本文選題：副豬嗜血桿菌 + 穩(wěn)定表達蛋白��； 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：副豬嗜血桿菌(Haemophilus parasuis)屬于巴斯德菌科嗜血桿菌屬,是一種非溶血性的、NAD依賴的、形態(tài)多為球桿狀和棒桿狀的細小革蘭氏陰性菌。該菌是豬Glasser' s病的病原,該病以多發(fā)性漿膜炎、關(guān)節(jié)炎和腦膜炎為主要特征。副豬嗜血桿菌的感染能夠引發(fā)豬群的高致病率和死亡率,給養(yǎng)豬業(yè)造成巨大的經(jīng)濟損失。目前,已經(jīng)鑒定出了 15種不同毒力的血清型,但仍有很大一部分分離株不能分型。由于不同菌株的毒力不同以及宿主自身免疫力的差異,急性和慢性病例所表現(xiàn)的臨床癥狀也有所不同。這給副豬嗜血桿菌病的診斷和防控帶來了很大困難。本實驗室在完成了對ZJ0906(12型)副豬嗜血桿菌全基因組測序的基對上,對AH1108(4型)、JS1023(5型)和ZJ0906(12型)三株副豬嗜血桿菌進行了細菌蛋白組學(xué)分析。根據(jù)分析結(jié)果篩選出在不同血清型中穩(wěn)定表達的3種抗原,ELISA結(jié)果和動物免疫實驗證明三個穩(wěn)定表達蛋白能夠檢測出不同血清型的副豬嗜血桿菌的感染,并對各個血清型的副豬嗜血桿菌產(chǎn)生交叉保護力。具體研究如下:1副豬嗜血桿菌不同血清型穩(wěn)定表達蛋白的篩選及鑒定以12型副豬嗜血桿菌菌株提取的DNA為模板擴增了 3種穩(wěn)定表達蛋白的基因,連入pET-28a(+)表達載體,重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21感受態(tài)細胞,IPTG誘導(dǎo),表達的蛋白利用Ni-NTA親和層析樹脂進行純化。之后通過免疫小鼠制備多克隆抗體,制備的多克隆抗體經(jīng)Protein A親和層析樹脂純化。對3種血清型的副豬嗜血桿菌株進行SDS-PAGE電泳,然后轉(zhuǎn)印至NC膜,以純化小鼠多抗為一抗,通過western-blot分析3種蛋白分別在3種血清型菌株中的表達量。結(jié)果顯示:表達純化后的3種穩(wěn)定蛋白濃度高、雜蛋白含量低,免疫小鼠后的抗體水平高,western-blot顯示3種蛋白在3種血清型的副豬嗜血桿菌中表達量基本一致。這3種穩(wěn)定蛋白的表達為建立能夠檢測多種血清型的副緒嗜血桿菌ELISA方法及篩選具有交叉保護力的疫苗候選因子奠定了基礎(chǔ)。2副豬嗜血桿菌穩(wěn)定表達蛋白間接ELISA檢測方法的建立及流行病學(xué)調(diào)查以3種原核表達蛋白作為候選抗原和全菌裂解物同時包被酶標板,對臨床豬血清進行副豬嗜血桿菌抗體檢測,發(fā)現(xiàn)以蛋白manganese-dependent superoxide dismutase(MnSOD)作為包被抗原得到的結(jié)果最接近用副豬嗜血桿菌全菌裂解物進行ELISA檢測的結(jié)果,故確定以蛋白MnSOD為抗原建立間接ELISA方法。通過矩陣法確定ELISA最佳反應(yīng)條件:抗原包被濃度為1.5μg/mL,4℃過夜;5%脫脂乳37℃封閉2h;待檢血清稀釋度為1:160,37℃作用90min;酶標二抗稀釋度為1:15000,37℃作用45min;底物顯色時間為37℃6min�？贵w臨界值OD450nm≥ 0.2451判為陽性,OD450nm0.2083判為陰性,介于兩者之間為可疑。特異性和重復(fù)性試驗證明,該方法與豬繁殖與呼吸綜合征病毒、豬瘟病毒、豬偽狂犬病毒、豬圓環(huán)病毒2型、豬口蹄疫病毒、紅斑丹毒絲菌、馬鏈球菌、豬鏈球菌、腸炎沙門氏菌血清抗體無交叉反應(yīng)。重復(fù)性試驗證明批內(nèi)、批間重復(fù)性良好。建立的間接ELISA方法對114份臨床血清進行檢測,與4、5、12型全菌裂解物ELISA檢測結(jié)果比較,陽性符合率分別為80.77%、80.00%、90.00%,總陽性符合率為90.63%。用建立的ELISA方法對來自8個不同省(區(qū))的1610份豬血清進行副豬嗜血桿菌的流行病學(xué)調(diào)查,結(jié)果顯示,總陽性檢出率高達54.41%,其中江蘇地區(qū)最高,江西地區(qū)最低,且秋冬季節(jié)的陽性檢出率較高。證明該方法可用于4、5、12型副豬嗜血桿菌的抗體檢測和流行病學(xué)調(diào)查。3副豬嗜血桿菌重組亞單位疫苗候選因子的篩選豬Glasser' s病的病原體是副豬嗜血桿菌。目前,廣泛應(yīng)用商業(yè)化的死菌苗和自家苗來保護豬群。然而,副豬嗜血桿菌眾多的血清型使得疫苗的交叉保護力受到了限制。本研究選用了 3種在4、5、12型副豬嗜血桿菌中穩(wěn)定表達的蛋白作為候選因子,應(yīng)用15A佐劑乳化蛋白,采用皮下注射免疫豚鼠,每兩周免疫一次,共免疫兩次。通過ELISA方法測定豚鼠血清抗體效價。二免后第二周,用副豬嗜血桿菌不同毒力毒株AH1108(4型)、JS1023(5型)和ZJ0906(12型)進行攻毒。結(jié)果顯示,所有試驗組豚鼠的血清抗體效價均顯著高于陰性對照組。與此同時,通過SYBR Green實時熒光定量PCR對六種細胞因子進行檢測,試驗組的細胞因子水平均顯著高于陰性對照組。攻毒試驗中,蛋白CyaY對三株副豬嗜血桿菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保護率分別為60%、40%和80%;蛋白MnSOD對三株副豬嗜血桿菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保護率為40%、40%和80%;蛋白GPD對三株副豬嗜血桿菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保護率為60%、40%和80%。研究表明,3種穩(wěn)定表達的蛋白對于AH1108(4型)、JS1023(5型)和ZJ0906(12型)的感染有交叉保護力,有作為副豬嗜血桿菌重組亞單位疫苗候選因子的可行性。
[Abstract]:Haemophilus parahaemophilus (Haemophilus parasuis) belongs to the genus Haemophilus Pasteur. It is a non hemolytic and NAD dependent type of small gram-negative bacilli. The pathogen is the pathogen of porcine Glasser's disease. The disease is characterized by multiple serotis, arthritis and meningitis. Infection can cause high morbidity and mortality of pigs and cause huge economic losses to the pig industry. At present, 15 serotypes of different virulence have been identified, but there are still a large number of isolates that can not be typed. The acute and chronic cases of different strains have different virulence and the difference of the host's own immunity. The clinical symptoms are also different. This has brought great difficulties to the diagnosis and control of Haemophilus porcine. In this laboratory, the whole genome sequence of ZJ0906 (type 12) Haemophilus accessory Haemophilus porcine was sequenced. The bacterial proteomics analysis of AH1108 (type 4), JS1023 (type 5) and ZJ0906 (type 12) of Haemophilus vice was analyzed. Results the 3 antigens expressed steadily in different serotypes were screened. The results of ELISA and animal immunization showed that three stable proteins could detect the infection of Haemophilus accessory pigs with different serotypes, and the cross protective ability to Haemophilus accessory Haemophilus porcine in various serotypes. The specific study was as follows: different sera of Haemophilus porcine 1 Screening and identification of the stable expression protein, the DNA extracted from Haemophilus pariae 12 was used as a template to amplify the gene of 3 stable expression proteins. The recombinant plasmid was linked to the expression vector of pET-28a (+), the recombinant plasmid was transformed into the BL21 receptive cell of Escherichia coli, and the protein expressed by IPTG was purified by Ni-NTA affinity chromatography resin. After that, the protein was purified by Ni-NTA affinity chromatography. The polyclonal antibody was prepared by the pestilence mice. The polyclonal antibody was purified by Protein A affinity chromatography resin. 3 serotypes of Haemophilus accessory Haemophilus porcine were electrophoretic and then transferred to NC membrane to purify the mouse polyclonal antibody, and the expression of the 3 proteins in 3 serotype strains were analyzed by Western-blot. The results showed: table The purified 3 stable proteins were high in concentration, low protein content and high antibody level after immunized mice. The expression of 3 proteins in the 3 serotypes of Haemophilus accessory Haemophilus porcine was basically the same. The expression of the 3 stable proteins was the establishment of a ELISA method for the establishment of Haemophilus accessory Haemophilus accessory, which was able to detect a variety of serotypes. The vaccine candidate factor of the fork protective force laid the basis for the establishment of the indirect ELISA detection method for the stable expression protein of Haemophilus.2, and the epidemiological investigation was carried out with 3 prokaryotic expression proteins as candidate antigens and whole bacterial lysates. The result of Se-dependent superoxide dismutase (MnSOD) as the result of the antigen of the envelope was closest to the result of ELISA detection with the whole bacterial lysate of Haemophilus accessory. Therefore, the indirect ELISA method was established with protein MnSOD as antigen. The optimum reaction condition of ELISA was determined by matrix method: the concentration of antigen inclusion was 1.5 u g/mL, 4 degrees centigrade overnight; 5% skimmed milk 37 centigrade was closed for 2h; the test serum dilution was 1:160,37 C 90min, the dilution of enzyme two was 1:15000,37 C and 45min was 45min; the color time of the substrate was 37 C 6min. antibody critical value OD450nm > 0.2451 as positive, OD450nm0.2083 was negative between the two. The specificity and repeatability test proved that this method was breeding with pigs and pigs. Respiratory syndrome virus, swine fever virus, porcine pseudorabies virus, porcine circovirus 2, pig foot and mouth disease virus, erythematous erysipelas, Streptococcus equine, Streptococcus suis, sera of Salmonella enteritis without cross reaction. Repeated tests have proved that in batch, the reproducibility is good. 114 clinical sera were detected by the established indirect ELISA method, and 4, The positive coincidence rate of 5,12 type total bacterial lysate ELISA was 80.77%, 80%, 90% respectively. The total positive coincidence rate was an epidemiological survey of 1610 porcine Haemophilus porcine from 8 different provinces (regions) using the ELISA method established by 90.63%.. The results showed that the total positive rate was up to 54.41%, of which the Jiangsu region was the most It is the lowest in Jiangxi area and the positive detection rate in the autumn and winter season. It is proved that this method can be used for the antibody detection and epidemiological investigation of Haemophilus pariae 4,5,12 and the candidate factor of recombinant subunit vaccine of Haemophilus pariae.3. The pathogen of porcine Glasser's disease is Haemophilus pariae. The commercial dead bacteria are widely used at present. However, a large number of serotypes of Haemophilus parahaemophilus have restricted the cross protection of the vaccine. 3 kinds of protein, which are expressed steadily in the Haemophilus parahaemophilus 4,5,12, are selected as candidate factors. The emulsified protein of 15A adjuvant is used and immunized in guinea pigs by subcutaneous injection, once every two weeks. The titer of serum antibody of guinea pig was measured by ELISA method. After second weeks of two immunization, AH1108 (type 4), JS1023 (type 5) and ZJ0906 (type 12) of Haemophilus accessory pigs were used to attack poison. The results showed that the serum antibody titer of all guinea pigs in all test groups was significantly higher than that of negative control group. At the same time, SYBR Green was in real time. Six cytokines were detected by fluorescence quantitative PCR, and the level of cytokines in the test group was significantly higher than that in the negative control group. In the attack test, the protective rates of protein CyaY to three strains of Haemophilus accessory swine AH1108 (type 4), JS1023 (type 5) and ZJ0906 (12 type) were 60%, 40% and 80%, and protein MnSOD to three strains of Haemophilus porcine AH1108 (4), JS1023 (5). The protective rates of type 12 and ZJ0906 (type 12) were 40%, 40% and 80%; protein GPD protected three strains of Haemophilus accessory pigs, JS1023 (type 4), JS1023 (type 5) and ZJ0906 (12), and 40% and 80%. studies showed that the 40% stable proteins had cross protective effects on AH1108 (4), JS1023 (5) and ZJ0906 (12) infection, as a Haemophilus vice. The feasibility of group subunit vaccine candidate factors.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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