AvBD2在毕赤酵母中的组成型表达及其发酵条件优化
发布时间：2019-03-16 15:48
【摘要】：鸡β-防御素2(Chicken beta-defensins 2,AvBD2)是一个富含半胱氨酸阳离子的小分子多肽,其成熟肽由39个氨基酸残基组成,在鸡的消化道组织中广泛表达,且抗菌谱广、抗菌活性较强,对鸡的先天性免疫和获得性免疫起着重要的作用。因此,AvBD2有望成为传统抗生素的替代品而应用于畜禽养殖业。本实验以AvBD2为研究对象,利用甘油醛-3-磷酸脱氢酶组成型表达载体pGHKα,构建了鸡AvBD2的毕赤酵母组成型表达系统,并研究其表达产物活性;同时,采用响应面法优化摇瓶发酵的接种量、装液量和发酵温度,为AvBD2的发酵生产及其产业化应用提供理论依据,主要研究内容如下:1、AvBD2在毕赤酵母中的重组与组成型表达依据NCBI中报道的AvBD2 cDNA编码区序列(登录号:NM_204992),参考毕赤酵母的密码子偏爱性,合成pUC57-AvBD2克隆载体。使用EcoRⅠ与NotⅠ双酶切pUC57-AvBD2和载体pGHKα,回收目的片段并连接,构建重组质粒pGHKα-AvBD2。经SacⅠ和BglⅡ依次酶切pGHKα-AvBD2,使其线性化,去除其中Amp抗性基因。将线性化片段转化至毕赤酵母GS115,根据其G418抗性及蛋白表达量,挑选出高表达菌株。表达上清经Tricine-SDS-PAGE和Western Blot分析显示,重组菌能够分泌5.8kDa的AvBD2蛋白。2、重组AvBD2的活性分析将高表达菌株接种于发酵培养基中,过夜培养,浓缩发酵液上清进行抑菌和溶血活性分析。琼脂扩散法抑菌实验表明,表达的重组AvBD2蛋白对大肠杆菌、绿脓假单胞菌、产气肠杆菌、粪肠球菌、巴氏杆菌、金黄色葡萄球菌均有较强的抑制作用;浓度为100、250和500mg/L的重组AvBD2总蛋白对2%-3%鸡红细胞的溶血率分别为0.74%、1.12%和2.17%。3、重组AvBD2发酵条件的优化将得到的高表达菌株于YPD培养基培养,测定其生长曲线,结果表明培养20h的重组菌,活力最强。首先对重组毕赤酵母发酵温度、接种量、装液量三个因素进行独立优化,分别得到最优的单因素水平;继而通过响应面法优化,得出在14.44%的接种量、8.43%的装液量、27.8℃的发酵温度条件下,重组菌的表达量最大,较优化前提高20%。本研究以组成型表达载体pGHKα为基础,构建重组AvBD2毕赤酵母工程菌;Tricine-SDS-PAGE和Western blot结果表明,重组酵母成功表达出目的蛋白;抗菌活性和溶血活性结果表明,重组酵母发酵产物的上清液对多种病原菌有抑制作用,并且在500mg/L浓度下的溶血活性仅为2.17%;通过响应面法优化发酵条件,得到重组酵母最佳表达条件为:接种量14.44%、装液量8.43%、发酵温度27.8℃,蛋白质浓度较优化前提高20%。本研究为AvBD2的生物合成、发酵生产及产业化应用提供了基础。
[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 伪, 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 伪 were digested by EcoR 鈪，
本文编号：2441674
[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 伪, 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 伪 were digested by EcoR 鈪，
本文编号：2441674
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