【作者】 江玲丽；

【导师】 方维焕；

【作者基本信息】 浙江大学 ， 预防兽医学， 2006， 博士

【摘要】 单核细胞增多性李斯特菌（简称单增李斯特菌，Lm）为重要的食源性病原菌，能引起人的败血症、脑炎、脑膜炎和胃肠炎，虽然发生率不高，但死亡率可达30％。不同来源Lm的致病性差异较大，有些菌株致病性很强，而另一些菌株毒力较弱甚至无致病性。将Lm减毒后作为携带外源基因的基因工程疫苗载体是近年来国外学者研究的热点之一。国内在Lm分离株的生物学特性、遗传多样性、基因结构与功能及其疫苗载体的研究方面鲜见报道。本研究以小鼠和鸡胚LD₅₀试验及体外培养鼠成纤维细胞的空斑形成试验比较了20株Lm食品及环境分离株的毒力。结果表明：大多数Lm分离株毒力与参考菌株10403S相当，对小鼠的LD₅₀在10^3.86-10^6.74之间，对鸡胚的LD₅₀在10^1.23-10^3.35之间，为强毒株；分离株H4对小鼠和鸡胚的LD₅₀分别为10^8.14和10^6.73，为弱毒株。空斑形成试验与LD₅₀结果基本一致，大多数单增李斯特菌分离株感染孔均有清晰的空斑形成。相对于参考菌株而言，它们的大小在83.7％-108.3％之间，而弱毒株H4感染孔无可见空斑。选取Lm的毒力基因actA，InlA和InlB高变区域外测保守区设计引物进行PCR扩增，T-A克隆至pMD18-T载体后进行测序，对20个Lm的食品及其环境分离株进行多位点序列分型。基于actA和InlA序列的分型结果基本一致，可将21个菌株分成6个亚型，与基于InlB的序列分型略有差异。综合上述3个毒力基因的分型结果可将所有菌株分成8个序列亚型，分别为S1-S8，其中菌株0194、10403S和H4各自成一亚型。用SmaⅠ对上述菌株基因组DNA进行酶切，以脉冲场凝胶电泳（PFGE）方法将21个菌株划分为6个亚型，分辨力与序列分型结果相当，弱毒株H4自成P6亚型。此外，分离自同一加工厂的海产品分离株1056，1057及其环境分离株6属于同一序列或PFGE亚型，表明该加工企业成品中的Lm来自其加工环境。消毒奶分离株320与其环境分离株690共为同一序列或PFGE亚型，表明成品奶中污染的Lm来自加工环境，而非奶牛场。为探讨Lm H4株弱毒的分子基础，对其主要毒力相关基因（iap，prfA，plcA，hly，mpl，actA，plcB，InlA和InlB）进行了测序和系统分析。体外试验表明：分离株H4与参考菌株10403S相比，具有相似的溶血活性和细胞侵袭力，但具有更强的细胞黏附力、更高的细胞内增殖水平和更强的溶脂活性。而H4株对小鼠及鸡胚的LD₅₀比菌株10403S毒力分别低2.7和4.8个对数。菌株H4与10403S及EGD的prfA、plcA和mpl具有很高同源性（＞98％）；与10403S菌株的hly基因核苷酸同源性为96.9％，氨基酸同源性为98.7％，体外溶血活性相似；与10403S菌株的plcB基因核苷酸同源性为95.4％，包含ORF第1位碱基及其上游序列-26位的突变（A-G，C-T），有可能使ORF从-27位开始形成，这一变化可能导致H4的强溶脂活性。H4株在肌动蛋白ActA的脯氨酸富集区有35个氨基酸缺失。本研究结果表明：单增李斯特菌H4株对小鼠和鸡胚的低毒力可能与actA中脯氨酸富集区的部分缺失而引起的细胞间迁移能力下降或丧失有关。为探明单增李斯特菌弱毒株H4强溶脂活性的分子基础，根据前期对plcB ORF及其上游序列分析结果，首先设计引物扩增plcB同源区（包含上游序列），定向克隆至pUC18，构建pUC18-plcB。设计3对点突变引物，以pUC18-plcB为模板进行PCR扩增，分别将plcB上游第26位（-26位）及ORF第1位双突变或-26位单突变为参考菌株10403S的相应碱基，测序确认后命名为pUC18-△plcB1、pUC18-△plcB2和pUC18-△plcB3。继而亚克隆至pKSV7，构建重组穿梭质粒pKSV7-△plcB1、pKSV7-△plcB2和pKSV7-△plcB3，并将其电转入H4株的感受态细胞中，利用同源重组技术实现目的碱基的置换，构建H4的突变株H4-△plcB1、H4-△plcB2和H4-△plcB3。目的片段的PCR扩增及序列分析表明各突变株构建成功。在含5％的卵黄琼脂平板中，各突变株均无溶脂活性，表明H4菌株强溶脂活性由plcB上游第26位碱基的突变（C-T）、致其ORF从-27位开始形成有关，比参考菌株10403S多9个氨基酸。这一突变对H4株plcB基因表达的增强作用机制还有待进一步探索。另外，小鼠LD₅₀试验表明突变株H4-△plcB1毒力比其亲本菌株H4提高1个Log，初步表明H4菌株中两个膜裂解相关基因hly与plcB的高效表达及协同作用可能增强了细菌对宿主细胞的毒性，并暴露于宿主的免疫系统而被清除，从而降低了对宿主的致病性。单增李斯特菌为侵袭性胞内菌，可在专职和非专职吞噬细胞内存活并繁殖，是目前疫苗载体研究的热点之一。以绿色荧光蛋白基因gfp为模式外源基因，以单增李斯特菌毒力基因hly为靶基因，通过基因切割-重叠延伸PCR法及同源重组技术构建表达GFP的重组单增李斯特菌。首先构建了pKSV7质粒衍生体，此质粒含有hly的启动子和信号肽序列，gfp片段及hly的同源区，通过电穿孔法把该质粒转入野生型Lm 10403S株，经两次等位基因交换将gfp片段整合入Lm染色体基因组中。目的片段的PCR、序列测定及该重组菌在显微镜下发出的绿色荧光表明该重组子的构建成功。溶血试验表明hly基因中gfp的插入后溶血活性消失，对Hela细胞的黏附和侵袭力降低。此外，小鼠及鸡胚的LD₅₀试验表明该重组菌比其亲本菌株毒力下降了2个Log。以新城疫病毒（NDV）编码融合蛋白的截短片段Fa为外源基因，通过基因切割-重叠延伸PCR法将其插入到单增李斯特菌毒力基因plcB信号肽序列下游，定向克隆至穿梭质粒pKSV7，并电转入单增李斯特菌10403S，通过同源重组技术构建携带NDV Fa片段的重组单增李斯特菌。PCR扩增结果表明重组菌Lm-△plcB-Fa构建成功。RT-PCR结果表明整合的外源基因能在重组菌中转录，SDS-PAGE及Western blot分析表明融合蛋白Fa能在重组菌中表达，与NDV阳性血清具有免疫反应性。毒力试验表明重组菌对小鼠与鸡胚的毒力降低，LD₅₀比其亲本菌株下降1.7-2.3个Log。重组菌Lm-△plcB-Fa对细胞的黏附和侵袭力均低于亲本株10403S（P＜0.05）。然而，重组菌口服或腹腔免疫SPF鸡后，均未能有效诱导针对NDV和融合蛋白Fa的特异性抗体，也未能保护NDV强毒的攻击。其可能原因是重组单增李斯特菌在鸡体内持续存在时间短、增殖率低，进而导致外源基因的低水平表达。本试验结果为深入探索单增李斯特菌的致病机理、对食品污染的控制和基因工程疫苗载体的研究奠定了良好基础。更多还原

【Abstract】 Listeria monocytogenes is an important food-borne pathogen that can cause septicaemia, encephalitis, meningitis and gastroenteritis in humans. L. monocytogenes encompasses a diversity of strains with varying pathogenic potential. While many L. monocytogenes strains could be of high pathogenicity, others are less virulent or even avirulent and produce little harm in the host. Recombinant bacterial vaccine vectors using L. monocytogenes as the model strain is one of the heated research areas in genetically engineered vaccine development. Currently, there are few studies in China on the biodiversity of L. monocytogenes, control strategies on its contamination along the food-processing lines, its gene structure and functions as well as its utilization as vaccine vectors upon proper attenuation.Mouse or chicken embryo based 50% lethal dose assays (LD₅₀) and plaque forming assay in cell monolayers were used to compare the virulence of twenty L. monocytogenes food and environment isolates. Most L. monocytogenes isolates were as virulent as the reference strain 10403S with the mouse LD₅₀ varying from 10^3.86 to 10^6.74 and the chicken embryonated egg LD₅₀ from 10^1.23 to 10^3.35. The L monocytogenes isolate H4 was of low pathogenicity as revealed by its LD₅₀ of 10^8.14 in mice and 10^6.73 in embryoniate chicken eggs. Most L. monocytogenes isolates formed clear plaques with plaque size varying from 83.7% to 108.3% relative to the reference strain 10403S. There was no visible plaque formed in isolate H4 infected cell monolayers, which was consistent with its low virulence as described above.Twenty L. monocytogenes strains isolated from food and environment sources together with the reference strain 10403S were characterized by pulsed-field gel electrophoresis （PFGE） using Sma I for genomic DNA digestion as well as by multilocus sequence typing （MLST） based on the hypervariable region of actA, InlA and InlB. All L. monocytogenes isolates could be divided into six subgroups using actA or InlA-based sequence typing, while the InlB -based sequence typing exhibited patterns slightly different from those of actA ox InlA. All L. monocytogenes isolates could be grouped into subtypes from S1 to S8 when the above three genes were considered together, with strains 0194, 10403S and H4 belonging to an individual subgroup. With PFGE, there were six subtypes of all L. monocytogenes isolates similar to MLST, with the low pathogenicity isolate H4 forming a separated subtype. Moreover, isolates 1056, 1057 and 6 exhibited the same sequence or PFGE subtype, indicating that the isolates 1056 and 1057 from the seafood products were contaminated by the environmental isolate 6. Isolates 320 and 690 shared the same sequence or PFGE subtype, illustrating that the isolate 320 from the milk product was contaminated by the environmental strain 690 other than from the dairy farm.A low pathogenicity isolate of Listeria monocytogenes from milk, as revealed in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Its corresponding virulence genes （iap, prfA, plcA, hly, mpl, actA, plcB, InlA and InlB） were compared with the reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S with LD₅₀ 10^8.14 vs 10^5.49 in mice and 10^6.73 vs 10^1.9 in chicken embryos). The genes prfA, plcA and mpl were generally homologous among L. monocytogens strains H4, 10403S and EGD （>98%）. The gene hly between strain 10403S and isolate H4 had 96.9% identity at the nucleotide level, but 98.7% at the amino acid level. The nucleotide identity of plcB between strains 10403S and H4 was 95.4%, including one important mutation from A to G at position 1 of the plcB ORF and another important mutation from C to T at position -26. The act A gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acids deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.A pair of primers was designed to amplify the plcB homologous region containing its upstream sequence according to plcB sequence from the Listeria monocytogenes milk isolate H4. The plcB fragment was then purified and cloned into pUC18 to construct the recombinant plasmid pUC18-plcB. Three pairs of primers were then designed to introduce mutations of the plcB gene at positions -26 and +1 using pUC18-plcB as the template such that G mutated to A at +1 and T to C at -26 or single mutation from T to C at -26. The mutated plasmids confirmed by sequencing were subcloned into the shuttle vector pKSV7 to construct recombinant plasmids pKSV7-ΔplcB1, pKSV7-ΔplcB2 and pKSV7-ΔplcB3. After electroporation of three recombinant shuttle vectors into competent L. monocytogenes H4 cells, homologous recombination was initiated, resulting in replacement of the target nucleotide（s）. PCR amplification of the target genes and sequencing confirmed successful construction of the mutated strains H4-ΔplcBl, H4-ΔplcB2 and H4-ΔplcB3. There was no phospholipase activity of three mutants as revealed by the egg yolk agar assay, indicating that high phospholipase activity of isolate H4 might be related to the mutation at position -26 （from C to T） with its ORF starting from position -27 relative to that in strain 10403S together with 9 extra amino acids. However, mechanisms of apparent expression of plcB due to extension of amino acids at the N terminus remain further exploration. The mutant strain H4-ΔplcB1 had increased virulence to mice of about 1 log in LD₅₀ as compared with the wild parent isolate H4, indicating that high expression of both membrane damaging genes plcB and hly might result in synergistic cytotoxic activity, exposing listerial cells to the host immune response with their resultant elimination and reduced pathogenicity to the host.L. monocytogenes is an invasive intracellular bacterium that can survive and replicate in both professional and nonprofessional phagocytic cells, and becomes an attractive vaccine vector. To construct a recombinant strain of L, monocytogenes for expression of heterologous genes, homologous recombination was utilized for insertional mutation targeting its listeriolysin O gene （hly）. The gene encoding green fluorescent protein （GFP） was used as the indicator of heterologous gene expression. The gene gfp was inserted into hly downstream to its promoter and signal sequence by overlapping extension polymerase chain reaction, which was then cloned into the shuttle plasmid pKSV7 for allelic exchange with L. monocytogenes chromosome. Homologous recombination was achieved by growing the electro-transformed L. monocytogenes cells on chloramphenicol plates at non-permissive temperature. Sequencing analysis indicated correct insertion of the target gene in-frame with the signal sequence. The recombinant strain expressed GFP constitutively as revealed by fluorescence microscopy. The mutant strain L. monocytogenesΔhly-gfp lost its hemolytic activity as visualized on the blood agar or analyzed with the culture supernatant samples and also exhibited reduced adherence and invasiveness ability to HeLa cells. Such insertional mutation resulted in reduced virulence of about 2 logs less than its parent strain 10403S as shown by the 50% lethal dose assays in the mouse and chicken embryonated egg models.Recombinant Listeria monocyogenes mutant carrying the truncated fragment Fa of the Newcastle disease virus （NDV） fusion protein gene was constructed by homologous recombination. The fragment Fa was integrated into plcB downstream of its signal sequence. Correct orientation of the inserted fragment was verified by PCR amplification. The inserted Fa was transcribed and expressed in the recombinant Lm-ΔplcB-Fa as shown by RT-PCR, SDS-PAGE and western blot respectively. The recombinant mutant exhibited reduced virulence to embryonated eggs and mice by about 1.7-2.3 logs, as compared with the parent wild strain 10403S. It was also less adherent or invasive than strain 10403S （P<0.05）. Chickens receiving the recombinant strain Lm-ΔplcB-Fa orally or intraperitoneally were partially protected from virulent NDV challenge possibly due to enhancement of non-specific immunity because the antibody titers against the homologous virus strain or the recombinant truncated fusion protein were marginal. Further research is needed in other animal models to see if the low antibody response results from insufficient expression of the heterologous genes as a result of failure of L. monocytogenes or its recombinants to persist or replicate in chickens.In summary, results from these studies have laid good foundation for further exploration of the pathogenesis of L. monocytogenes, control of its contamination along the food-processing lines as well as its potential as vaccine vectors for other passenger antigens.更多还原