【作者】 王琰；

【导师】 陆承平； 孙建和；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 猪链球菌病是一种重要的人兽共患传染病,能导致仔猪脑膜炎、败血症、关节炎、心内膜炎、肺炎和人的脑膜炎,对公共卫生尤其是相应从业人员的生命安全构成威胁。因抗生素滥用,导致细菌耐药,给该病的防控带来了困难。由噬菌体基因编码的裂解酶——裂解效率高、与抗生素具协同作用,且不易产生抗性菌株,是一种颇具潜力的杀菌制剂。本文通过重组表达的方法获得纯化的猪链球菌噬菌体裂解酶LySMP,研究了该酶的杀菌作用特点、酶不同结构区域Lys1、Lys2、Lys3和Lys4的杀菌活性以及应用裂解酶全酶治疗感染猪链球菌的斑马鱼的保护作用。为了获得具有生物学活性的猪链球菌噬菌体裂解酶LySMP,采用重组表达技术,PCR扩增噬菌体SMP的裂解酶基因,插入pET-28a(+)中,构建出重组表达载体pET-lys。将该载体导入大肠杆菌BL21中诱导表达,获得55kDa的融合LySMP蛋白。酶谱试验表明该蛋白具有裂解宿主菌SS2-H的活性。表达产物经纯化后,浓度可达1-3mg/ml。裂解酶LySMP的体外生物学活性的研究发现纯化的LySMP需加0.8%β-巯基乙醇方可发挥裂解作用。同时,细菌用PMSF和溶菌酶处理后也具有更好的裂解效果。平板裂解试验结果显示,LySMP的超声破碎粗提物裂解谱比原始噬菌体广。噬菌体的宿主菌仅有2株猪链球菌2型,但在17株猪链球菌2型中,有15株可被裂解酶裂解,此外,所测的猪链球菌7型、9型,马兽疫链球菌亚种以及金黄色葡萄球菌,也可被裂解,不过目前尚未发现其可裂解大肠杆菌和沙门氏菌。LySMP在37℃,pH5.2的条件下,裂解效率最高。LySMP可使所测细菌浊度下降约1.2-98%不等。为了进一步研究LySMP发挥裂解功能的区域,并比较各功能区与全酶的裂解效率,同样通过大肠杆菌原核表达技术,针对全酶LySMP上的催化区和结合区,进行了四种不同组合的区域表达。各片段位于LySMP 1-243、1-279、2-300和151-443位氨基酸,表达产物命名为Ly1, Lys2,Lys3,Lys4。表达后产物大小分别为32kDa、36kDa、39kDa和37kDa。酶谱试验显示,Lys1和Lys2蛋白具有裂解宿主菌SS2-H的活性。而Lys3和Lys4未形成可见的透明条带。表达产物Lys1, Lys2经纯化后,浓度可达1-3mg/ml。对有较强裂解活性的区段Lys1和Lys2进行了体外生物学活性测定和条件优化。超声波处理的Lys1, Lys2粗提物的裂解谱均比原始噬菌体广,但比全酶LySMP的裂解谱窄。在17株猪链球菌2型中,7株可被Lys1和Lys2分别裂解,金黄色葡萄球菌也可被裂解,形成透明的抑菌圈。7型菌株、9型菌株、马兽疫链球菌亚种、大肠杆菌和沙门氏菌均不被裂解,不形成抑菌圈。Lys1中加入0.3%β-巯基乙醇或加入20mM半胱氨酸后酶活性最高;而Lys2中则需加入0.8%β-巯基乙醇或加入15mM半胱氨酸方可使酶活性达到最高。Lys1和Lys2的最佳作用温度均为37℃,Lys1和Lys2分别在pH5.2和pH 6.8的条件下裂解效率最高。浓度均为100mM的Lys1、Lys2和LySMP对SS2-4菌株的裂解试验结果显示,LySMP使细菌浊度下降最多,达到33.7%;其次是Lys2,下降27.2%;Lys1最低,只有8.9%。为了研究裂解酶的体内抗菌效果,选用裂解活性较强的LySMP,以斑马鱼作为模式动物,统计裂解酶处理组和细菌攻击对照组斑马鱼的存活率,从而揭示裂解酶的体内抗感染效果。试验研究了LySMP在不同感染阶段对猪链球菌HA9801的抗感染效果。将斑马鱼分为7组,每组30尾。裂解酶处理组1为在50LD50猪链球菌HA9801攻击前30min,用2μg LySMP腹腔注射斑马鱼;裂解酶处理组2为用50LD50的猪链球菌HA9801与2μg lySMP的混合物(1:1)腹腔注射斑马鱼;裂解酶处理组3为在50LD50猪链球菌HA9801攻击30min后,用2μg LySMP腹腔注射斑马鱼;抗生素处理组用2μg阿莫西林,于50LD50猪链球菌HA9801攻击后30min腹腔注射斑马鱼,计算各组斑马鱼的存活率。同时设猪链球菌HA9801攻击对照组、PBS对照组、LySMP对照组。结果表明,裂解酶处理组1、2、3均对猪链球菌有抗感染作用,斑马鱼的存活率分别可达70%、66.7%和70%,而HA9801攻击组存活率仅有6.7%。抗生素治疗组存活率可达96.7%,PBS对照组、LySMP对照组存活率均为100%,表明裂解酶对斑马鱼具有抗感染效果。为了评估LySMP对猪链球菌不同血清型、不同菌株的体内抗菌活性,选用猪链球菌2型SS2-H、SS2-3、猪链球菌9型SH040817和猪链球菌7型SH040805作为攻击菌株,分别腹腔注射斑马鱼各30尾,30min后再注射2μg LySMP,计算斑马鱼的存活率。结果显示,LySMP注射后能有效抵御猪链球菌SS2-H、SS2-3、SH040817及SH040805的攻击,存活率分别达到90%、93.3%、86.7%及60%。LySMP对SS2-H、SS2-3、SH040805有较强的抗感染效果,与攻击对照组相比,存活率分别提高了36.7%、66.6%、40%。斑马鱼感染猪链球菌后通过注射LySMP可显著提高存活率的事实提示,LySMP可作为防控猪链球菌病的一种高效抗感染制剂。猪链球菌烈性噬菌体SMP宿主谱很窄,仅可感染猪链球菌SS2-H和SS2-6形成噬斑,因而制约了噬菌体在防控猪链球菌上的应用。为了阐明噬菌体与猪链球菌相互作用的物质基础,本研究通过化学和酶法以及原生质体吸附等方法确定了噬菌体在猪链球菌上的受体成分。通过提取细菌细胞壁,将细胞壁稀释后用蛋白酶K、SDS、变溶菌素处理的方法,分别破坏蛋白或多糖成分,再与噬菌体共孵育,发现采用变溶菌素处理的细胞壁可使噬菌体的吸附率降低至23.1%;制备SS2-H原生质体,作原生质球吸附试验,结果显示噬菌体对原生质体吸附率为0。分别用甘露糖、半乳糖、核糖、鼠李糖、氨基葡萄糖、N-乙酰氨基葡萄糖和葡萄糖等组成噬菌体磷壁酸的成分进行结合阻断试验,结果表明,甘露糖、鼠李糖、氨基葡萄糖和N-乙酰氨基葡萄糖对SMP分别有32.3%、41.9%、50%和25.8%的阻断作用。在可逆性吸附试验中,分别在30min、60min和90min时,检测噬菌体对细胞壁的吸附率,结果呈现不可逆吸附,在90min时即达到完全吸附。上述结果表明,参与噬菌体SMP受体结合的细菌成分为多糖,极有可能为肽聚糖或磷壁酸,蛋白质未参与其吸附作用。SMP识别细菌细胞表面的氨基葡萄糖、N-乙酰葡萄糖胺、甘露糖及鼠李糖成分,而葡萄糖和半乳糖不参与噬菌体识别,噬菌体对受体的识别是不可逆的。更多还原

【Abstract】 Streptococcus suis is an important zoonotic pathogen that causes meningitis, sepsis, arthritis, endocarditis and pneumonia in piglet or human meningitis. It is a big threaten to public hygiene and human life. Worse more, the severe antibiotics resistance of bacteria make it difficult to the prevention and treatment of this disease. In this thesis, recombinant S. suis phage lysin, named LySMP and truncated segments Lys1, Lys2, Lys3, Lys4 were expressed and purified to eliminate the clonization of bacteria. For animal test, LySMP was chosed for anti-infectious therapy of S. suis infected zebrafish.Purified active LySMP was obtained by recombinant expression in Escherichia coli. The DNA of SMP was extracted before a pair of primers was designed according to the lysin gene, designated LySMP. After amplification by PCR, the products were then inserted into a prokaryotic expression plasmid, pET-28a(+) to obtain the recombinant plasmid pET-lys. It was introduced into E. coli BL21 competent cells and induced for expression. The expression product of about 55 kDa was obtained. The zymogram testified that it could lyse host strain SS-H. The purified product of recombinant LySMP protein was 1-3mg/ml.The in vitro characterization of LySMP was analyzed. Chromatographically purified LySMP treated with 0.8% of b-mercaptoethanol showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Plate assay showed an extensive lysin spectrum of lysin lysate than those of whole phage against bacteria investigated. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and S. suis serotype 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. LySMP exerted efficient lysis activity at 37℃, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2–68%.To investigate and choose the active lytic LySMP domains, catalyse and binding segments of LySMP together was expressed in E. coli. Four domains of LySMP at amino acid position of 1-243, 1-279, 2-300, 151-443 were expressed and the recombinant proteins were named as Lys1, Lys2, Lys3, Lys4. The resulting product were about 32kDa,36kDa,39kDa,37kDa, respectively. By zymogram, it deduced that both of Lys1 and Lys2 could lyse the host strain SS2-H. However, for Lys3 and Lys4, no band were found. The purified product of Lys1 and Lys2 protein were 1-3mg/ml.Characterization of truncated Lys1, Lys2 in vitro was tested and optimun conditions for lysis were chosed. Lys1, Lys2 lysate exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. Seven of seventeen strains of S. suis serotype 2 could be lysed, as well as S. aureus. But S. suis serotype 7 and S. suis serotype 9, S. equi ssp. zooepidemicus,E. coli and S. enterica were not affected.. Purified Lys1 showed high degrading efficiency when added 0.3% ofβ-mercaptoethanol or 20mM cysteine; while purified Lys2 showed high degrading efficiency when added 0.8% ofβ-mercaptoethanol or 15mM cysteine. Lys1 exerted efficient lysis activity at 37℃, pH 5.2 and Lys2 exerted efficient lysis activity at 37℃, pH 6.8. 100mM LySMP exerted a higher degradation activity compared to Lys1 and Lys2 under the same concentration which can obtain a reduced turbidity of 33.7% verse 27.2% and 8.9%.The in vivo anti-infective effect of LySMP was tested by using zebrafish as the infection model of S. suis serotype 2. Survival rate of lysin treated groups and S. suis challenging group were compared to investigate the anti-infective effect of LySMP. Zebrafish were fist divided into seven groups for the anti-infective test of S. suis HA9801 with 30 fish in each group. Lysin treated group 1 were inoculated with 2μg LySMP in peritoneal before challenging with 50 LD50 HA9801. In group 2, 2μg LySMP and 50LD50 HA9801 were equally mixed and inoculated to zebrafish. In group 3, 2μg LySMP was adminstered post infection, and then the survival rate was calculated. PBS, LySMP controlling groups and antibiotic group were respectively inoculated with PBS, LySMP and amoxicillin. Result showed that three lysin treated groups have anti-infective effect toward HA9801 challenging, the survival rate were 70%、66.7% and 70%. For challenging group, the survival rate was 6.7%. Survival rate of antibiotic group was 96.7%, PBS and LySMP controlling groups were 100%.Different strains of S. suis were adminstered to zebrafish to evaluate the anti-infective effect of LySMP. Two SS2 stains, SS2-H and SS2-3, one SS7 strain, SH040805, one SS9 strain, SH040817 were respectively inoculated to zebrafish and for 30min after, 2μg LySMP were again inoculated for the rescue of fish. Survival rate of four groups were 90%, 93.3%, 86.7%, 60%. Result showed that LySMP could rescue fish from high mortality rate, and LySMP could be a candidate therapy agent for S. suis disease.S. suis phage SMP has a limited host rang that can only infect SS2-H and SS2-6 strains and form plague on agar plate, which limited the use of phage. To elucidate interaction of phage and its host bacteria, chemically and enzymatically treated cell wall and extracted protoplasts were tested to confirm gradients invovled in phage adsorption. In this sturdy, SS2-H cell wall were extracted and diluted to a proper concentration. Diluted cell walls were treated with SDS, proteinase K and mutanolysin respectively. The mutanolysin treated cell wall made a reduction rate of adsorption to phages to 23.1%. SS2-H protoplasts were prepared and adsorption assay was done to find no adsorption result of protoplasts with phages. Numerous saccharides, including mannose, galactose, glucose, glucosamine, N- acetylglucosamine, rhamnose and ribose, were tested. Mannose, rhamnose, glucosamine and N- acetylglucosamine could adsorb to phage with an adsorption rate of 32.3%, 41.9%, 50% and 25.8%. In reversible adsorption assay, phages were incubated with SS2-H cell walls. At intervals of 30min, 60min, 90min, dissociated phages were separated and counted to find a gradually reduction until 100% adsorption to cell wall at 90min. It demonstrated that saccharides ingredients was the receptor of SMP instead of protein and probable ingredients were peptidoglycan or teichoic acid. Mannose, rhamnose, glucosamine and N- acetylglucosamine could be the surface binding sites of SS2-H to SMP and the binding of cell walls to SMP were irreversible.更多还原