發(fā)布時間：2018-05-22 19:13

  本文選題：無乳鏈球菌 + LuxS活性分析��； 參考：《華南農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：無乳鏈球菌(Streptococcus agalactiae)屬于B族鏈球菌(GBS),具有廣泛的宿主范圍,能感染人類、哺乳動物、爬行動物、兩棲動物和魚類等宿主。自2009年以來,在我國南方羅非魚養(yǎng)殖區(qū)呈大規(guī)模爆發(fā),病害發(fā)生的范圍較廣,羅非魚受感染率與死亡率均較高,個別發(fā)病率超過50%,發(fā)病魚死亡率超過95%,造成了嚴重的經(jīng)濟損失。該病已成為影響我國羅非魚養(yǎng)殖業(yè)最為嚴重的細菌性疾病之一,但對其病原的研究知之甚少,目前認為該病的發(fā)生是魚體健康狀況、病原菌、水質(zhì)三者相互作用的結(jié)果。雖然抗生素治療具有一定效果,但長期使用可導(dǎo)致細菌產(chǎn)生耐藥性。據(jù)報道,細菌耐藥性的產(chǎn)生以及致病作用與環(huán)境有著密切的關(guān)系,細菌密度感應(yīng)系統(tǒng)調(diào)控某些基因的表達以適應(yīng)外界環(huán)境的變化,為此本論文對無乳鏈球菌密度感應(yīng)系統(tǒng)關(guān)鍵酶-luxS進行了研究,為無乳鏈球菌致病機制的研究提供了理論依據(jù)。再者長期使用抗生素并不是最為科學(xué)的防治方法,疫苗免疫仍然是控制該病的最佳手段。由于魚體免疫方式的限制,口服疫苗必然是水產(chǎn)疫苗研究的重點方向。生物可降解PLGA納米/微米球及其衍生物作為載體用于疫苗免疫、基因治療已有大量研究報道。殼聚糖自身帶正電的特性,能夠增強與帶負電質(zhì)粒的吸附,并且增強細胞滲透的作用,因而作為口服免疫載體也被認為有潛在優(yōu)勢。主要研究內(nèi)容如下:1.羅非魚無乳鏈球菌分離鑒定。本論文從2011~2014年間從廣東省廣州市、惠州市、江門市三個地區(qū)送樣的病死或頻臨死亡的100多份羅非魚樣品中,分離無乳鏈球菌47株,通過革蘭氏染色、生化鑒定、PCR技術(shù)鑒定為無乳鏈球菌。通過分子血清型分析分離菌株均為血清Ia型。通過密碼子RSCU值分析,無乳鏈球菌在進化關(guān)系上不受宿主的限制。長期進化過程中,無乳鏈球菌是獨立的復(fù)制單位,不像病毒一樣受宿主的限制。為我國羅非魚無乳鏈球菌流行病學(xué)調(diào)查提供了基礎(chǔ)資料。2.無乳鏈球菌S-核糖基高半胱氨酸酶基因(lux S)活性分析。LuxS是密度感應(yīng)系統(tǒng)的關(guān)鍵酶編碼基因,研究luxS基因?qū)�抗生素抗性、毒力的影�?將為無乳鏈球菌致病機理提供理論基礎(chǔ)。利用pSET4S自殺載體,通過體外構(gòu)建具有上下游同源臂和氯霉素選擇標簽的重組自殺性質(zhì)粒,電轉(zhuǎn)化入無乳鏈球菌,經(jīng)同源重組,獲得了luxS基因缺失突變株,PCR鑒定luxS基因被氯霉素選擇標簽所代替。通過細菌生長曲線測定,luxS基因缺失突變株生長速度與野生株沒有明顯區(qū)別。哈維氏弧菌弧菌測定生物發(fā)光能力,發(fā)現(xiàn)缺失株喪失了生物發(fā)光能力。缺失株對諾氟沙星、頭孢拉定敏感性降低。對羅非魚攻毒試驗證實luxS缺失突變株毒力明顯降低。對上皮細胞的粘附力明顯降低。對酸耐受性明顯降低。外源添加7.4nM AI-2是回復(fù)缺失株功能的最佳濃度。3.無乳鏈球菌免疫原性蛋白的篩選。根據(jù)生物信息學(xué)預(yù)測的結(jié)果,選擇無乳鏈球菌免疫原性蛋白-表面免疫相關(guān)蛋白sip、莢膜多糖糖基轉(zhuǎn)移酶cpsE、Ⅶ型分泌系統(tǒng)分泌蛋白ESAT6作為研究對象。利用PCR分別從無乳鏈球菌血清型Ⅰa菌株ZX1中擴增編碼這3種蛋白的基因,電泳結(jié)果顯示,分別獲得的基因片段大小與預(yù)期的相符合,sip蛋白編碼基因全長1305bp,cpsE蛋白編碼基因全長450bp,ESAT6蛋白編碼基因全長294bp。利用體外克隆技術(shù),將3個蛋白分別連入原核表達載體pET32a質(zhì)粒中。采用原核表達方式,經(jīng)IPTG誘導(dǎo),3種蛋白均能在大腸桿菌中成功表達,其中sip蛋白和ESAT6蛋白以可溶性蛋白形式表達,cpsE蛋白以包涵體蛋白形式表達。3種蛋白經(jīng)純化后與鼠多克隆抗體均具有良好的免疫原性,為進一步在動物體內(nèi)進行免疫效力研究奠定了基礎(chǔ)。4.殼聚糖-PLGA包裹無乳鏈球菌口服疫苗及其免疫研究。分別構(gòu)建了真核表達重組質(zhì)粒pcDNA-sip、pcDNA-cpsE、pcDNA-sip-cpsE、pcDNA-IL8-sip-cpsE、pcDNA-sip-IL8-cpsE。殼聚糖-PLGA包裹無乳鏈球菌口服核酸疫苗的最佳工藝為2mg/mL PLGA,0.3 mg/mL殼聚糖,2 mg/mL PVA,其平均微球直徑為846.9 nm,平均Z電位為48.0 mV。以口服核酸疫苗20μg/尾、50μg/尾、100μg/尾劑量免疫羅非魚,每隔7 d采集血清,測定了口服疫苗ELISA效價,口服疫苗血清效價高于注射核酸疫苗組�？诜�疫苗血清ELISA效價最高值在21 d出現(xiàn)。每隔7 d采集各組免疫羅非魚肝、脾、腎、鰓、心臟、腸組織,測定口服疫苗在羅非魚各組織臟器的表達情況,各口服疫苗組在羅非魚均能有效表達。免疫30 d后,以2LD50(2×108 cfu/mL)劑量無乳鏈球菌攻毒羅非魚,口服疫苗免疫保護效果高于注射核酸疫苗組。其相對免疫保護率在25%~100%之間。上述研究結(jié)果表明,sip、cpsE、ESAT6蛋白具有良好的免疫原性,可以作為無乳鏈球菌新型亞單位疫苗的候選蛋白,殼聚糖-PLGA包裹口服核酸疫苗對羅非魚具有較強的免疫保護率,具有重要的臨床實踐意義。
[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.
【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S943

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