携带IBV基因真核表达质粒的大肠杆菌工程菌发酵工艺研究
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摘要
本研究以本课题组构建的携带鸡传染性支气管炎病毒S1、M、N基因真核表达质粒(pVAX1-S1、pVAX1-M、pVAX1-N、VR1020-S1、VR1020-M、VR10200N)的大肠杆菌基因工程菌为研究对象,对其发酵工艺进行了研究。
采用单因子分析和正交设计,摇瓶发酵对各重组大肠杆菌的培养基条件进行了研究,确定优化了培养基的成份。发酵后,以优化的碱裂解法提取质粒,产量最高可达27.8mg/L,纯度达到1.88,是未优化前质粒产量的2.62倍。
研究了携带鸡传染性支气管炎病毒真核表达质粒基因工程菌在5L发酵罐中的发酵工艺。优化了上罐发酵接种量、培养温度、初始pH及溶氧等培养条件。确定了优势菌株pVAX1-M,以优化的碱裂解法提取质粒产量可达57mg/L,纯度达到1.88,是未优化前质粒产量的4.3倍。
对发酵液用热激法和碱裂解法两种不同的提取方法进行粗提,对碱裂解法进行优化,对裂解时间和裂解反应液的比例进行研究。再用聚乙二醇和硅藻土两种方法进行纯化,建立了以碱裂解法粗提,硅藻土法纯化的质粒DNA提纯工艺。该工艺可去除大部分RNA、基因组DNA和蛋白质等杂质,提取的质粒DNA纯度可达到1.88,超螺旋比例达到98.4%,符合世界卫生组织关于质粒产品的质量要求。对发酵罐发酵的菌体以本工艺提纯,质粒产量可达到57mg/L。与传统纯化方法比较,生产成本降低了20-40%。
本研究降低了基因工程菌发酵和质粒提取的生产成本,为DNA疫苗的大规模生产提供了理论和实验依据。
The fermentation parameters of recombinant E.coli which contains the eukaryoticexpression plasmid pVAX1-S1, pVAX1-M, pVAX1-N, VR1020-S1, VR1020-M and VR1020-N were investigated.
By a single factor experiment and orthogonal test, the fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in shake flask were investigated. In the experiment, the optimum medium was carried out and definited the medium ingredient. After fermentating, the optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 27.8mg/L, also the purification was 1.88, which was 2.62-fold in comparison with using conventional LB culture.
The fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in 5L fermentor were investigated. Inoculum size, temperature, pH and dissolved oxygen were optimized in the fermatator. pVAX1-M was definited to be the predominant strains. The optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 57mg/L, also the purification was 1.88, which was 4.3-fold in comparison with using conventional LB culture.
The fermentation broth was extracted by alkaline lysis and schizolysis. The alkaline lysis, such as schizolysis time and the ratio of the lysate were optimized. Then, PEG and Diatomaceous Earth were used to purificatd and compare. The extraction and purification technology was founded based on alkaline lysis and Diatomaceous Earth. This technology can move out foreign matter, such as most of the RNA, genome DNA and proteins. Meanwhile, the purification of the plasmid DNA can reach 1.88 and the ratio of superhelix DNA was 98.4%, which were consistent with the plasmid quality equirement of WHO. The thalli cultured in the fermentator were extracted and purificated by this technology, as a result, the yield of plasmid DNA can reach 57mg/L. The cost had cut down by 20-40% in comparison with traditional technology.
This study cut down the production costs of fermentation medium and plasmid extraction. It provides an efficient and economical method for large-scale production of DNA vaccine.
采用单因子分析和正交设计,摇瓶发酵对各重组大肠杆菌的培养基条件进行了研究,确定优化了培养基的成份。发酵后,以优化的碱裂解法提取质粒,产量最高可达27.8mg/L,纯度达到1.88,是未优化前质粒产量的2.62倍。
研究了携带鸡传染性支气管炎病毒真核表达质粒基因工程菌在5L发酵罐中的发酵工艺。优化了上罐发酵接种量、培养温度、初始pH及溶氧等培养条件。确定了优势菌株pVAX1-M,以优化的碱裂解法提取质粒产量可达57mg/L,纯度达到1.88,是未优化前质粒产量的4.3倍。
对发酵液用热激法和碱裂解法两种不同的提取方法进行粗提,对碱裂解法进行优化,对裂解时间和裂解反应液的比例进行研究。再用聚乙二醇和硅藻土两种方法进行纯化,建立了以碱裂解法粗提,硅藻土法纯化的质粒DNA提纯工艺。该工艺可去除大部分RNA、基因组DNA和蛋白质等杂质,提取的质粒DNA纯度可达到1.88,超螺旋比例达到98.4%,符合世界卫生组织关于质粒产品的质量要求。对发酵罐发酵的菌体以本工艺提纯,质粒产量可达到57mg/L。与传统纯化方法比较,生产成本降低了20-40%。
本研究降低了基因工程菌发酵和质粒提取的生产成本,为DNA疫苗的大规模生产提供了理论和实验依据。
The fermentation parameters of recombinant E.coli which contains the eukaryoticexpression plasmid pVAX1-S1, pVAX1-M, pVAX1-N, VR1020-S1, VR1020-M and VR1020-N were investigated.
By a single factor experiment and orthogonal test, the fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in shake flask were investigated. In the experiment, the optimum medium was carried out and definited the medium ingredient. After fermentating, the optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 27.8mg/L, also the purification was 1.88, which was 2.62-fold in comparison with using conventional LB culture.
The fermentation parameters of recombinant E.coli which contains the eukaryotic expression plasmid with IBV genes in 5L fermentor were investigated. Inoculum size, temperature, pH and dissolved oxygen were optimized in the fermatator. pVAX1-M was definited to be the predominant strains. The optimum alkaline lysis was used to extract the plasmid DNA, and the yield could reach 57mg/L, also the purification was 1.88, which was 4.3-fold in comparison with using conventional LB culture.
The fermentation broth was extracted by alkaline lysis and schizolysis. The alkaline lysis, such as schizolysis time and the ratio of the lysate were optimized. Then, PEG and Diatomaceous Earth were used to purificatd and compare. The extraction and purification technology was founded based on alkaline lysis and Diatomaceous Earth. This technology can move out foreign matter, such as most of the RNA, genome DNA and proteins. Meanwhile, the purification of the plasmid DNA can reach 1.88 and the ratio of superhelix DNA was 98.4%, which were consistent with the plasmid quality equirement of WHO. The thalli cultured in the fermentator were extracted and purificated by this technology, as a result, the yield of plasmid DNA can reach 57mg/L. The cost had cut down by 20-40% in comparison with traditional technology.
This study cut down the production costs of fermentation medium and plasmid extraction. It provides an efficient and economical method for large-scale production of DNA vaccine.
引文
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