抗病饵料藻及酵母的构建与活性分析

发布时间：2018-03-19 08:21

本文选题：抗菌肽　切入点：pisL9K22WK　出处：《深圳大学》2017年硕士论文　论文类型：学位论文

【摘要】：水产及畜牧业面临着各类病害频繁发生的难题,抗生素是防治养殖病害的传统方法,但抗生素会导致生物安全性与环境污染问题。抗菌肽由于其独特的抗菌机理,使细菌不易对其产生耐药性,且因代谢速度快而不会在机体中产生有害残留,被认为是潜在的传统抗生素替代品。由于抗菌肽的人工合成成本较高,又很难直接从生物体内分离获得大批量抗菌肽,采用基因工程技术重组表达抗菌肽是目前最可行的抗菌肽制备途径。本研究利用抗菌肽NZ2114和pisL9K22WK的基因序列,构建了受FLD1启动子调控的抗菌肽重组表达载体,并分别转入Pichiapink酵母中,获得了能高效表达抗菌肽的基因工程菌株。与此同时,本研究还将经密码子优化的抗菌肽NZ2114转入莱茵衣藻的叶绿体基因组内,构建了利用叶绿体遗传系统表达抗菌肽的基因工程藻。具体研究结果如下:1.以甲醛脱氢酶启动子FLD1替换pPink-LC质粒中的醇氧化酶启动子AOX1,构建了可由氯化胆碱诱导的新质粒。并以此为基础,分别插入密码子优化过的抗菌肽pisL9K22WK基因和NZ2114基因,构建了FPLC和FNLC两个酵母表达载体。此外,还采用串联方式构建了含有α分泌信号肽的pisL9K22WK基因四串联酵母胞外表达载体αFPHLC和含有α分泌信号肽的NZ2114基因四串联酵母胞外表达载体αFNHLC。2.上述表达质粒分别电转化Pichiapink酵母,经PAD平板上筛选、PCR验证和甲醇诱导初筛,最终获得了能在细胞内高效表达抗菌肽基因pisL9K22WK和抗菌肽基因NZ2114的酵母工程菌PH6和NZ6,以及能在胞外高效分泌表达抗菌肽基因pisL9K22WK和NZ2114的酵母工程菌PIS3和NZ2114-9。3.为了避免使用甲醇诱导酵母表达外源重组蛋白时所带来了安全隐患及甲醇残留问题,本研究利用饲料添加剂氯化胆碱代替甲醇作为诱导物,探索酵母工程菌PH6和NZ6的发酵条件。抑菌实验结果均表明,氯化胆碱成功诱导PH6和NZ6表达出了抗菌肽pisL9K22WK及抗菌肽NZ2114,而基因工程酵母的细胞总蛋白对金黄色葡萄球菌具有明显抑制作用。进一步对氯化胆碱诱导PH6及NZ6发酵产抗菌肽的最佳条件研究发现:氯化胆碱诱导PH6的最佳诱导浓度为1%(w/v),最佳诱导时间为120 h,而氯化胆碱诱导NZ6的最佳诱导浓度为2%(w/v),最佳诱导时间为72 h。4.根据莱茵衣藻叶绿体基因的密码子偏好性优化了抗菌肽NZ2114的基因,并构建了含有16s/psbA杂合启动子的莱茵衣藻叶绿体表达质粒p322-NZ;通过“基因枪法”将该质粒和表达壮观霉素抗性基因的辅助质粒p228共同转化衣藻叶绿体,经多次同质化筛选最终获得能成功表达抗菌肽NZ2114的基因工程藻株16。抑菌实验结果显示:工程藻16的细胞粗提液对金黄色葡萄球菌有明显的抑制效果,在24 h内均保留抑菌活性。Tris-Tricine SDS PAGE电泳结果表明,工程藻16的细胞粗提液在4 kDa处出现蛋白条带,与NZ2114的预期大小基本一致。本研究获得了能表达抗菌肽的Pichiapink酵母工程菌,探索了用氯化胆碱替代有毒甲醇进行重组抗菌肽诱导表达的新途径;构建了能利用衣藻叶绿体遗传系统表达抗菌肽的基因工程藻株,实现了抗菌肽NZ2114在莱茵衣藻内的有效表达,为抗病饵料藻的开发与应用打下了重要基础。
[Abstract]:Aquatic animal husbandry and the problems facing the frequent occurrence of various diseases, antibiotics are traditional methods for prevention and treatment of disease in breeding, but the antibiotics will lead to biological safety and environmental pollution. Because of its unique antibacterial peptide, antibacterial mechanism, which is not easy to produce drug resistance of the bacteria, and because of the metabolic speed in the body does not produce harmful the residual, is considered to be potential alternatives to traditional antibiotics. Due to high cost of synthetic antimicrobial peptides, it is difficult to obtain large quantities of direct separation of antimicrobial peptides from organisms, using genetic engineering technology, recombinant expression of antimicrobial peptides is currently the most feasible way of preparation of antibacterial peptide. The gene sequence of antibacterial peptide NZ2114 and pisL9K22WK, constructed by promoter FLD1 antimicrobial peptide recombinant expression vector and then transformed into Pichiapink yeast, to obtain a high expression of antimicrobial peptide gene engineering bacteria Plant chloroplast genome. At the same time, this study will also by codon optimization of the antimicrobial peptide NZ2114 into Rhine Chlamydomonas, construct the expression of antimicrobial peptides by genetic engineering alga chloroplast genetic system. The results are as follows: 1. to replace formaldehyde dehydrogenase promoter FLD1 alcohol oxidase promoter in the plasmid pPink-LC AOX1, constructed a new the plasmid can be induced by choline chloride. And on this basis, were inserted into the codon optimized antibacterial peptide pisL9K22WK gene and NZ2114 gene, constructed FPLC and FNLC two yeast expression vector. In addition, the series also developed a FPHLC pisL9K22WK gene expression vector containing a secretion signal peptide four series of extracellular yeast and containing alpha secretion signal peptide NZ2114 gene four series of yeast expression vector of the extracellular alpha FNHLC.2. expression plasmids were electroporated into Pichiapink yeast by PAD plate screening PCR, validation and methanol induction screening, finally obtained high expression of antimicrobial peptide gene pisL9K22WK and antibacterial peptide gene NZ2114 in yeast PH6 and NZ6 in the cell, and the secretory expression of antibacterial peptide gene pisL9K22WK and NZ2114 in yeast PIS3 and NZ2114-9.3. in order to avoid the use of methanol induced expression of exogenous recombinant protein in yeast what brings security risks and problems of methanol residue in the extracellular, the feed additive choline chloride instead of methanol as the inducer, the fermentation conditions of exploration engineering yeast strain PH6 and NZ6. The bacteriostatic experiment results indicated that choline chloride induced PH6 and NZ6 successfully expressed antimicrobial peptide pisL9K22WK and antimicrobial peptide NZ2114 gene engineering yeast cell total protein has obvious inhibitory effect on Staphylococcus aureus. The choline chloride induced antibacterial peptide PH6 and the best fermentation NZ6 The study found that: the best conditions of inducing concentration of choline chloride induced by PH6 was 1% (w/v), the best induction time was 120 h, and the optimal concentration of choline chloride induced by NZ6 was 2% (w/v), the best induction time is 72 h.4. according to the Rhine Chlamydomonas chloroplast gene codon optimization of antibacterial peptide NZ2114 gene. And constructed with 16s/psbA hybrid promoter of Rhine Chlamydomonas chloroplast expression plasmid p322-NZ by gene gun "; the expression plasmid and helper plasmid P228 spectinomycin resistance gene into chloroplast, after repeated homogeneity obtained successful gene engineering expression of antimicrobial peptide NZ2114 in 16. strains of algae showed antibacterial experiment screening: 16 cell engineering algae extract has obvious inhibiting effect on Staphylococcus aureus, within 24 h were retained antibacterial activity of.Tris-Tricine SDS PAGE electrophoresis results showed that the project was 16 The cell extracts at 4 kDa protein bands, consistent with the expected size of NZ2114. This research has obtained the expression of Pichiapink in yeast antibacterial peptide, to explore a new way of recombinant antibacterial peptide induced expression of choline chloride to replace toxic methanol; construction of the genetic engineering strain of antimicrobial peptides expression of the Chlamydomonas chloroplast genetic system, realize the effective expression of antimicrobial peptide NZ2114 in Rhine in the Chlamydomonas, an important foundation for the development and application of microalgae resistance to fight.

【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q943.2

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