本文關鍵詞：泰山松花粉多糖對畢赤酵母表達的禽波氏桿菌OmpA的免疫增強效果研究　出處：《山東農業(yè)大學》2016年碩士論文　論文類型：學位論文

  更多相關文章： 禽波氏桿菌 外膜蛋白A 畢赤酵母表達 亞單位疫苗 泰山松花粉多糖 免疫增強

【摘要】：禽波氏桿菌病是由禽波氏桿菌(Bordetella avium,B.avium)引起的一種家禽上呼吸道性傳染性疾病,既可水平傳播,也可垂直傳播,主要導致死胚率升高,孵化率降低和雛雞的急性死亡�；疾‰u生長遲緩、飼料報酬低,給養(yǎng)禽業(yè)造成了巨大的經濟損失。有研究表明,本菌還是人體的一種機會致病菌。目前,由于藥物殘留的影響以及禽波氏桿菌耐藥性的形成,通過抗生素治療本病的難度越來越大。關于禽波氏桿菌病的預防,國外僅對火雞波氏桿菌病疫苗開展了研究。目前用于預防火雞波氏桿菌病的疫苗包括溫度敏感變異株活菌苗和全細胞菌素,但這兩種疫苗只能適當降低病變的嚴重程度以及延緩臨床癥狀的出現,并不能從根本上防止本病的傳播。因此急需開辟新的途徑,研發(fā)新型疫苗。佐劑可以激活機體的先天免疫系統(tǒng)進而增強機體對特異性抗原的適應性免疫,從而使機體產生高效持久的免疫反應,因此,疫苗佐劑在免疫過程中是不可或缺的。本實驗室前期的研究表明,泰山松花粉多糖(Taishan Pinus massoniana pollen polysaccharide,TPPPS)作為滅活疫苗和亞單位疫苗佐劑具有良好的免疫增強效果,但其對真核表達的外膜蛋白A(outer membrane protein A,ompA)的免疫效果研究未見報道。因此,本研究以畢赤酵母表達系統(tǒng)為基礎,分泌表達重組的禽波氏桿菌ompA,采用TPPPS為疫苗佐劑,并評估其對重組禽波氏桿菌ompA亞單位疫苗的免疫增強效果。根據GenBank中禽波氏桿菌ompA基因序列設計一對引物,以實驗室分離得到的禽波氏桿菌DNA為模板,PCR擴增目的基因。將PCR擴增產物連接到pMD18-T克隆載體上,經測序驗證正確后,重組克隆載體進行EcoR I和Not I雙酶切,膠回收產物連接到相同酶切的表達載體pPIC9上,重組表達載體線性化后轉化到畢赤酵母GS115感受態(tài)細胞中,RDB板篩選禽波氏桿菌ompA畢赤酵母陽性轉化子,PCR鑒定并進行基因測序。同時,轉化空質粒pPIC9并篩選畢赤酵母轉化子作為陰性對照。采用甲醇誘導法誘導禽波氏桿菌ompA畢赤酵母陽性轉化子分泌表達ompA,同時對空質粒畢赤酵母轉化子進行誘導表達,作為陰性對照。于誘導后24、48、72、96 h分別離心收集上清,通過SDS-PAGE和Western blot分析進行檢測。經鑒定,在SDS-PAGE凝膠中有一條大小為21 kDa的條帶,其分子量大小與目的蛋白分子量大小相一致,而陰性對照中并沒有該蛋白條帶。鎳柱純化的蛋白經SDS-PAGE分析只檢測到一條大小為21 kDa蛋白條帶。Western blot分析中也出現大小為21 kDa的特異性條帶,與SDS-PAGE檢測結果相一致。動物試驗中,將180只SPF雞隨機分為6組,3日齡首免,分別頸部皮下接種0.2 m L20、40、60 mg/m L TPPPS佐劑ompA亞單位疫苗,不完全弗氏佐劑ompA亞單位疫苗,單純的ompA亞單位疫苗和PBS,于首免后7和14 d分別進行加強免疫。首免后3、7、14、21、28、35、42、49 d,每組隨機取3只雞采血,并檢測其血清抗體效價,血清白細胞介素-4(IL-4)濃度,外周血CD4+和CD8+T淋巴細胞百分含量,T淋巴細胞轉化率。三免后1周,每組(除對照組)隨機取20只雞進行動物保護試驗,連續(xù)7 d觀察記錄臨床癥狀和存活率。結果表明,單純的ompA亞單位疫苗能有效誘導機體產生抗-ompA抗體,分泌IL-4,提高CD4+T淋巴細胞百分含量和T淋巴細胞轉化率,并能提供71.67%的保護率。此外,TPPPS佐劑疫苗能顯著提高機體免疫反應水平,且60 mg/m L TPPPS佐劑疫苗效果最好。研究表明,含TPPPS的重組禽波氏桿菌ompA亞單位疫苗具有廣闊的免疫調理應用前景,為禽波氏桿菌亞單位疫苗研究提供研究基礎。
[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.

【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S859.7

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