猪圆环病毒2型Cap蛋白毕赤酵母分泌表达及发酵条件优化

发布时间：2018-08-07 12:37

【摘要】：为实现猪圆环病毒2型(PCV2)Cap蛋白在酵母中的高效表达,本研究将去除信号肽并经序列优化后的PCV2 ORF2片段插入pPICZαA表达载体,将重组表达质粒克隆转化巴斯德毕赤酵母(Pichia pastoris)X33感受态细胞中,应用Zeocin抗性和PCR法对转化子进行初步筛选和表型鉴定,利用"双膜法"筛选出PCV2 Cap表达量相对较高的酵母。采用正交试验对优选的酵母进行发酵试验,分析培养基pH、甲醇诱导时间、甲醇诱导浓度和诱导时酵母浓度对PCV2重组Cap蛋白(rCap)分泌表达的影响。结果显示酵母分泌表达PCV2 rCap的优化条件为:培养基pH为5.0,发酵液OD600nm至1.0,加入终浓度1.0%甲醇诱导96 h。优化后的PCV2 rCap分泌量可达207.58μg/m L。中和试验结果显示,发酵产物PCV2 rCap免疫小鼠产生的抗体可有效中和PCV2。本研究为利用酵母表达PCV2 Cap进行疫苗研制奠定了基础。
[Abstract]:In order to achieve high expression of porcine circovirus type 2 (PCV2) Cap protein in yeast, the signal peptide was removed and sequenced PCV2 ORF2 fragment was inserted into pPICZ 伪 A expression vector. The recombinant expression plasmid was cloned into Pichia pastoris (Pichia pastoris) X33 competent cells. The transformants were preliminarily screened and phenotypic identified by Zeocin resistance and PCR method. Yeast with relatively high PCV2 Cap expression was screened by "double membrane method". Orthogonal experiment was used to study the effects of medium pH, methanol induction time, methanol concentration and yeast concentration on the secretion and expression of PCV2 recombinant Cap protein (rCap). The results showed that the optimal conditions for yeast secretion and expression of PCV2 rCap were as follows: medium pH 5.0, fermentation broth OD600nm to 1.0, and final concentration 1.0% methanol for 96 h. The optimized PCV2 rCap secretion can reach 207.58 渭 g / mL. Neutralization test showed that the antibody produced by PCV2 rCap immunized mice was effective in neutralizing PCV2. This study laid a foundation for vaccine development using yeast expressed PCV2 Cap.
【作者单位】：广东海大畜牧兽医研究院;
【基金】：基金项目:猪用疫苗生产新工艺及产业化关键技术的研发(2017YFD0500604)
【分类号】：S852.651

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