【作者】 王楠；

【导师】 刘德虎；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 本研究首次尝试在毕赤酵母细胞中高效表达和生产小分子抗菌肽～假黑盘菌素。首先按照毕赤酵母所偏爱的密码子,人工拼接出一个完整的、编码假黑盘菌素成熟短肽的基因;通过在基因两端添加合适的限制性酶切位点,将该人工序列定向插入到毕赤酵母诱导型分泌表达载体pPIC9K中的多克隆位点内。为了实现该小片段外源基因在毕赤酵母细胞中的高效表达,又将3个串联后的芜菁花叶病毒-人巨细胞病毒复合增强子序列插入到假黑盘菌素成熟短肽基因终止密码子的下游,由此得到了一个全新的酵母诱导型分泌表达载体3ENple-9K。利用线性化处理后的3ENple-9K质粒DNA对毕赤酵母GS115菌株进行遗传转化,并随后使用选择性培养基、G418抗性及摇瓶发酵,对获得的大量重组毕赤酵母进行筛选,得到了一个能够高效和稳定表达假黑盘菌素抗菌肽的工程菌株PleI。在中试规模(50L发酵罐)基础上,对该工程菌的发酵和外源蛋白高效表达条件进行了优化,包括营养成分、发酵温度、菌体密度、pH值、溶氧量、接种量、甲醇浓度和蛋白诱导表达时间等参数,由此使假黑盘菌素抗菌肽重组蛋白的表达量达到0.14g/L。此外,还对重组假黑盘菌素抗菌肽的提取、纯化等工艺也进行了初步的研究,摸索出一套简便、快捷和比较经济的方案,即将目标蛋白的提取、纯化工艺分成了以下四个阶段,分别是:1)发酵液的初处理;2)发酵上清液的再处理;3)发酵上清液的高度浓缩;4)重组蛋白的高度纯化。对各阶段所需的实验材料和工艺等影响重组蛋白回收率的条件也都进行了探索。在对纯化出的重组假黑盘菌素抗菌肽进行体外抑菌活性试验时发现,它不仅对一些常见的革兰氏阳性病原细菌具有较强的杀灭作用,而对某些革兰氏阴性细菌也具有一定的杀灭和抑制作用。活体动物试验表明,重组假黑盘菌素抗菌肽对感染马链球菌兽疫亚种的小鼠具有一定的疗效。本研究首次尝试在汉逊酵母细胞中高效表达和生产T4溶菌酶重组蛋白。通过在基因两端添加的合适的限制性酶切位点,将一个改构的T4溶菌酶基因定向插入到毕赤酵母诱导型分泌表达载体pPIC9K中多克隆位点内,并在该表达载体中添加了来源于汉逊酵母的rDNA序列作为同源整合序列,由此得到了一个全新的酵母诱导型分泌表达载体T4-rD-9K。利用线性化处理后的T4-rD-9K质粒DNA对汉逊酵母A16菌株进行遗传转化,并随后使用选择性培养基(G418抗性)及摇瓶发酵,对获得的大量重组汉逊酵母进行筛选,得到了一个能够高效和稳定表达改构T4溶菌酶重组蛋白的工程菌株H5。在上述研究基础上,本研究构建了一个含假黑盘菌素抗菌肽和改构T4溶菌酶基因的双元表达体系,实现了两种外源蛋白在毕赤酵母细胞中的同步和高效表达,并且这两种重组蛋白在发酵液中能协同发挥杀菌或抑菌活性,由此更进一步扩大了它们医药、食品和饲料等工业等领域的应用潜力。为了进一步提高外源蛋白基因在宿主细胞中表达效率,本研究还尝试构建一个高效的瑞氏木霉表达体系。首先通过PCR的方法,以瑞氏木霉基因组DNA为模板,从中克隆出其纤维二糖水解酶基因启动子-纤维二糖水解酶分泌信号肽编码序列以及纤维二糖水解酶基因终止子序列。然后利用上述序列,分别与改构的T4溶菌酶和假黑盘菌素抗菌肽编码序列进行拼接,形成了两个新的外源基因表达框。以毕赤酵母诱导型表达载体pPIC9K为出发载体,通过添加合适的限制性酶切位点,将改构的T4溶菌酶基因或假黑盘菌素抗菌肽基因表达框分别定向插入到pPIC9K载体的多克隆位点内,又获得了T4-hT-9K和ple-hT-9K两种瑞氏木霉诱导型分泌表达载体。此外,在构建上述载体的过程中,还插入了潮霉素抗性基因作为将来木霉重组子的筛选标记。更多还原

【Abstract】 This paper attempted to express the low molecular weight antimicrobial peptide～plectasin in Pichia pastoris. Firstly, the gene encoding mature plectasin was synthesized according to yeast codon usage bias, and then directionally inserted into the MCS of the expression vector pPIC9K with restriction endonuclease and T4 ligase, which is inducible expression with methanol as the inducer in P. Pastoris. In order to express efficiently the gene encoding heterologuos low molecular weight of protein in P. Pastoris, an artificial enhancer with 3 times tandem repeating sequence, derived from TYMV3’UTR and CMVp enhancers was inserted into the downstream of terminator codons of the plectasin gene, forming a new inductive and secretary expression vector of P. Pastoris designated 3ENple-9k. The 3ENple-9K plasmid DNA linearized with Sal I was transformed into the GS115 strain of P.pastoris. By using selective medium, G418 resistance and shaking-flask culture, one transformant designated PleI was selected from hundreds of transformants, which can express effectively and stably the recombinant plectasin peptides. Based on the pilot test (50L fermenter), a lot of parameter relating to the conditions of fermentation and protein expression were optimized, including nutrient component, temperature, cell density, pH, dissolved oxygen, inoculum size, methanol concentration and inductive time. The expression level up to 0.14g/L of the recombinant plectasin was realized by using the strategy motioned above. In addition, an extracting and purifying process of the recombinant plectasin was investigated and a simple, convenient and economic method was established, which involved in four stages: 1) pretreatment of the fermentation broth; 2) retreatment of the supernatant of fermentation; 3) high enrichment of the supernatant of fermentation by nano-filtration; 4) high purification of the recombinant protein by molecular-sieve chromatography. Meanwhile, conditions of experimental material and methods affecting recovery of the recombinant plectasin were studied. Antibacterial activity analysis of the recombinant plectasin in vitro shows that not only can it inhibit strongly growth of some common gram-positive pathogenic bacteria, but also inhibit some gram-negtive bacteria. In vivo inhibiting experiment indicated that the recombinant plectasin are able to partly cure the infection of mice caused by Streptococcus Equi Subsp. Zooepidemicus.A modified recombinant T4 lysozyme was firstly attempted to express and produce in Hansenula polymorpha. The gene encoding the modified T4 lysozyme according to yeast codon usage bias was inserted directionally into the MCS of a modified expression vector pPIC9K containing the rDNA sequence from H.polymorpha as a homologous recombination sequence, which is inducible expression with methanol as the inducer in H. polymorpha. The expression vector designated T4-rD-9K was linearized and transformed into the A16 strain of H.polymorpha. By using selective medium G418 resistance and shaking-flask culture, one transformant designated H5 was selected from hundreds of transformants, which can express effectively and stably the recombinant T4 lysozyme. Based on the results mentioned above, a dual-gene expression system designated pleT4-9K was constructed and transformed into the GS115 strain of P.pastoris. One transformant named PTI was obtained by screening, which can simultaneously express both the plectasin and T4 lysozyme genes in P.pastoris. Antibacterial activity analysis in vitro of the fermentation broth shows that both the recombinant proteins can play a role simultaneously, which predicts a broad application of the recombinant proteins at medicine, food, forage industry field in the near future.For further increase expression efficiency of the heterologous genes in host strain,a high expression system was also constructed, which will be used in Trichoderma reesie. The Pcbh1-sig and Tcbh1 DNA sequences were prepared by PCR approach, using genomic DNA of T. ressei as the template. The Pcbh1-sig and Tcbh1 sequences were added to 5’end and 3’end of the gene encoding plectasin or T4 lysozyme respectively, which formed two new expression cassettes. The plectasin or T4 lysozyme expression cassette was into the MCS of a modified expression vector pPIC9K containing a hygromycin resistance gene as the screening marker, forming two expression vectors designated T4-hT-9K and ple-hT-9K, which are inductive and secretary with cellulose as the inducer in T. reesie.更多还原