本文選題：多頭帶絳蟲　切入點：TmAdh　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：腦多頭蚴病(Cerebral coenurosis)是由多頭帶絳蟲(Taenia multiceps)的中絳期幼蟲腦多頭蚴(Coenurus cerebralis)引起的一種人畜共患寄生蟲病。腦多頭蚴可寄生于牛、羊等家畜的腦、脊髓等中樞神經(jīng)系統(tǒng),使宿主產(chǎn)生神經(jīng)癥狀,甚至導(dǎo)致死亡,人作為中間宿主亦可偶發(fā)腦多頭蚴病。因此,該病不僅給畜牧業(yè)造成巨大的經(jīng)濟損失,而且威脅著人體健康。隨著分子生物學(xué)和免疫學(xué)技術(shù)的發(fā)展,使用疫苗防治絳蟲蚴病取得了一定的成效,成為目前研究的熱點。本研究以多頭帶絳蟲基因組DNA為模板,PCR擴增Tm Adh基因家族成員基因組序列,獲得6個含有N端信號肽和1個Fn III結(jié)構(gòu)域的基因序列。利用生物信息學(xué)軟件對Tm Adh家族成員進行預(yù)測分析,發(fā)現(xiàn)其都由3個外顯子和2個內(nèi)含子組成;序列比對顯示其蛋白序列一致性在44.9-99.2%;進化分析發(fā)現(xiàn)該蛋白屬于帶科絳蟲免疫保護性抗原家族的一個新的亞類。以腦多頭蚴c DNA為模板擴增出Tm Adh1、Tm Adh2和Tm Adh4 c DNA基因片段,Tm Adh3基因片段人工合成,將Tm Adh1-4基因片段連接至原核表達載體p GEX4T-1進行原核表達。成功表達出產(chǎn)物大小約為39.0、38.1、38.9和37.8 ku的重組蛋白。利用Western-blot分析Tm Adh1-4重組蛋白的反應(yīng)原性,結(jié)果表明腦多頭蚴自然感染綿羊血清與Tm Adh3重組蛋白反應(yīng)明顯,而與Tm Adh1、Tm Adh2和Tm Adh4重組蛋白反應(yīng)不明顯。細胞黏著活性實驗結(jié)果表明重組蛋白Tm Adh1-4對中國倉鼠卵巢細胞(CHO)和正常鼠腎細胞(NRK)都有黏著活性。免疫組化實驗結(jié)果表明,Tm Adh1主要分布在腦多頭蚴原頭節(jié)的上皮、頂突(主要在小鉤)、組織內(nèi)部有零星分布;Tm Adh2主要分布在腦多頭蚴原頭節(jié)的組織內(nèi)部、頂突、小鉤、上皮有少量分布;Tm Adh3主要分布在腦多頭蚴原頭節(jié)的頂突、小鉤、上皮、組織內(nèi)部零星分布;Tm Adh4大量分布在腦多頭蚴原頭節(jié)的組織內(nèi)部、頂突和小鉤上。重組蛋白的動物免疫保護實驗結(jié)果顯示Tm Adh1、Tm Adh3和Tm Adh4重組蛋白免疫綿羊后抗體水平都顯著上升,三免后達到峰值,攻蟲后抗體水平開始逐漸下降。用多頭帶絳蟲蟲卵感染綿羊后,Tm Adh1重組蛋白免疫組綿羊的腦包囊數(shù)較對照組減少61.71%,Tm Adh3的減蟲率為53.17%,Tm Adh4的減蟲率為50.76%,從而表明Tm Adh1、Tm Adh3和Tm Adh4重組抗原可以誘導(dǎo)綿羊?qū)Χ囝^帶絳蟲蟲卵感染產(chǎn)生一定的抵抗力。本研究為進一步研制腦多頭蚴病疫苗奠定基礎(chǔ)。
[Abstract]:Cerebral coenurosis (Cerebral Coenensis) is a zoonotic parasitic disease caused by the middle stage larvae of Taenia multicepsm (Taenia multiceps), which can parasitize the central nervous system, such as the brain, spinal cord, and so on of cattle, sheep and other domestic animals. It causes neurotic symptoms and even death of the host. As an intermediate host, human may occasionally develop polycephalosis. Therefore, the disease not only causes great economic losses to animal husbandry, With the development of molecular biology and immunology, the use of vaccine to prevent and treat tapeworm echinococcosis has made some progress. In this study, genomic DNA of Taenia multiplex was used as template to amplify genomic sequence of TM Adh gene family. Six sequences containing N-terminal signal peptide and one Fn III domain were obtained. The members of TM Adh family were predicted and analyzed by bioinformatics software. It was found that all of them were composed of 3 exons and 2 introns. Sequence alignment showed that the protein sequence was consistent in 44.9-99.2. Evolutionary analysis showed that the protein belonged to a new subclass of the family of immune protective antigens of Taenia solium. TmAdh1Tm Adh2 and TM Adh4 c DNA were amplified by using c DNA as template. The gene fragment of TM Adh3 was synthesized by artificial synthesis. The TM Adh1-4 gene fragment was ligated to the prokaryotic expression vector p GEX4T-1 for prokaryotic expression. The recombinant proteins with the sizes of 39.0U 38.1U 38.9 and 37.8 ku were successfully expressed. The reactivity of TM Adh1-4 recombinant protein was analyzed by Western-blot. The results showed that the recombinant protein of TM Adh3 in sheep serum infected naturally with cerebral polycephalosis was significantly reactivity. The results of cell adhesion assay showed that the recombinant protein TM Adh1-4 had adhesion activity to both Chinese hamster ovary cells (Cho) and normal rat renal cells (NRK). TM Adh1 was mainly distributed in the epithelium of the procephalic ganglion of the cerebrum polycephalosis. In the parietal process (mainly in the small uncinate, in the tissue there are scattered distribution of TM Adh2 mainly in the tissue of the cerebrum metacercaria, the parietal process, the small uncinate, the epithelium have a little distribution of TM Adh3, mainly distributed in the parietal process, the small hook, the epithelium of the cerebrum polycephalosis, the parietal process, the small hook, the epithelium. Tm Adh4 was distributed sporadically in the tissues of cerebrum polycephalosis, parietal process and uncinate. The results of animal immunological protection test showed that the antibody levels of TmAdh1Tm Adh3 and TM Adh4 recombinant protein increased significantly after immunization with sheep, and the results showed that TmAdh1Tm Adh3 and TM Adh4 recombinant protein increased significantly after immunizing sheep with TmAdh1Tm Adh3 and TM Adh4 recombinant protein. After three exemptions, the peak value was reached. The antibody level began to decrease gradually after infection with Taenia multiceps eggs in sheep. The number of brain cyst of sheep immunized with Tm Adh1 recombinant protein was 61.71% lower than that of control group, and the worm reduction rate of TM Adh3 was 53.17%. The decrease rate of TM Adh4 was 50.76%, which indicated that TM Adh1TM was reduced. The recombinant antigens of Adh3 and TM Adh4 could induce the resistance of sheep to egg infection of Taenia multicephalus, which laid a foundation for the further development of the vaccine against cerebrum multicephalic echinococcosis.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.7

【參考文獻】

中國期刊全文數(shù)據(jù)庫 前1條

1 王安;雙龍;李宏圖;僧格仁欽;德格吉日乎;陶雪蘋;董湘清;白明珠;伊茹;卓甘格圖;;呼盟牧區(qū)羊多頭蚴病綜合防治措施研究[J];中國農(nóng)業(yè)大學(xué)學(xué)報;1998年S2期

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本文編號：1572303