本文選題：偽旋毛蟲 + 新生幼蟲cDNA文庫　； 參考：《吉林大學(xué)》2012年碩士論文

【摘要】：旋毛蟲病(trichinosis/trichinellosis)是一種人獸共患性食源性寄生蟲病，由旋毛線蟲感染引起，分布范圍極其廣泛，集中發(fā)生在東南亞欠發(fā)達國家和有打獵習(xí)慣的少數(shù)發(fā)達國家。近年來有關(guān)該病的報道大幅度提升，原因可能是隨著生活水平的提高，人們對肉類食品的需求量也有所增加，跨區(qū)域性旅游等行為也導(dǎo)致該病的分布范圍擴散。在北美洲國家、歐洲國家和澳大利亞等地，牛肉、馬肉和熊肉等其它肉類的消費產(chǎn)品也是導(dǎo)致人類旋毛蟲感染的重要原因。在中國東北三省，廣西，西藏及云南等地，由于當(dāng)?shù)仫嬍沉?xí)慣而引發(fā)的發(fā)病報道也時有發(fā)生，對社會造成嚴重的恐慌，由此造成的社會影響極其嚴重。 在旋毛線蟲屬中已報道十二個種中，偽旋毛蟲是唯一一種既可以感染哺乳動物也可以感染鳥類的旋毛線蟲，流動性較大，，難防難控，有著異于其它種的特殊的流行分布及感染特點。成蟲時期在宿主腸道侵襲時間持續(xù)長達30d，所產(chǎn)的新生幼蟲有長達4個月的肌肉侵襲期，并且感染整個肌肉細胞，給感染者帶來長期持久的疼痛。其診斷方式，主要是直接肌肉壓片鏡檢或肌肉消化后用光學(xué)顯微鏡觀察，由于偽旋毛蟲在肌肉感染時期不形成包囊或僅形成包囊較薄，所以傳統(tǒng)的肌肉壓片鏡檢方法很難檢測出該病原；另外由于道德倫理等方面的限制，在人體做肌肉切片檢查也難以實現(xiàn)。 偽旋毛蟲發(fā)育不同時期體表抗原和分泌抗原各不相同，均能誘導(dǎo)宿主產(chǎn)生不同程度的免疫力。做為宿主免疫系統(tǒng)應(yīng)答是別的主要針對時期，在血液中游走的新生幼蟲是其整個生活史中唯一不在宿主細胞內(nèi)寄生于的發(fā)育階段,該時期蟲體對宿主的免疫應(yīng)答反應(yīng)有著重要調(diào)節(jié)作用。偽旋毛蟲肌肉幼蟲在宿主肌肉細胞內(nèi)生存時間長達數(shù)年甚至更久，在宿主體內(nèi)產(chǎn)生的抗體可以用以診斷。而如果該時期的抗體能同時識別新生幼蟲時期和成蟲時期的蟲體抗原，那么將尋找到一種識別蟲體整個生活周期的特異性基因。本實驗所用的偽旋毛蟲(ISS13)新生幼蟲cDNA文庫，經(jīng)過滴度測定，確定其滴度為4.25×107pfu/mL，完全符合實驗標準。在對表達文庫進行免疫篩選之前，必須首先擴增非特異性藍斑并測定擴增后的藍斑滴度，在達到一定的數(shù)量級后（109pfu/mL），用假篩選法對血清中非特異性的抗噬菌體蛋白抗體和抗大腸桿菌抗體進行有效的吸附，以確保偽旋毛蟲新生幼蟲cDNA文庫免疫學(xué)篩選的特異性。第一輪篩選到陽性克隆54個，以第一輪篩選結(jié)果為基礎(chǔ)，進行第二輪篩選，最終得到陽性克隆10個，并對其進行測序和序列分析，并對其生物學(xué)特性進行相關(guān)闡述。在10個測序的基因中，有1條已知基因，將之命名為HW7,與ABL09499.2基因同源達到100%。有2條未知基因，將之命名為HW1，HW9。另外7條基因分別命名為HW1、HW2、HW4、HW5、 HW6、 HW8、 HW10：其中HW3與編碼蛋白為神經(jīng)膠細胞瘤病原相關(guān)蛋白-1(glioma pathogeneis-related protein1[Trichinella spiralis],XP_003378739.1)的同源性為84%;HW2與編碼假定蛋白質(zhì)(conserved hypothetical protein，ZP_06571682.1)同源性為36%；HW4與編碼細胞色素C氧化酶I-型亞基(cytochrome c oxidase subunit I [Trichinella spiralis]，NP_077265.1)同源性為82%；HW5與編碼II型脫氧核糖核酸酶超家族(deoxyribonuclease II superfamily [Trichinella spiralis],XP_003373067.1)同源性為81%,與新生幼蟲期特異性DNaseII-12(newborn larvae-specific DNase II-12[T r i c h i n e l l a s p i r a l i s], A A X22752.1)同源性為79%; H W6抑制素(prohibitin[Trichinella spiralis], XP_003373863.1)同源性為96%; HW8與編碼鈣激活鉀通道蛋白slo-1(calcium-activated potassium channel slo-1[Trichinellaspiralis],XP_003370272.1)同源性為93%; HW10與編碼神經(jīng)元鈣傳感蛋白質(zhì)類-1(neuronal calcium sensor1[Trichinella spiralis],XP_003380590.1)同源性為92%。其中HW3具有完整的開放閱讀框架(ORF),可編碼蛋白的堿基序列最長。在篩選到的陽性克隆中，發(fā)現(xiàn)有期特異性HW5，編碼蛋白與旋毛蟲DNaseII超家族蛋白有很高的同源性，II型核酸酶在引發(fā)了肌細胞膠原蛋白基因的異常轉(zhuǎn)錄與表達，與偽旋毛蟲體外形成了的薄膠原蛋白包囊有直接關(guān)系。 目前本實驗室已分別完成偽旋毛蟲感染后26d、60d后抗血清對新生幼蟲時期cDNA文庫的的免疫學(xué)篩選，本論文成功使用感染后32d的抗血清對新生幼蟲時期cDNA文庫的免疫學(xué)篩選，并得到相關(guān)抗原性基因。該研究可進一步對所篩選到的目的基因采用原核表達系統(tǒng)在體外進行大量表達，建立敏感、特異的偽旋毛蟲病的早期免疫學(xué)檢測方法，以及為偽旋毛蟲病的早期診斷和疫苗研發(fā)及功能基因的研究奠定基礎(chǔ)。
[Abstract]:Trichinelliasis (trichinosis/trichinellosis) is a zoonotic foodborne parasitic disease caused by Trichinella infection, the distribution range is extremely wide, concentrated in less developed countries and some developed countries have the habit of hunting in Southeast Asia. In recent years the disease report is greatly improved, the reason may be with the improvement of living standards, people the demand for meat also increased, cross regional tourism behavior also caused the disease to spread. The distribution range in North America, Europe and Australia, beef, horse meat and other meat meat products is an important cause of human Trichinella infection. China in Northeast China, Guangxi Tibet, and Yunnan, reported the incidence caused by local eating habits have also occurred, causing serious social impact of social panic, which caused the It's extremely serious.
In Schistosoma japonicum have been reported in the twelve species, t. pseudospiralis is the only one that can infect mammals can also infect birds of Trichinella spiralis, greater mobility, difficult to prevent and control, is different from other kinds of special epidemic distribution and infection characteristics. Adult period lasted for 30d in the host gut the invasion time, newborn larvae produced a 4 month long muscle invasion period, and the whole muscle cells to infection, infection bring long lasting pain. The diagnosis, mainly direct muscle or muscle tabletting microscopy after digestion by optical microscope, due to Trichinella pseudospiralis infection period not formed cysts in the muscle or only the formation of cysts is thin, so the traditional method of microscopic examination of muscle compression is difficult to detect the pathogen; in addition due to the limitation of moral and ethical aspects of the muscle biopsy in the human body is also difficult to achieve.
T.pseudospiralis in different development stages of surface antigen and secretory antigen is different, can induce different degrees of immunity. As the host immune response is mainly aimed at other times, newborn larvae travel in the blood is the life cycle not only in the initial stage of development in the host cells are parasitic, an important role in regulating the immune response of the host parasite. During the period of pseudo Trichinella spiralis muscle larva survival in host muscle cells for several years or even longer, antibody production in vivo can be used for diagnosis. If the antibody in this period can also identify newborn larvae and adult period period worm antigen specific. You will find a gene for identifying worms in the entire life cycle. T.pseudospiralis used in this experiment (ISS13) of newborn larvae cDNA library, after the titer, determine its titer 4.25 * 107pfu/mL, totally accord with the standard. Before the immunoscreening of expression library, we must first amplification of nonspecific blue spots and determination of the titer of blue spots, to reach a certain magnitude after (109pfu/mL), with a false screening for serum non-specific phage antibody and protein effectively. The adsorption of antibodies against Escherichia coli, in order to ensure the specificity of screening the cDNA Library of immunology pseudo newborn larvae of Trichinella spiralis. The first round of screening to 54 positive clones, with the first round of screening based on the results of the second round of screening, finally obtained 10 positive clones, was sequenced and analyzed the related elaboration on and the biological characteristics. Among the 10 sequenced genes, 1 known genes, named HW7, homologous with ABL09499.2 gene to 100%. 2 unknown genes, named HW1, HW9. and other 7 genes Don't be named HW1, HW2, HW4, HW5, HW6, HW8, HW10: HW3 and encoding a protein of glial cell tumor pathogenesis related protein -1 (glioma pathogeneis-related protein1[Trichinella spiralis], XP_003378739.1) 84% homology with HW2; encoding putative proteins (conserved hypothetical protein, ZP_06571682.1) 36% homology with HW4; encoding cytochrome C oxidase subunit I- (cytochrome c oxidase subunit I [Trichinella spiralis], NP_077265.1) homology is 82%; HW5 and II encoding type deoxyribonuclease superfamily (deoxyribonuclease II superfamily [Trichinella spiralis], XP_003373067.1) 81% homology with newborn larvae stage specific DNaseII-12 (newborn larvae-specific DNase II-12[T r i c h i n e l l a s p I r a l i s] A A X22752.1), the homology was 79%; H W6 prohibitin[Trichinella spiralis (hormone inhibition ], XP_003373863.1) 96% homology with HW8 encoding; calcium activated potassium channel protein slo-1 (calcium-activated potassium channel slo-1[Trichinellaspiralis], XP_003370272.1) 93% homology with HW10 encoding; neuron calcium sensor protein -1 (neuronal calcium sensor1[Trichinella spiralis], XP_003380590.1) with homology to 92%. in which HW3 has a complete open reading frame (ORF). The nucleotide sequence encoding the protein can be long. In the positive clones screened, found to have stage specific HW5, encoding protein and DNaseII family protein of Trichinella spiralis have very high homology, II nuclease in triggering abnormal transcription of muscle cells and collagen gene expression, there is a direct relationship between thin collagen and cyst in vitro formation of Trichinella pseudospiralis.
At present, the laboratory of Trichinella pseudospiralis 26D after infection respectively, after 60d antiserum on newborn larvae cDNA library Immunoscreen the period, the successful use of antiserum after 32D infection on the immune period of newborn larvae cDNA library screening, and get the antigenicity of gene. The study can further target gene of the screened by the prokaryotic expression system for expression, established in vitro sensitive early immunological method to detect specific pseudo trichinosis, and lay the foundation for study on early diagnosis and vaccine development and functional genes for pseudo trichinosis.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1

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