副猪嗜血杆菌重组蛋白ELISA检测方法的建立及亚单位疫苗候选因子的筛选

发布时间：2018-05-26 10:47

本文选题：副猪嗜血杆菌 + 稳定表达蛋白　；参考：《南京农业大学》2015年硕士论文

【摘要】：副猪嗜血杆菌(Haemophilus parasuis)属于巴斯德菌科嗜血杆菌属,是一种非溶血性的、NAD依赖的、形态多为球杆状和棒杆状的细小革兰氏阴性菌。该菌是猪Glasser' s病的病原,该病以多发性浆膜炎、关节炎和脑膜炎为主要特征。副猪嗜血杆菌的感染能够引发猪群的高致病率和死亡率,给养猪业造成巨大的经济损失。目前,已经鉴定出了 15种不同毒力的血清型,但仍有很大一部分分离株不能分型。由于不同菌株的毒力不同以及宿主自身免疫力的差异,急性和慢性病例所表现的临床症状也有所不同。这给副猪嗜血杆菌病的诊断和防控带来了很大困难。本实验室在完成了对ZJ0906(12型)副猪嗜血杆菌全基因组测序的基对上,对AH1108(4型)、JS1023(5型)和ZJ0906(12型)三株副猪嗜血杆菌进行了细菌蛋白组学分析。根据分析结果筛选出在不同血清型中稳定表达的3种抗原,ELISA结果和动物免疫实验证明三个稳定表达蛋白能够检测出不同血清型的副猪嗜血杆菌的感染,并对各个血清型的副猪嗜血杆菌产生交叉保护力。具体研究如下:1副猪嗜血杆菌不同血清型稳定表达蛋白的筛选及鉴定以12型副猪嗜血杆菌菌株提取的DNA为模板扩增了 3种稳定表达蛋白的基因,连入pET-28a(+)表达载体,重组质粒转化大肠杆菌BL21感受态细胞,IPTG诱导,表达的蛋白利用Ni-NTA亲和层析树脂进行纯化。之后通过免疫小鼠制备多克隆抗体,制备的多克隆抗体经Protein A亲和层析树脂纯化。对3种血清型的副猪嗜血杆菌株进行SDS-PAGE电泳,然后转印至NC膜,以纯化小鼠多抗为一抗,通过western-blot分析3种蛋白分别在3种血清型菌株中的表达量。结果显示:表达纯化后的3种稳定蛋白浓度高、杂蛋白含量低,免疫小鼠后的抗体水平高,western-blot显示3种蛋白在3种血清型的副猪嗜血杆菌中表达量基本一致。这3种稳定蛋白的表达为建立能够检测多种血清型的副绪嗜血杆菌ELISA方法及筛选具有交叉保护力的疫苗候选因子奠定了基础。2副猪嗜血杆菌稳定表达蛋白间接ELISA检测方法的建立及流行病学调查以3种原核表达蛋白作为候选抗原和全菌裂解物同时包被酶标板,对临床猪血清进行副猪嗜血杆菌抗体检测,发现以蛋白manganese-dependent superoxide dismutase(MnSOD)作为包被抗原得到的结果最接近用副猪嗜血杆菌全菌裂解物进行ELISA检测的结果,故确定以蛋白MnSOD为抗原建立间接ELISA方法。通过矩阵法确定ELISA最佳反应条件:抗原包被浓度为1.5μg/mL,4℃过夜;5%脱脂乳37℃封闭2h;待检血清稀释度为1:160,37℃作用90min;酶标二抗稀释度为1:15000,37℃作用45min;底物显色时间为37℃6min。抗体临界值OD450nm≥ 0.2451判为阳性,OD450nm0.2083判为阴性,介于两者之间为可疑。特异性和重复性试验证明,该方法与猪繁殖与呼吸综合征病毒、猪瘟病毒、猪伪狂犬病毒、猪圆环病毒2型、猪口蹄疫病毒、红斑丹毒丝菌、马链球菌、猪链球菌、肠炎沙门氏菌血清抗体无交叉反应。重复性试验证明批内、批间重复性良好。建立的间接ELISA方法对114份临床血清进行检测,与4、5、12型全菌裂解物ELISA检测结果比较,阳性符合率分别为80.77%、80.00%、90.00%,总阳性符合率为90.63%。用建立的ELISA方法对来自8个不同省(区)的1610份猪血清进行副猪嗜血杆菌的流行病学调查,结果显示,总阳性检出率高达54.41%,其中江苏地区最高,江西地区最低,且秋冬季节的阳性检出率较高。证明该方法可用于4、5、12型副猪嗜血杆菌的抗体检测和流行病学调查。3副猪嗜血杆菌重组亚单位疫苗候选因子的筛选猪Glasser' s病的病原体是副猪嗜血杆菌。目前,广泛应用商业化的死菌苗和自家苗来保护猪群。然而,副猪嗜血杆菌众多的血清型使得疫苗的交叉保护力受到了限制。本研究选用了 3种在4、5、12型副猪嗜血杆菌中稳定表达的蛋白作为候选因子,应用15A佐剂乳化蛋白,采用皮下注射免疫豚鼠,每两周免疫一次,共免疫两次。通过ELISA方法测定豚鼠血清抗体效价。二免后第二周,用副猪嗜血杆菌不同毒力毒株AH1108(4型)、JS1023(5型)和ZJ0906(12型)进行攻毒。结果显示,所有试验组豚鼠的血清抗体效价均显著高于阴性对照组。与此同时,通过SYBR Green实时荧光定量PCR对六种细胞因子进行检测,试验组的细胞因子水平均显著高于阴性对照组。攻毒试验中,蛋白CyaY对三株副猪嗜血杆菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保护率分别为60%、40%和80%;蛋白MnSOD对三株副猪嗜血杆菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保护率为40%、40%和80%;蛋白GPD对三株副猪嗜血杆菌AH1108(4型)、JS1023(5型)和ZJ0906(12型)的保护率为60%、40%和80%。研究表明,3种稳定表达的蛋白对于AH1108(4型)、JS1023(5型)和ZJ0906(12型)的感染有交叉保护力,有作为副猪嗜血杆菌重组亚单位疫苗候选因子的可行性。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

【参考文献】