泰山松花粉多糖对毕赤酵母表达的禽波氏杆菌OmpA的免疫增强效果研究

发布时间：2018-01-02 12:42

本文关键词：泰山松花粉多糖对毕赤酵母表达的禽波氏杆菌OmpA的免疫增强效果研究　出处：《山东农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：禽波氏杆菌病是由禽波氏杆菌(Bordetella avium,B.avium)引起的一种家禽上呼吸道性传染性疾病,既可水平传播,也可垂直传播,主要导致死胚率升高,孵化率降低和雏鸡的急性死亡。患病鸡生长迟缓、饲料报酬低,给养禽业造成了巨大的经济损失。有研究表明,本菌还是人体的一种机会致病菌。目前,由于药物残留的影响以及禽波氏杆菌耐药性的形成,通过抗生素治疗本病的难度越来越大。关于禽波氏杆菌病的预防,国外仅对火鸡波氏杆菌病疫苗开展了研究。目前用于预防火鸡波氏杆菌病的疫苗包括温度敏感变异株活菌苗和全细胞菌素,但这两种疫苗只能适当降低病变的严重程度以及延缓临床症状的出现,并不能从根本上防止本病的传播。因此急需开辟新的途径,研发新型疫苗。佐剂可以激活机体的先天免疫系统进而增强机体对特异性抗原的适应性免疫,从而使机体产生高效持久的免疫反应,因此,疫苗佐剂在免疫过程中是不可或缺的。本实验室前期的研究表明,泰山松花粉多糖(Taishan Pinus massoniana pollen polysaccharide,TPPPS)作为灭活疫苗和亚单位疫苗佐剂具有良好的免疫增强效果,但其对真核表达的外膜蛋白A(outer membrane protein A,ompA)的免疫效果研究未见报道。因此,本研究以毕赤酵母表达系统为基础,分泌表达重组的禽波氏杆菌ompA,采用TPPPS为疫苗佐剂,并评估其对重组禽波氏杆菌ompA亚单位疫苗的免疫增强效果。根据GenBank中禽波氏杆菌ompA基因序列设计一对引物,以实验室分离得到的禽波氏杆菌DNA为模板,PCR扩增目的基因。将PCR扩增产物连接到pMD18-T克隆载体上,经测序验证正确后,重组克隆载体进行EcoR I和Not I双酶切,胶回收产物连接到相同酶切的表达载体pPIC9上,重组表达载体线性化后转化到毕赤酵母GS115感受态细胞中,RDB板筛选禽波氏杆菌ompA毕赤酵母阳性转化子,PCR鉴定并进行基因测序。同时,转化空质粒pPIC9并筛选毕赤酵母转化子作为阴性对照。采用甲醇诱导法诱导禽波氏杆菌ompA毕赤酵母阳性转化子分泌表达ompA,同时对空质粒毕赤酵母转化子进行诱导表达,作为阴性对照。于诱导后24、48、72、96 h分别离心收集上清,通过SDS-PAGE和Western blot分析进行检测。经鉴定,在SDS-PAGE凝胶中有一条大小为21 kDa的条带,其分子量大小与目的蛋白分子量大小相一致,而阴性对照中并没有该蛋白条带。镍柱纯化的蛋白经SDS-PAGE分析只检测到一条大小为21 kDa蛋白条带。Western blot分析中也出现大小为21 kDa的特异性条带,与SDS-PAGE检测结果相一致。动物试验中,将180只SPF鸡随机分为6组,3日龄首免,分别颈部皮下接种0.2 m L20、40、60 mg/m L TPPPS佐剂ompA亚单位疫苗,不完全弗氏佐剂ompA亚单位疫苗,单纯的ompA亚单位疫苗和PBS,于首免后7和14 d分别进行加强免疫。首免后3、7、14、21、28、35、42、49 d,每组随机取3只鸡采血,并检测其血清抗体效价,血清白细胞介素-4(IL-4)浓度,外周血CD4+和CD8+T淋巴细胞百分含量,T淋巴细胞转化率。三免后1周,每组(除对照组)随机取20只鸡进行动物保护试验,连续7 d观察记录临床症状和存活率。结果表明,单纯的ompA亚单位疫苗能有效诱导机体产生抗-ompA抗体,分泌IL-4,提高CD4+T淋巴细胞百分含量和T淋巴细胞转化率,并能提供71.67%的保护率。此外,TPPPS佐剂疫苗能显著提高机体免疫反应水平,且60 mg/m L TPPPS佐剂疫苗效果最好。研究表明,含TPPPS的重组禽波氏杆菌ompA亚单位疫苗具有广阔的免疫调理应用前景,为禽波氏杆菌亚单位疫苗研究提供研究基础。
[Abstract]:B.avium disease is caused by Bordetella avium (Bordetella avium, B.avium) caused a poultry respiratory infectious disease, which can also be horizontal transmission, vertical transmission, mainly caused the death rate of embryos increased, the hatching rate and reduce the acute death of chickens. The prevalence of chicken growth retardation, low feed conversion, the poultry industry has caused tremendous economic losses. Studies have shown that a chance of the bacteria or human pathogenic bacteria. At present, due to the influence of drug residues and drug resistance of Salmonella avian wave, through antibiotic treatment of this disease has become more and more difficult. The prevention of b.avium disease, only to Turkey abroad bordetellosis vaccine research. At present for the prevention of Turkey bordetellosis vaccine includes a temperature sensitive mutant vaccine and whole cell bacteria, but these two vaccines can only be appropriate to reduce the severity of the disease and delay Clinical symptoms, and can prevent the spread of the disease from the root. So there is an urgent need to open up new avenues of research and development of new vaccines. The innate immune system can activate the body's adjuvant and enhance adaptive immune responses to specific antigen, which causes immune responses, efficient and durable so, is indispensable in vaccine adjuvant in the immune process. Our previous study showed that Taishan pine pollen polysaccharide (Taishan Pinus massoniana pollen polysaccharide, TPPPS) as inactivated vaccine and subunit vaccine immune adjuvant has good reinforcing effect, but its eukaryotic expression of outer membrane protein A (outer membrane protein A, ompA) on the immune effect has not been reported. Therefore, in this study, Pichia pastoris expression system as the foundation, the expression and secretion of recombinant b.avium ompA, using TPPPS as a vaccine adjuvant, and evaluate its effect on restructuring The immune enhancement of b.avium vaccine of ompA. A pair of primers designed according to the GenBank of b.avium ompA gene sequence in laboratory isolated b.avium DNA as template, PCR amplification of the target gene. The PCR product was ligated into pMD18-T cloning vector and sequenced correctly, recombinant EcoR I and Not cloning vector I digested expression vector pPIC9 gel recovery products connected to the same enzyme digestion, the recombinant expression vector was linearized and transformed into Pichia pastoris GS115 competent cells, RDB plate screening of b.avium ompA Pichia pastoris transformants, PCR identification and sequencing of the gene at the same time, transformed the empty plasmid pPIC9 and screening of Pichia pastoris transformants as negative control. With methanol induction induced b.avium ompA positive transformants of Pichia pastoris expression and secretion of ompA, while the empty plasmid transforming Pichia The son was induced and expressed as negative control. After the induction of 24,48,72,96 in h were collected by centrifugation through SDS-PAGE and Western blot analysis were detected. After identification, there is a band size of 21 kDa in SDS-PAGE gel, consistent with its molecular weight and protein molecular weight, and negative control and none of the protein bands. Ni NTA purified protein was detected by SDS-PAGE analysis of a size of 21 kDa protein bands.Western blot analysis also appeared in the size of specific bands of 21 kDa, consistent with the SDS-PAGE results. The animal experiment, 180 SPF chickens were randomly divided into 6 groups at the age of 3 days, the first free, respectively subcutaneous inoculation of 0.2 m L20,40,60 mg/m L TPPPS ompA subunit vaccine adjuvant, incomplete Freund's adjuvant ompA subunit vaccine, ompA subunit vaccine and pure PBS, in 7 after the first immunization and 14 d respectively with strong immune. Free 3,7,14,21,28,35,42,49 D, each group were randomly selected 3 chicken blood and detect the serum antibody titer, serum interleukin -4 (IL-4) concentration, peripheral blood CD4+ and CD8+T lymphocyte percentage, T lymphocyte transformation rate of three. 1 weeks after immunization, each group (except control group) were randomly selected from 20 chickens animal protection test, 7 d observed the clinical symptoms and the survival rate continuously. The results showed that the ompA subunit vaccine alone can effectively induce the secretion of anti -ompA antibody, IL-4, to improve the percentage of CD4+T lymphocyte and T lymphocyte transformation rate, and can provide 71.67% protection rate. In addition, the TPPPS adjuvant vaccine significantly to improve the immune response level, and the 60 mg/m L TPPPS vaccine had the best effect. The study shows that the recombinant b.avium subunit ompA vaccine containing TPPPS has a broad application prospect for immunomodulation, b.avium subunit vaccine Research provides the basis for research.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S859.7

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