本文關(guān)鍵詞：豬溶菌酶的重組表達(dá)及其增效研究　出處：《江南大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 豬溶菌酶 抗生素 抗菌肽 膜滲透 程序性細(xì)胞死亡

【摘要】：隨著食品安全問題的日益突出以及飼用抗生素濫用問題的愈發(fā)嚴(yán)重,尋找安全無污染、高效穩(wěn)定、廣譜抗菌且能夠規(guī)模生產(chǎn)的抗生素替代品已經(jīng)迫在眉睫。作為動(dòng)物機(jī)體免疫系統(tǒng)的組成部分,溶菌酶是天然的抗生素替代品,目前已被普遍應(yīng)用在醫(yī)藥、生物科研、食品以及飼料等領(lǐng)域,前景非常廣闊。然而,作為畜牧行業(yè)最重要的成員之一,豬還沒有專用的溶菌酶產(chǎn)品。豬溶菌酶(Sus scrofa lysozyme,SSL)作為豬體內(nèi)抵抗細(xì)菌性疾病的重要屏障,不僅具備傳統(tǒng)C型溶菌酶的抗菌功能,還能抵抗胃蛋白酶的降解作用,是最理想的豬用抗生素替代品。然而,由于SSL的來源有限(主要通過豬胃組織的提取),加之其抗革蘭氏陰性菌的能力較差,目前還沒其相關(guān)應(yīng)用的報(bào)道。本文利用基因工程的手段,通過微生物發(fā)酵的方法規(guī)�；a(chǎn)SSL產(chǎn)品;同時(shí)利用胰蛋白酶對(duì)SSL水解結(jié)合一定的分離純化技術(shù),獲得了可以殺滅革蘭氏陰性菌的抗菌肽產(chǎn)品;然后利用基因修飾的方法,將SSL中抗菌肽的作用強(qiáng)化,得到了既保留SSL抗革蘭氏陽(yáng)性菌的性能,又可以殺滅革蘭氏陰性菌的新型SSL產(chǎn)品。此外,還對(duì)新型SSL產(chǎn)品的抗菌機(jī)理進(jìn)行了探究,為其工業(yè)化應(yīng)用奠定理論基礎(chǔ)。(1)根據(jù)NCBI網(wǎng)站的基因序列,化學(xué)合成了SSL的編碼基因,并對(duì)其進(jìn)行大腸桿菌宿主的密碼子優(yōu)化,在BL21(DE3)中成功得到了誘導(dǎo)表達(dá)。在最優(yōu)的誘導(dǎo)條件(25℃,前培養(yǎng)時(shí)間3 h,利用0.8 mmol·L-1的IPTG誘導(dǎo)8 h)發(fā)酵后,通過對(duì)包涵體的洗滌、溶解、透析以及濃縮,最終獲得了166.91±3.37 mg·L-1的SSL,比酶活力可達(dá)7950.42±226.67 U·mg-1。利用畢赤酵母X-33(pPICZαA)成功地表達(dá)了SSL基因,并比較了兩種密碼子的優(yōu)化方法對(duì)SSL基因在畢赤酵母中表達(dá)的影響,發(fā)現(xiàn)“全局隨機(jī)優(yōu)化法”對(duì)其表達(dá)量的提高較為明顯,最終獲得了189.28±4.16 mg·L-1比酶活力為2845.38±124.19 U·mg-1的SSL產(chǎn)品,總蛋白表達(dá)量是原始基因表達(dá)量的2.63倍,而“一對(duì)一”優(yōu)化法對(duì)其表達(dá)無明顯改善。此外,組成型表達(dá)載體pGAPZαA和蛋白酶缺陷型宿主SMD1168H均無法達(dá)到野生型(誘導(dǎo)表達(dá)載體)X-33(pPICZαA)的表達(dá)水平。通過親和層析或SSL的復(fù)性過程,結(jié)合超濾的純化方法,得到了電泳純的SSL產(chǎn)品。對(duì)其酶學(xué)性質(zhì)和抗菌特性進(jìn)行了研究,發(fā)現(xiàn)重組SSL的性質(zhì)與其理論值基本一致:最適反應(yīng)溫度35℃、最適反應(yīng)pH 6.0,說明利用微生物發(fā)酵的方法生產(chǎn)SSL是切實(shí)可行的。而通過對(duì)大腸桿菌和畢赤酵母表達(dá)系統(tǒng)生產(chǎn)SSL的過程進(jìn)行比較分析,發(fā)現(xiàn)大腸桿菌表達(dá)系統(tǒng)的生產(chǎn)周期更短,得到的產(chǎn)品比酶活力更高,有助于對(duì)其性質(zhì)開展進(jìn)一步研究。(2)通過對(duì)SSL進(jìn)行蛋白酶水解,產(chǎn)物的SDS-PAGE分析以及抗菌活性測(cè)定,發(fā)現(xiàn)SSL對(duì)胃蛋白酶的水解具有抗性;而胰蛋白酶的水解物具有最強(qiáng)的抗革蘭氏陰性菌活性,其抗菌系數(shù)可達(dá)2.81,可殺滅99%以上的測(cè)試菌。利用凝膠過濾色譜和反相分離色譜的分離純化,結(jié)合液質(zhì)聯(lián)用的分離與鑒定,得到了兩種對(duì)革蘭氏陰性菌具有抗菌活性的肽,其氨基酸序列分別為:G-V-S-L-A-N-W-V-C-L-A-K(LP)和A-W-V-A-W-K(SP)。經(jīng)化學(xué)合成以后進(jìn)行抗菌譜的測(cè)定,發(fā)現(xiàn)LP對(duì)革蘭氏陰性菌具有抗菌活性,但是對(duì)革蘭氏陽(yáng)性菌卻沒有相應(yīng)的作用;而SP既可以殺滅革蘭氏陰性菌,也基本上保留了SSL的抗革蘭氏陽(yáng)性菌的能力。通過圓二色譜(Circular dichroism,CD)分析,Swiss-modeling結(jié)構(gòu)模擬以及AFM檢測(cè),推測(cè)LP和SP的作用方式與具備螺旋-回環(huán)-螺旋(Helix-loop-helix,HLH)結(jié)構(gòu)域的抗菌肽類似:主要是通過改變靶細(xì)胞細(xì)胞膜的通透性來殺死細(xì)胞。但是它們作用的具體方式不同,LP可能是通過地毯式模型的作用方式,而SP則是可能借助于其α-螺旋結(jié)構(gòu)在細(xì)胞膜上形成孔洞。(3)將本研究中分離得到的抗菌效果較好的抗菌肽SP的編碼基因、含有SP的HLH的編碼基因以及SP的六拷貝的6SP編碼基因分別與SSL的編碼序列的N端或C端進(jìn)行融合,構(gòu)建表達(dá)載體后發(fā)酵得到了既能保留SSL殺滅革蘭氏陽(yáng)性菌的能力,又具備SP的抗革蘭氏陰性菌活性的新型溶菌酶產(chǎn)品。同等條件下,SSL的N端融合產(chǎn)物的抗菌活性要明顯高于C端的融合產(chǎn)物。通過CD與Swiss-modeling分析發(fā)現(xiàn),融合SSL的N端距離SSL的活性中心更近,可能與其作時(shí)用的底物結(jié)合有關(guān)。其中,N端融合產(chǎn)物6SP-SSL的抗菌活性最高。6SP的存在一方面提高了SP與靶細(xì)胞的接觸幾率,另一方面也提高了融合產(chǎn)物的疏水性,增加了其與靶細(xì)胞的結(jié)合能力。此外,SSL與SP的協(xié)同作用也有所體現(xiàn),特別是作用于金黃色葡萄球菌Staphylococcus aureus ATCC 25923和大腸桿菌Escherichia coli ATCC 25922時(shí)。6SP-SSL的獲得為C型溶菌酶的增效研究提供了一種簡(jiǎn)單、高效且廣譜的途徑。(4)通過原子力顯微鏡以及膜電勢(shì)的檢測(cè)發(fā)現(xiàn),6SP-SSL的作用可以使靶細(xì)胞E.coli ATCC 10798的細(xì)胞壁結(jié)構(gòu)發(fā)生破壞,其通透性得到改變從而使細(xì)胞內(nèi)溶物流出,導(dǎo)致細(xì)胞死亡。這也是SSL與SP共同的作用機(jī)理,我們推測(cè)6SP-SSL作用于靶細(xì)胞時(shí),首先通過SP的疏水性或其特殊的α螺旋構(gòu)象在靶細(xì)胞的細(xì)胞壁上形成孔洞,讓SSL有機(jī)會(huì)接觸到其底物結(jié)合位點(diǎn),然后發(fā)揮其溶菌機(jī)制。此外,基于相關(guān)試劑盒以及流式細(xì)胞儀的檢測(cè)發(fā)現(xiàn),6SP-SSL作用后的靶細(xì)胞中有細(xì)胞凋亡現(xiàn)象的發(fā)生,并且發(fā)生細(xì)胞凋亡的細(xì)胞比例與融合SSL的作用時(shí)間及其濃度有關(guān)。說明除了直接殺菌機(jī)理之外,6SP-SSL還可以通過誘發(fā)靶細(xì)胞的程序性細(xì)胞死亡(如細(xì)胞凋亡),從而達(dá)到殺滅靶細(xì)胞的效果。通過進(jìn)一步的研究發(fā)現(xiàn),6SP-SSL可以通過下調(diào)靶細(xì)胞(E.coli ATCC 10798)中mazE基因的轉(zhuǎn)錄而影響其正常表達(dá),使得毒素-抗毒素系統(tǒng)中的毒素成分maz F得以積累,從而誘發(fā)細(xì)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
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;S816.7

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