罗非鱼无乳链球菌luxS功能鉴定及口服疫苗的研制

发布时间：2018-05-22 19:13

本文选题：无乳链球菌 + LuxS活性分析　；参考：《华南农业大学》2016年博士论文

【摘要】：无乳链球菌(Streptococcus agalactiae)属于B族链球菌(GBS),具有广泛的宿主范围,能感染人类、哺乳动物、爬行动物、两栖动物和鱼类等宿主。自2009年以来,在我国南方罗非鱼养殖区呈大规模爆发,病害发生的范围较广,罗非鱼受感染率与死亡率均较高,个别发病率超过50%,发病鱼死亡率超过95%,造成了严重的经济损失。该病已成为影响我国罗非鱼养殖业最为严重的细菌性疾病之一,但对其病原的研究知之甚少,目前认为该病的发生是鱼体健康状况、病原菌、水质三者相互作用的结果。虽然抗生素治疗具有一定效果,但长期使用可导致细菌产生耐药性。据报道,细菌耐药性的产生以及致病作用与环境有着密切的关系,细菌密度感应系统调控某些基因的表达以适应外界环境的变化,为此本论文对无乳链球菌密度感应系统关键酶-luxS进行了研究,为无乳链球菌致病机制的研究提供了理论依据。再者长期使用抗生素并不是最为科学的防治方法,疫苗免疫仍然是控制该病的最佳手段。由于鱼体免疫方式的限制,口服疫苗必然是水产疫苗研究的重点方向。生物可降解PLGA纳米/微米球及其衍生物作为载体用于疫苗免疫、基因治疗已有大量研究报道。壳聚糖自身带正电的特性,能够增强与带负电质粒的吸附,并且增强细胞渗透的作用,因而作为口服免疫载体也被认为有潜在优势。主要研究内容如下:1.罗非鱼无乳链球菌分离鉴定。本论文从2011~2014年间从广东省广州市、惠州市、江门市三个地区送样的病死或频临死亡的100多份罗非鱼样品中,分离无乳链球菌47株,通过革兰氏染色、生化鉴定、PCR技术鉴定为无乳链球菌。通过分子血清型分析分离菌株均为血清Ia型。通过密码子RSCU值分析,无乳链球菌在进化关系上不受宿主的限制。长期进化过程中,无乳链球菌是独立的复制单位,不像病毒一样受宿主的限制。为我国罗非鱼无乳链球菌流行病学调查提供了基础资料。2.无乳链球菌S-核糖基高半胱氨酸酶基因(lux S)活性分析。LuxS是密度感应系统的关键酶编码基因,研究luxS基因对抗生素抗性、毒力的影响,将为无乳链球菌致病机理提供理论基础。利用pSET4S自杀载体,通过体外构建具有上下游同源臂和氯霉素选择标签的重组自杀性质粒,电转化入无乳链球菌,经同源重组,获得了luxS基因缺失突变株,PCR鉴定luxS基因被氯霉素选择标签所代替。通过细菌生长曲线测定,luxS基因缺失突变株生长速度与野生株没有明显区别。哈维氏弧菌弧菌测定生物发光能力,发现缺失株丧失了生物发光能力。缺失株对诺氟沙星、头孢拉定敏感性降低。对罗非鱼攻毒试验证实luxS缺失突变株毒力明显降低。对上皮细胞的粘附力明显降低。对酸耐受性明显降低。外源添加7.4nM AI-2是回复缺失株功能的最佳浓度。3.无乳链球菌免疫原性蛋白的筛选。根据生物信息学预测的结果,选择无乳链球菌免疫原性蛋白-表面免疫相关蛋白sip、荚膜多糖糖基转移酶cpsE、Ⅶ型分泌系统分泌蛋白ESAT6作为研究对象。利用PCR分别从无乳链球菌血清型Ⅰa菌株ZX1中扩增编码这3种蛋白的基因,电泳结果显示,分别获得的基因片段大小与预期的相符合,sip蛋白编码基因全长1305bp,cpsE蛋白编码基因全长450bp,ESAT6蛋白编码基因全长294bp。利用体外克隆技术,将3个蛋白分别连入原核表达载体pET32a质粒中。采用原核表达方式,经IPTG诱导,3种蛋白均能在大肠杆菌中成功表达,其中sip蛋白和ESAT6蛋白以可溶性蛋白形式表达,cpsE蛋白以包涵体蛋白形式表达。3种蛋白经纯化后与鼠多克隆抗体均具有良好的免疫原性,为进一步在动物体内进行免疫效力研究奠定了基础。4.壳聚糖-PLGA包裹无乳链球菌口服疫苗及其免疫研究。分别构建了真核表达重组质粒pcDNA-sip、pcDNA-cpsE、pcDNA-sip-cpsE、pcDNA-IL8-sip-cpsE、pcDNA-sip-IL8-cpsE。壳聚糖-PLGA包裹无乳链球菌口服核酸疫苗的最佳工艺为2mg/mL PLGA,0.3 mg/mL壳聚糖,2 mg/mL PVA,其平均微球直径为846.9 nm,平均Z电位为48.0 mV。以口服核酸疫苗20μg/尾、50μg/尾、100μg/尾剂量免疫罗非鱼,每隔7 d采集血清,测定了口服疫苗ELISA效价,口服疫苗血清效价高于注射核酸疫苗组。口服疫苗血清ELISA效价最高值在21 d出现。每隔7 d采集各组免疫罗非鱼肝、脾、肾、鳃、心脏、肠组织,测定口服疫苗在罗非鱼各组织脏器的表达情况,各口服疫苗组在罗非鱼均能有效表达。免疫30 d后,以2LD50(2×108 cfu/mL)剂量无乳链球菌攻毒罗非鱼,口服疫苗免疫保护效果高于注射核酸疫苗组。其相对免疫保护率在25%~100%之间。上述研究结果表明,sip、cpsE、ESAT6蛋白具有良好的免疫原性,可以作为无乳链球菌新型亚单位疫苗的候选蛋白,壳聚糖-PLGA包裹口服核酸疫苗对罗非鱼具有较强的免疫保护率,具有重要的临床实践意义。
[Abstract]:Streptococcus agalactiae, which belongs to B Streptococcus (GBS), has extensive host range and can infect humans, mammals, reptiles, amphibians and fish and other hosts. Since 2009, a large-scale outbreak of tilapia in the south of China, the wide range of disease occurrence, the infection rate and mortality rate of tilapia. The disease has become one of the most serious bacterial diseases that affect the breeding industry of tilapia, but the disease has become one of the most serious bacterial diseases that affect the breeding industry of tilapia. However, little is known about its pathogen. At present, the disease is considered to be the health of the fish body, the pathogen and the water quality of three people interact with each other. The results. Although antibiotic treatment has a certain effect, long-term use can lead to bacterial resistance. It is reported that the production of bacterial resistance and the pathogenic effect are closely related to the environment. The bacterial density induction system regulates the expression of certain genes to adapt to the changes in the environment. Therefore, the density of Streptococcus lactis has been studied in this paper. The key enzyme -luxS of the induction system has been studied, which provides a theoretical basis for the study of the pathogenic mechanism of Streptococcus lactis. Moreover, the long-term use of antibiotics is not the most scientific method of prevention and control. Vaccine immunization is still the best means to control the disease. Due to the restriction of the fish body immunity, oral vaccine is the key point of the study of aquatic vaccine. The biodegradable PLGA nano / micron spheres and their derivatives are used as carriers for vaccine immunization. Gene therapy has been widely reported. The characteristics of chitosan itself with positive electricity can enhance the adsorption of negative electric plasmids and enhance the effect of cell infiltration. Therefore, as an oral immune carrier, it is also considered to have potential advantages. The research contents are as follows: 1. the isolation and identification of Streptococcus lactis (1.). In this paper, from three regions of Guangzhou, Huizhou, Jiangmen, Guangdong Province, more than 100 samples of tilapia without Streptococcus were isolated from three regions of Huizhou, Jiangmen City, and 47 strains of Streptococcus Lactococcus were isolated, and through Gram staining, biochemical identification and PCR technique identification of Streptococcus nactis. The isolates were all serotype I a type a. Through the RSCU value of codon, Streptococcus free streptococcus was not restricted by the host. In the long course of evolution, Streptococcus lactis was an independent replicating unit, unlike the virus, which was not limited by the host. Basic data.2. activity analysis of S- ribonucleic homocysteine (Lux S) gene of Streptococcus lactis (Lux S) is a key enzyme encoding gene in the density induction system. The study of the effect of luxS gene on antibiotic resistance and virulence will provide a theoretical basis for the pathogenesis of Streptococcus free Streptococcus. The pSET4S suicide vector is used to build up the upstream and downstream in vitro. The recombinant human arm and chloramphenicol selected recombinant suicidal particles were converted into Streptococcus lactis without Streptococcus, and the luxS gene deletion mutant was obtained by homologous recombination. The PCR identification luxS gene was replaced by the chloramphenicol selection label. The growth rate of the luxS gene deletion mutant was not significantly different from that of the wild strain by the bacterial growth curve. Vibrio bacteria detected the bioluminescence ability and found that the missing strains lost their bioluminescence ability. The missing strains were less sensitive to norfloxacin and Cefradine. The toxicity of luxS missing mutant strain was obviously reduced. The adhesion force to epithelial cells was significantly reduced. The tolerance to acid was significantly reduced. Exogenous 7.4nM AI-2 was a response to it. The optimum concentration of the function of the deletion strain.3. was screened by Streptococcus lactis immunogenic protein. According to the results of bioinformatics prediction, the immunogenic protein of Streptococcus lactis immunogenic protein sip, the capsular polysaccharide glycosyl transferase cpsE, and the secretory protein ESAT6 of the VII type secretory system were selected as the research object. PCR was used as the non milk Streptococcus. The gene of these 3 proteins was amplified in the bacterial serotype I a strain ZX1. The electrophoresis results showed that the size of the gene fragment was in accordance with the expectation, the total length of the SIP protein encoding gene was 1305bp, the cpsE protein encoding gene was 450bp, and the ESAT6 protein encoding gene was full length 294bp. in vitro cloning technology, and the 3 proteins were connected to the prokaryotic table respectively. In the vector pET32a plasmid, the 3 proteins can be expressed successfully in Escherichia coli by using prokaryotic expression and IPTG. The SIP protein and ESAT6 protein are expressed in the form of soluble protein. The cpsE protein expresses.3 protein in the form of inclusion body protein and has good immunogenicity. The study of immunization in animals laid a foundation for the basic.4. chitosan -PLGA encapsulated Streptococcus lactis oral vaccine and its immunological study. The best recombinant plasmid pcDNA-sip, pcDNA-cpsE, pcDNA-sip-cpsE, pcDNA-IL8-sip-cpsE, and pcDNA-sip-IL8-cpsE. chitosan -PLGA encapsulated the best oral nucleic acid vaccine of Streptococcus free Streptococcus were constructed. The process was 2mg/mL PLGA, 0.3 mg/mL chitosan and 2 mg/mL PVA with the average microsphere diameter of 846.9 nm, the average Z potential was 48 mV., and the oral nucleic acid vaccine 20 micron tail, 50 mu g/ tail and 100 mu g/ tail dose immunized the tilapia. The serum titer of oral vaccine was measured every 7 D, and the oral vaccine serum titer was higher than the injection nucleic acid vaccine group. Oral vaccine was higher than the injection nucleic acid vaccine group. Oral vaccine was higher than the injection nucleic acid vaccine group. Oral vaccination was higher than that of the injection nucleic acid vaccine group. Oral vaccine was higher than the injection of nucleic acid vaccine. Oral vaccination was higher than the oral vaccine. The highest value of ELISA potency appeared at 21 d. Each group was immunized with the liver, spleen, kidney, gill, gill, heart and intestinal tissue every 7 d, and the oral vaccine was expressed in the organs of the tilapia. The oral vaccine group was effectively expressed in the tilapia. After immunization 30 d, the dose of Streptococcus lactis (2 x 108 cfu/mL) was used to attack the tilapia. The immune protection effect of the vaccine was higher than that of the injected nucleic acid vaccine group. The relative immune protection rate was between 25%~100%. The results showed that SIP, cpsE, ESAT6 protein had good immunogenicity, and could be used as a candidate protein for the new subunit vaccine of Streptococcus nactis. The oral nucleic acid vaccine of chitosan -PLGA was stronger for tilapia. The rate of immune protection is of great significance in clinical practice.
【学位授予单位】：华南农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S943

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