摘要】：雞β-防御素2(Chicken beta-defensins 2,AvBD2)是一個富含半胱氨酸陽離子的小分子多肽,其成熟肽由39個氨基酸殘基組成,在雞的消化道組織中廣泛表達,且抗菌譜廣、抗菌活性較強,對雞的先天性免疫和獲得性免疫起著重要的作用。因此,AvBD2有望成為傳統(tǒng)抗生素的替代品而應(yīng)用于畜禽養(yǎng)殖業(yè)。本實驗以AvBD2為研究對象,利用甘油醛-3-磷酸脫氫酶組成型表達載體pGHKα,構(gòu)建了雞AvBD2的畢赤酵母組成型表達系統(tǒng),并研究其表達產(chǎn)物活性;同時,采用響應(yīng)面法優(yōu)化搖瓶發(fā)酵的接種量、裝液量和發(fā)酵溫度,為AvBD2的發(fā)酵生產(chǎn)及其產(chǎn)業(yè)化應(yīng)用提供理論依據(jù),主要研究內(nèi)容如下:1、AvBD2在畢赤酵母中的重組與組成型表達依據(jù)NCBI中報道的AvBD2 cDNA編碼區(qū)序列(登錄號:NM_204992),參考畢赤酵母的密碼子偏愛性,合成pUC57-AvBD2克隆載體。使用EcoRⅠ與NotⅠ雙酶切pUC57-AvBD2和載體pGHKα,回收目的片段并連接,構(gòu)建重組質(zhì)粒pGHKα-AvBD2。經(jīng)SacⅠ和BglⅡ依次酶切pGHKα-AvBD2,使其線性化,去除其中Amp抗性基因。將線性化片段轉(zhuǎn)化至畢赤酵母GS115,根據(jù)其G418抗性及蛋白表達量,挑選出高表達菌株。表達上清經(jīng)Tricine-SDS-PAGE和Western Blot分析顯示,重組菌能夠分泌5.8kDa的AvBD2蛋白。2、重組AvBD2的活性分析將高表達菌株接種于發(fā)酵培養(yǎng)基中,過夜培養(yǎng),濃縮發(fā)酵液上清進行抑菌和溶血活性分析。瓊脂擴散法抑菌實驗表明,表達的重組AvBD2蛋白對大腸桿菌、綠膿假單胞菌、產(chǎn)氣腸桿菌、糞腸球菌、巴氏桿菌、金黃色葡萄球菌均有較強的抑制作用;濃度為100、250和500mg/L的重組AvBD2總蛋白對2%-3%雞紅細胞的溶血率分別為0.74%、1.12%和2.17%。3、重組AvBD2發(fā)酵條件的優(yōu)化將得到的高表達菌株于YPD培養(yǎng)基培養(yǎng),測定其生長曲線,結(jié)果表明培養(yǎng)20h的重組菌,活力最強。首先對重組畢赤酵母發(fā)酵溫度、接種量、裝液量三個因素進行獨立優(yōu)化,分別得到最優(yōu)的單因素水平;繼而通過響應(yīng)面法優(yōu)化,得出在14.44%的接種量、8.43%的裝液量、27.8℃的發(fā)酵溫度條件下,重組菌的表達量最大,較優(yōu)化前提高20%。本研究以組成型表達載體pGHKα為基礎(chǔ),構(gòu)建重組AvBD2畢赤酵母工程菌;Tricine-SDS-PAGE和Western blot結(jié)果表明,重組酵母成功表達出目的蛋白;抗菌活性和溶血活性結(jié)果表明,重組酵母發(fā)酵產(chǎn)物的上清液對多種病原菌有抑制作用,并且在500mg/L濃度下的溶血活性僅為2.17%;通過響應(yīng)面法優(yōu)化發(fā)酵條件,得到重組酵母最佳表達條件為:接種量14.44%、裝液量8.43%、發(fā)酵溫度27.8℃,蛋白質(zhì)濃度較優(yōu)化前提高20%。本研究為AvBD2的生物合成、發(fā)酵生產(chǎn)及產(chǎn)業(yè)化應(yīng)用提供了基礎(chǔ)。
[Abstract]:Chicken 尾-defensin 2 (Chicken beta-defensins-2, AvBD2) is a small molecular polypeptide rich in cysteine cations. Its mature peptide, composed of 39 amino acid residues, is widely expressed in the digestive tract of chickens, and has a broad antibacterial spectrum and strong antibacterial activity. It plays an important role in innate immunity and acquired immunity in chickens. Therefore, AvBD2 is expected to become a substitute for traditional antibiotics and be used in livestock and poultry breeding. The expression system of chicken AvBD2 by Pichia pastoris was constructed by using glyceraldehyde-3-phosphate dehydrogenase group expression vector pGHK 偽, and the activity of the expressed product was studied by using AvBD2 as the research object. At the same time, the response surface method was used to optimize the inoculation quantity, the volume of liquid and the fermentation temperature of shaking flask fermentation, which provided the theoretical basis for the fermentation production of AvBD2 and its industrial application. The main contents of the study were as follows: 1, The recombinant and constitutive expression of AvBD2 in Pichia pastoris was based on the sequence of AvBD2 cDNA coding region (accession number: NM_204992) reported in NCBI, referring to the codon preference of Pichia pastoris to synthesize pUC57-AvBD2 clone vector. The pUC57-AvBD2 and vector pGHK 偽 were digested by EcoR 鈪，

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