發(fā)布時間：2018-03-02 16:41

  本文選題：細胞因子　切入點：Blec　出處：《山西農業(yè)大學》2014年碩士論文　論文類型：學位論文

【摘要】：雞主要組織相容性復合體(MHC)長期以來被認為是識別對疾病易感和耐受的免疫反應系統(tǒng),尤其與馬立克氏病(MD)密切相關。哈爾濱獸醫(yī)研究所國家禽類實驗動物種子中心根據(jù)MHC-B區(qū)域中的4個微衛(wèi)星位點等位基因,篩選出B13、B15、B2、B5、B21和B19六種單倍型,這是我國唯一具有自主知識產權的無特定病原體雞群。本試驗用B15、B19和B21單倍型來作為實驗動物進行試驗。 本研究對2月齡B15單倍型的胸腺組織提取RNA,經RT-PCR擴增ChBLec基因,并以pET-30a(+)為原核表達載體成功構建重組質粒pET-30a-ChBLec,并在BL21(DE3)中經IPTG (0.02mmol/L)誘導表達,獲得大小約為17kDa以包涵體形式存在的ChBLec重組蛋白。把重組蛋白所在凝膠研磨后免疫新西蘭兔,獲得兔抗血清。用western blot檢測后兔抗血清能檢測到pET-30a-ChBLec外源蛋白。結果表明：ChBLec基因在大腸桿菌成功表達,且成功制備了兔抗ChBLec血清。 為了了解雞外周血淋巴細胞(PBL)中BNK, BLec基因以及IFN-γ、IL-4、IL-10、IL-18細胞因子的轉錄水平與遺傳背景的聯(lián)系。本試驗通過肌肉注射的方法用MDV Md5細胞結合型病毒對1日齡雞進行攻毒,每羽800PFU,對照組接種無菌的PBS,在4、7、10、13、19dpi時采集抗凝血,用雞外周血淋巴細胞分離液分離PBL,然后用ConA(25uggmL)刺激,培養(yǎng)48h后收獲細胞提取RNA并且反轉錄,然后用雙重熒光定量PCR技術檢測BNK、BLec和四種細胞因子轉錄水平的動態(tài)變化。BNK和BLec在PBL的動態(tài)變化表明：在4、7dpi(第一次溶細胞感染期)和19dpi BNK和BLec大多表現(xiàn)出不同程度的降低；在10dpi(潛伏期)B19單倍型雞中BNK和BLec表現(xiàn)出不同程度的升高,B21單倍型雞中BNK升高；在13dpi(第二次溶細胞感染期)B21單倍型雞中BNK和BLec表現(xiàn)出不同程度的升高。四種細胞因子在PBL的動態(tài)變化表明：在4、7dpi(第一次溶細胞感染期)和19dpi四種細胞因子大多表現(xiàn)出不同程度的降低；在10dpi(潛伏期)B21單倍型雞中IFN-γ(19%)、I-4(5%)和I-18(32%)表現(xiàn)出不同程度的升高,而B19單倍型雞中IFN-γ(-16%)、IL-4(-29%)和IL,-18(-23%)表現(xiàn)出不同程度的降低；在13dpi(第二次溶細胞感染期)B21單倍型雞中IL-18(47%)和I-10(69%)表現(xiàn)出不同程度的升高,而B19單倍型雞中IL-18(-30%)和IL-10(-37%)表現(xiàn)出不同的降低。 根據(jù)文獻中對MD發(fā)病機理的闡述可知,在第一次溶細胞感染期淋巴器官會出現(xiàn)不同程度的萎縮,引起免疫抑制,這與本試驗在4、7dpi時BNK、BLec和四種細胞因子轉錄水平下降結果一致；在感染后8-14d為第二次溶細胞感染期,耐受雞一般不會經歷,與之相反易感雞會持續(xù)免疫抑制。以上結果說明這種變化與雞的遺傳背景是相關的,并且B21單倍型雞比B19單倍型雞對MD耐受。
[Abstract]:Chicken major histocompatibility complex (MHC) has long been considered as an immunoreactive system for identifying susceptibility and tolerance to disease. The National Laboratory Animal seed Center of Harbin Veterinary Research Institute screened six haplotypes B13, B15, B _ 2, B _ (5), B _ (21) and B _ (19) according to the alleles of four microsatellite loci in the MHC-B region. This is the only chicken flocks with independent intellectual property rights in China without specific pathogens. In this experiment, B15B19 and B21 haplotypes were used as experimental animals. In this study, ChBLec gene was amplified by RT-PCR from 2-month-old B15 haplotype thymus tissue. The recombinant plasmid pET-30a-ChBLecwas successfully constructed by using pET-30a () as prokaryotic expression vector, and was induced by IPTG 0.02mmolL / L in BL21DDE3. A recombinant ChBLec protein was obtained in the form of inclusion body of about 17kDa. The New Zealand rabbits were immunized by grinding the gel in which the recombinant protein was located. Rabbit antiserum was obtained and pET-30a-ChBLec exogenous protein was detected by western blot. The results showed that the pET-30a-ChBLec gene was successfully expressed in Escherichia coli and rabbit anti-#en3# serum was successfully prepared. In order to understand the relationship between genetic background and transcriptional level of BNK-, BLec gene and IL-10 IL-18 cytokines in peripheral blood lymphocytes of chicken, MDV Md5 cell-binding virus was injected intramuscularly to attack 1-day-old chicken. The control group was inoculated with aseptic PBSs. Anticoagulant blood was collected at 1319 dpi. PBLs were isolated from chicken peripheral blood lymphocytes, then stimulated with Cona 25uggm L. After 48 hours of culture, the RNA was extracted from the cells and reversed-transcripted. Then the dynamic changes of the transcription levels of BNK-BLec and four cytokines were detected by double fluorescence quantitative PCR. The dynamic changes of BNK and BLec in PBL showed that most of them were decreased in different degrees at 4dpiand 19dpi BNK and BLec. BNK and BLec increased in different degree in 10 d pii (incubation period B19 haplotype) and BNK increased in B21 haplotype. BNK and BLec increased in different degrees in 13dpi. the dynamic changes of four cytokines in PBL showed that the four cytokines were mostly expressed at 4dpi7dpiand 19dpi. Showing varying degrees of decline; IFN- 緯 ~ (19) and I-18 ~ (32) and IFN- 緯 ~ (19) and I-18 ~ (32) showed different degrees of elevation in 10dpi. while IFN- 緯 -16 + IL-4 ~ (-29) and IL-18-23 in B19 haplotypes showed different degrees of decrease, and increased at 13 d (IL-18 ~ (47) and I-10 ~ (69) in B21 haplotypes in the second stage of cytolytic infection). In B 19 haplotypes, IL-18-30) and IL-10-37) showed different decreases. According to the explanation of the pathogenesis of MD in the literature, the lymphoid organs of the first lysocytic infection stage will shrink in varying degrees, causing immunosuppression, which is consistent with the decrease of the transcription level of BNK-BLec and four cytokines at 4 ~ 7 dpi in this experiment. During the second cytolytic infection period from 8 to 14 days after infection, the tolerant chickens did not generally experience it, whereas the susceptible chickens continued to undergo immunosuppression. These results suggest that this change is related to the genetic background of the chickens. And B 21 haplotypes were more resistant to MD than B 19 haplotypes.
【學位授予單位】：山西農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：S858.31

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本文編號：1557329