猪溶菌酶的重组表达及其增效研究

发布时间：2017-12-27 17:37

本文关键词：猪溶菌酶的重组表达及其增效研究　出处：《江南大学》2017年博士论文　论文类型：学位论文

【摘要】：随着食品安全问题的日益突出以及饲用抗生素滥用问题的愈发严重,寻找安全无污染、高效稳定、广谱抗菌且能够规模生产的抗生素替代品已经迫在眉睫。作为动物机体免疫系统的组成部分,溶菌酶是天然的抗生素替代品,目前已被普遍应用在医药、生物科研、食品以及饲料等领域,前景非常广阔。然而,作为畜牧行业最重要的成员之一,猪还没有专用的溶菌酶产品。猪溶菌酶(Sus scrofa lysozyme,SSL)作为猪体内抵抗细菌性疾病的重要屏障,不仅具备传统C型溶菌酶的抗菌功能,还能抵抗胃蛋白酶的降解作用,是最理想的猪用抗生素替代品。然而,由于SSL的来源有限(主要通过猪胃组织的提取),加之其抗革兰氏阴性菌的能力较差,目前还没其相关应用的报道。本文利用基因工程的手段,通过微生物发酵的方法规模化生产SSL产品;同时利用胰蛋白酶对SSL水解结合一定的分离纯化技术,获得了可以杀灭革兰氏阴性菌的抗菌肽产品;然后利用基因修饰的方法,将SSL中抗菌肽的作用强化,得到了既保留SSL抗革兰氏阳性菌的性能,又可以杀灭革兰氏阴性菌的新型SSL产品。此外,还对新型SSL产品的抗菌机理进行了探究,为其工业化应用奠定理论基础。(1)根据NCBI网站的基因序列,化学合成了SSL的编码基因,并对其进行大肠杆菌宿主的密码子优化,在BL21(DE3)中成功得到了诱导表达。在最优的诱导条件(25℃,前培养时间3 h,利用0.8 mmol·L-1的IPTG诱导8 h)发酵后,通过对包涵体的洗涤、溶解、透析以及浓缩,最终获得了166.91±3.37 mg·L-1的SSL,比酶活力可达7950.42±226.67 U·mg-1。利用毕赤酵母X-33(pPICZαA)成功地表达了SSL基因,并比较了两种密码子的优化方法对SSL基因在毕赤酵母中表达的影响,发现“全局随机优化法”对其表达量的提高较为明显,最终获得了189.28±4.16 mg·L-1比酶活力为2845.38±124.19 U·mg-1的SSL产品,总蛋白表达量是原始基因表达量的2.63倍,而“一对一”优化法对其表达无明显改善。此外,组成型表达载体pGAPZαA和蛋白酶缺陷型宿主SMD1168H均无法达到野生型(诱导表达载体)X-33(pPICZαA)的表达水平。通过亲和层析或SSL的复性过程,结合超滤的纯化方法,得到了电泳纯的SSL产品。对其酶学性质和抗菌特性进行了研究,发现重组SSL的性质与其理论值基本一致:最适反应温度35℃、最适反应pH 6.0,说明利用微生物发酵的方法生产SSL是切实可行的。而通过对大肠杆菌和毕赤酵母表达系统生产SSL的过程进行比较分析,发现大肠杆菌表达系统的生产周期更短,得到的产品比酶活力更高,有助于对其性质开展进一步研究。(2)通过对SSL进行蛋白酶水解,产物的SDS-PAGE分析以及抗菌活性测定,发现SSL对胃蛋白酶的水解具有抗性;而胰蛋白酶的水解物具有最强的抗革兰氏阴性菌活性,其抗菌系数可达2.81,可杀灭99%以上的测试菌。利用凝胶过滤色谱和反相分离色谱的分离纯化,结合液质联用的分离与鉴定,得到了两种对革兰氏阴性菌具有抗菌活性的肽,其氨基酸序列分别为:G-V-S-L-A-N-W-V-C-L-A-K(LP)和A-W-V-A-W-K(SP)。经化学合成以后进行抗菌谱的测定,发现LP对革兰氏阴性菌具有抗菌活性,但是对革兰氏阳性菌却没有相应的作用;而SP既可以杀灭革兰氏阴性菌,也基本上保留了SSL的抗革兰氏阳性菌的能力。通过圆二色谱(Circular dichroism,CD)分析,Swiss-modeling结构模拟以及AFM检测,推测LP和SP的作用方式与具备螺旋-回环-螺旋(Helix-loop-helix,HLH)结构域的抗菌肽类似:主要是通过改变靶细胞细胞膜的通透性来杀死细胞。但是它们作用的具体方式不同,LP可能是通过地毯式模型的作用方式,而SP则是可能借助于其α-螺旋结构在细胞膜上形成孔洞。(3)将本研究中分离得到的抗菌效果较好的抗菌肽SP的编码基因、含有SP的HLH的编码基因以及SP的六拷贝的6SP编码基因分别与SSL的编码序列的N端或C端进行融合,构建表达载体后发酵得到了既能保留SSL杀灭革兰氏阳性菌的能力,又具备SP的抗革兰氏阴性菌活性的新型溶菌酶产品。同等条件下,SSL的N端融合产物的抗菌活性要明显高于C端的融合产物。通过CD与Swiss-modeling分析发现,融合SSL的N端距离SSL的活性中心更近,可能与其作时用的底物结合有关。其中,N端融合产物6SP-SSL的抗菌活性最高。6SP的存在一方面提高了SP与靶细胞的接触几率,另一方面也提高了融合产物的疏水性,增加了其与靶细胞的结合能力。此外,SSL与SP的协同作用也有所体现,特别是作用于金黄色葡萄球菌Staphylococcus aureus ATCC 25923和大肠杆菌Escherichia coli ATCC 25922时。6SP-SSL的获得为C型溶菌酶的增效研究提供了一种简单、高效且广谱的途径。(4)通过原子力显微镜以及膜电势的检测发现,6SP-SSL的作用可以使靶细胞E.coli ATCC 10798的细胞壁结构发生破坏,其通透性得到改变从而使细胞内溶物流出,导致细胞死亡。这也是SSL与SP共同的作用机理,我们推测6SP-SSL作用于靶细胞时,首先通过SP的疏水性或其特殊的α螺旋构象在靶细胞的细胞壁上形成孔洞,让SSL有机会接触到其底物结合位点,然后发挥其溶菌机制。此外,基于相关试剂盒以及流式细胞仪的检测发现,6SP-SSL作用后的靶细胞中有细胞凋亡现象的发生,并且发生细胞凋亡的细胞比例与融合SSL的作用时间及其浓度有关。说明除了直接杀菌机理之外,6SP-SSL还可以通过诱发靶细胞的程序性细胞死亡(如细胞凋亡),从而达到杀灭靶细胞的效果。通过进一步的研究发现,6SP-SSL可以通过下调靶细胞(E.coli ATCC 10798)中mazE基因的转录而影响其正常表达,使得毒素-抗毒素系统中的毒素成分maz F得以积累,从而诱发细胞的程序性死亡。
[Abstract]:With the increasingly prominent food safety problems and the increasingly serious abuse of feed antibiotics, it is urgent to find safe, pollution-free, efficient, stable, broad-spectrum antibacterial and large-scale production of antibiotic substitutes. As an important part of animal immune system, lysozyme is a natural substitute for antibiotics. It has been widely applied in medicine, biological research, food and feed, and has wide prospects. However, as one of the most important members of the animal husbandry industry, pigs have no special lysozyme products. Sus scrofa lysozyme (SSL) is an important barrier for pig to resist bacterial diseases. It not only has the antibacterial function of traditional C lysozyme, but also resists the degradation of pepsin. It is the best substitute for swine antibiotics. However, due to the limited source of SSL (mainly extracted from pig stomach tissue) and its ability to resist gram negative bacteria, there are no reports related to its application. In this paper, by the means of genetic engineering, by microorganism fermentation method of large-scale production of SSL products; at the same time using Trypsin Hydrolysis of SSL combined with the separation and purification technology, obtained can kill gram negative bacteria antibacterial peptide products; and then use the method of gene modification, the SSL will strengthen the role of antimicrobial peptides, are obtained keep SSL resistance of gram positive bacteria, and the new SSL products to kill gram negative bacteria. In addition, the antibacterial mechanism of the new SSL products was explored, and the theoretical basis for its industrial application was laid. (1) according to the gene sequence of NCBI website, we synthesized SSL encoding gene and optimized the codon of Escherichia coli host, and got the induced expression in BL21 (DE3) successfully. After the best induction conditions (25 h, 3 L-1) and 0.8 mmol. L-1 IPTG were used to induce 8 h, the SSL of 166.91 + 3.37 mg L-1 was obtained through washing, dissolving, dialysis and concentrating the inclusion body. The specific activity of the enzyme reached 7950.42 + 226.67 U / mg-1. The use of Pichia pastoris X-33 (pPICZ a A) the successful expression of the SSL gene, and compare the two kinds of codon optimization methods on expression of SSL gene in Pichia pastoris, found "stochastic global optimization method for its expression is obviously improved, eventually won the 189.28 + 4.16 Mg - L-1 ratio of enzyme activity was 2845.38 + 124.19 U - mg-1 SSL products, the total amount of protein expression is the original gene expression was 2.63 times, while the" one to one "optimization method on the expression of no significant improvement. In addition, the expression level of the wild type (inducible expression vector) X-33 (pPICZ alpha A) was not reached by the pGAPZ - alpha A and the proteinase deficient host SMD1168H. The electrophoretic pure SSL products were obtained by the combination of affinity chromatography or SSL, combined with ultrafiltration purification. The enzymatic properties and antibacterial properties of the recombinant SSL were studied. It is found that the properties of recombinant pH are basically the same with its theoretical values: the best reaction temperature is 35 C and the best reaction is pH 6, indicating that it is feasible to produce SSL by microbial fermentation. By comparing and analyzing the production process of SSL in E. coli and Pichia pastoris expression system, it is found that the production cycle of E.coli expression system is shorter, and the products obtained are higher than enzyme activity, which is helpful for further research on its properties. (2) by enzymatic hydrolysis of SSL products, SDS-PAGE analysis and determination of antibacterial activity, found that SSL is resistant to pepsin hydrolysis and hydrolysis; trypsin has the strongest activity against gram negative bacteria, the antibacterial factor is up to 2.81, more than 99% of the bacteria killing test. Using gel filtration chromatography and reversed-phase separation chromatography, we identified two kinds of peptides with antimicrobial activity against gram negative bacteria, and their amino acid sequences were G-V-S-L-A-N-W-V-C-L-A-K (LP) and A-W-V-A-W-K (SP) respectively. After the chemical synthesis, we found that LP has antibacterial activity against gram negative bacteria, but has no corresponding effect on Gram-positive bacteria. SP can not only kill Gram-negative bacteria, but also basically retain SSL's ability to resist gram positive bacteria. By round two chromatography (Circular dichroism CD) analysis, Swiss-modeling simulation and AFM test structure, mode of action that LP and SP and the helix loop helix with (Helix-loop-helix, HLH) domain: similar antibacterial peptide is mainly by changing the target cell membrane permeability to kill cells. But the specific way of their action is different. LP may be acted by carpet model, while SP may form holes on its cell membrane by means of its alpha helix structure. (3) the antibacterial effect were obtained in this study better antibacterial peptide SP gene encoding, containing six copies of the 6SP gene encoding the SP gene encoding HLH and SP respectively with the encoding sequence of SSL N or C terminal fusion expression vector was constructed after fermentation obtained can keep the ability SSL kill gram positive bacteria, new products with lysozyme activity against gram negative bacteria SP. Under the same condition, the antibacterial activity of the N end fusion product of SSL is obviously higher than that of the fusion product at the C end. Through CD and Swiss-modeling analysis, it is found that the N end of the fusion SSL is closer to the active center of SSL, which may be related to the substrate binding. Among them, the N end fusion product 6SP-SSL has the highest antibacterial activity. The existence of 6SP increased the contact probability between SP and target cells on the one hand. On the other hand, it also improved the hydrophobicity of fusion products and increased its binding ability to target cells. In addition, the synergistic effect of SSL and SP is also reflected, especially in Staphylococcus aureus Staphylococcus aureus ATCC 25923 and Escherichia coli Escherichia coli ATCC 25922. Study on the efficiency of 6SP-SSL for C type lysozyme
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q78;S816.7

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