發(fā)布時(shí)間：2018-03-19 08:21

  本文選題：抗菌肽　切入點(diǎn)：pisL9K22WK　出處：《深圳大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：水產(chǎn)及畜牧業(yè)面臨著各類病害頻繁發(fā)生的難題,抗生素是防治養(yǎng)殖病害的傳統(tǒng)方法,但抗生素會(huì)導(dǎo)致生物安全性與環(huán)境污染問題�？咕挠捎谄洫�(dú)特的抗菌機(jī)理,使細(xì)菌不易對其產(chǎn)生耐藥性,且因代謝速度快而不會(huì)在機(jī)體中產(chǎn)生有害殘留,被認(rèn)為是潛在的傳統(tǒng)抗生素替代品。由于抗菌肽的人工合成成本較高,又很難直接從生物體內(nèi)分離獲得大批量抗菌肽,采用基因工程技術(shù)重組表達(dá)抗菌肽是目前最可行的抗菌肽制備途徑。本研究利用抗菌肽NZ2114和pisL9K22WK的基因序列,構(gòu)建了受FLD1啟動(dòng)子調(diào)控的抗菌肽重組表達(dá)載體,并分別轉(zhuǎn)入Pichiapink酵母中,獲得了能高效表達(dá)抗菌肽的基因工程菌株。與此同時(shí),本研究還將經(jīng)密碼子優(yōu)化的抗菌肽NZ2114轉(zhuǎn)入萊茵衣藻的葉綠體基因組內(nèi),構(gòu)建了利用葉綠體遺傳系統(tǒng)表達(dá)抗菌肽的基因工程藻。具體研究結(jié)果如下:1.以甲醛脫氫酶啟動(dòng)子FLD1替換pPink-LC質(zhì)粒中的醇氧化酶啟動(dòng)子AOX1,構(gòu)建了可由氯化膽堿誘導(dǎo)的新質(zhì)粒。并以此為基礎(chǔ),分別插入密碼子優(yōu)化過的抗菌肽pisL9K22WK基因和NZ2114基因,構(gòu)建了FPLC和FNLC兩個(gè)酵母表達(dá)載體。此外,還采用串聯(lián)方式構(gòu)建了含有α分泌信號肽的pisL9K22WK基因四串聯(lián)酵母胞外表達(dá)載體αFPHLC和含有α分泌信號肽的NZ2114基因四串聯(lián)酵母胞外表達(dá)載體αFNHLC。2.上述表達(dá)質(zhì)粒分別電轉(zhuǎn)化Pichiapink酵母,經(jīng)PAD平板上篩選、PCR驗(yàn)證和甲醇誘導(dǎo)初篩,最終獲得了能在細(xì)胞內(nèi)高效表達(dá)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達(dá)抗菌肽基因pisL9K22WK和NZ2114的酵母工程菌PIS3和NZ2114-9。3.為了避免使用甲醇誘導(dǎo)酵母表達(dá)外源重組蛋白時(shí)所帶來了安全隱患及甲醇?xì)埩魡栴},本研究利用飼料添加劑氯化膽堿代替甲醇作為誘導(dǎo)物,探索酵母工程菌PH6和NZ6的發(fā)酵條件。抑菌實(shí)驗(yàn)結(jié)果均表明,氯化膽堿成功誘導(dǎo)PH6和NZ6表達(dá)出了抗菌肽pisL9K22WK及抗菌肽NZ2114,而基因工程酵母的細(xì)胞總蛋白對金黃色葡萄球菌具有明顯抑制作用。進(jìn)一步對氯化膽堿誘導(dǎo)PH6及NZ6發(fā)酵產(chǎn)抗菌肽的最佳條件研究發(fā)現(xiàn):氯化膽堿誘導(dǎo)PH6的最佳誘導(dǎo)濃度為1%(w/v),最佳誘導(dǎo)時(shí)間為120 h,而氯化膽堿誘導(dǎo)NZ6的最佳誘導(dǎo)濃度為2%(w/v),最佳誘導(dǎo)時(shí)間為72 h。4.根據(jù)萊茵衣藻葉綠體基因的密碼子偏好性優(yōu)化了抗菌肽NZ2114的基因,并構(gòu)建了含有16s/psbA雜合啟動(dòng)子的萊茵衣藻葉綠體表達(dá)質(zhì)粒p322-NZ;通過“基因槍法”將該質(zhì)粒和表達(dá)壯觀霉素抗性基因的輔助質(zhì)粒p228共同轉(zhuǎn)化衣藻葉綠體,經(jīng)多次同質(zhì)化篩選最終獲得能成功表達(dá)抗菌肽NZ2114的基因工程藻株16。抑菌實(shí)驗(yàn)結(jié)果顯示:工程藻16的細(xì)胞粗提液對金黃色葡萄球菌有明顯的抑制效果,在24 h內(nèi)均保留抑菌活性。Tris-Tricine SDS PAGE電泳結(jié)果表明,工程藻16的細(xì)胞粗提液在4 kDa處出現(xiàn)蛋白條帶,與NZ2114的預(yù)期大小基本一致。本研究獲得了能表達(dá)抗菌肽的Pichiapink酵母工程菌,探索了用氯化膽堿替代有毒甲醇進(jìn)行重組抗菌肽誘導(dǎo)表達(dá)的新途徑;構(gòu)建了能利用衣藻葉綠體遺傳系統(tǒng)表達(dá)抗菌肽的基因工程藻株,實(shí)現(xiàn)了抗菌肽NZ2114在萊茵衣藻內(nèi)的有效表達(dá),為抗病餌料藻的開發(fā)與應(yīng)用打下了重要基礎(chǔ)。
[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.

【學(xué)位授予單位】：深圳大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;Q943.2

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本文編號：1633421