本文選題：六鉤蚴 + M26�。� 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：細(xì)粒棘球絳蟲(chóng)(Echinococcus granulosus)主要寄生于犬、狼、狐貍等犬科動(dòng)物終末宿主體內(nèi),其蟲(chóng)卵和孕節(jié)隨糞便排出體外,污染草料和飲水,牛、羊以及人等中間宿主吞食蟲(chóng)卵后感染此病,進(jìn)入消化道的六鉤蚴鉆入腸壁經(jīng)血流或淋巴散布到體內(nèi)各處,主要在肝和肺臟引起病變導(dǎo)致生長(zhǎng)緩慢,嚴(yán)重危害人的健康和畜牧業(yè)的快速發(fā)展,給世界經(jīng)濟(jì)造成嚴(yán)重?fù)p失。因此,防治此病在人類健康和畜牧業(yè)發(fā)展方面非常重要。長(zhǎng)期以來(lái),各國(guó)研究者都在探索快速診斷此病的方法,但是并未在早期診斷方面取得突破性成果。綿羊是該寄生蟲(chóng)的中間宿主,若在綿羊感染早期,即在六鉤蚴進(jìn)入血液,早期分泌蛋白時(shí)診斷出此病,并采取有效治療措施,將對(duì)控制此病流行具有重要意義。目前,包蟲(chóng)病的診斷方式主要通過(guò)影像檢測(cè)(X線射片,CT,超聲,MR,DSA/SCA),皮內(nèi)試驗(yàn)及血象檢測(cè)等。但這些方法多為后期診斷(即形成包囊后)。而本試驗(yàn)的方法是采用六鉤蚴分泌蛋白為抗原通過(guò)ELISA法對(duì)包蟲(chóng)病進(jìn)行早期診斷,目前針對(duì)包蟲(chóng)病早期診斷的相關(guān)研究國(guó)內(nèi)外尚未有過(guò)報(bào)道。因此選擇特異性高的抗原,建立敏感性高以及特異性強(qiáng)的早期診斷方法十分必要。本研究為了探索在感染早期能夠檢測(cè)出此病的抗原,在實(shí)驗(yàn)室前期研究基礎(chǔ)上選擇了六鉤蚴差異表達(dá)蛋白——副肌球蛋白(M26)作為研究對(duì)象,經(jīng)原核表達(dá)制備多克隆抗體和篩選陽(yáng)性雜交瘤細(xì)胞,建立ELISA方法檢測(cè)感染早期宿主血清中的抗原,即六鉤蚴分泌的蛋白。為尋找有效的診斷抗原和建立高效、特異的診斷方法進(jìn)行了如下試驗(yàn)。1.細(xì)粒棘球六鉤蚴抗原基因的篩選及原核表達(dá):在本實(shí)驗(yàn)室以細(xì)粒棘球絳蟲(chóng)的成蟲(chóng),六鉤蚴,原頭蚴為材料,通過(guò)i TRAQ方法分離檢測(cè)差異蛋白,其中六鉤蚴的差異表達(dá)蛋白可作為早期診斷抗原的前期研究基礎(chǔ)上,本研究在這些差異表達(dá)蛋白中篩選出具有高效免疫保護(hù)性的副肌球蛋白(M26),擴(kuò)增其基因并成功構(gòu)建克隆載體和表達(dá)載體,經(jīng)原核表達(dá)得到大小約為41k Da的重組蛋白,蛋白濃度為0.5 mg/mL,純度達(dá)85%。2.M26多克隆抗體的制備與純化:以M26為抗原免疫新西蘭大白兔,成功制備了M26多克隆抗體,抗體效價(jià)為1:12800。經(jīng)過(guò)Western blot驗(yàn)證,有且只有一條帶,表明該抗體能夠特異性識(shí)別M26。采用間接ELISA法利用多克隆抗體檢測(cè)人工感染綿羊包蟲(chóng)病早期各時(shí)間點(diǎn)(5-10h,24 h和48 h)血清中副肌球蛋白,結(jié)果表明,當(dāng)多抗的稀釋倍數(shù)在1:3200-1:12800時(shí),各時(shí)間點(diǎn)的樣品孔與陰性孔的數(shù)據(jù)比值均大于或等于2.1,即為陽(yáng)性,說(shuō)明各時(shí)間點(diǎn)都有該蛋白。3.M26陽(yáng)性雜交瘤細(xì)胞篩選:以M26作為抗原免疫Balb/c小鼠,經(jīng)細(xì)胞融合、雜交瘤篩選和ELISA法,篩選出2株陽(yáng)性雜交瘤細(xì)胞,通過(guò)間接ELISA法利用此細(xì)胞株檢測(cè)人工感染綿羊細(xì)粒棘球絳蟲(chóng)早期各時(shí)間點(diǎn)(5-10 h,24 h和48 h)血清中副肌球蛋白,在10 h和24 h檢測(cè)出該蛋白,結(jié)果說(shuō)明此檢測(cè)方法成功建立,可以表明利用該蛋白作為抗原采用間接ELISA法能夠有效診斷出綿羊感染早期包蟲(chóng)病。
[Abstract]:Echinococcus granulosus (Echinococcus granulosus) mainly parasitized in the end host of canine, wolves, foxes and other canine terminal hosts. Its eggs and pregnancy were discharged from the feces, contaminated grass and drinking water, cattle, sheep and human intermediate hosts swallowed the eggs and infected the disease. The six hooks entered the intestinal wall and were drilled into the intestinal wall through blood flow or lymph distribution into the body. All places, mainly caused by the liver and lung disease causing slow growth, serious harm to human health and rapid development of animal husbandry, cause serious loss to the world economy. Therefore, prevention and control of the disease is very important in human health and animal husbandry development. For a long time, researchers in various countries have been exploring the method of rapid diagnosis of this disease, but it has not been found Breakthroughs have been achieved in early diagnosis. Sheep are the intermediate host of the parasite. If six cercariae enter the blood in the early stage of the sheep infection, the disease is diagnosed when the early secretory protein is secreted, and the effective treatment measures will be of great significance to control the epidemic. Shoot, CT, ultrasound, MR, DSA/SCA), intradermal tests and Hemogram detection, but these methods are mostly late diagnosis (that is, after the formation of the capsule). The method of this experiment is to use the six hook secretory protein as the antigen to diagnose hydatid disease by ELISA method, and the related research on the early diagnosis of hydatid disease has not been reported at home and abroad. This study is necessary to select high specific antigen and to establish an early diagnostic method with high sensitivity and specificity. In order to explore the antigen of this disease in the early stage of infection, we chose the differential expression protein of six hooks, the accessory myosin (M26), as the research object and the prokaryotic expression on the basis of the early laboratory study. To prepare the polyclonal antibody and screen positive hybridoma cells, the ELISA method was established to detect the antigen in the serum of the early infected host, that is, the protein secreted by the six cercariae. In order to find an effective diagnostic antigen and establish an efficient and specific diagnostic method, the following experiments were carried out to screen and express the antigen gene of the.1. fine spinous Echinococcus spinous Echinococcus. In the laboratory, the differentially expressed proteins were detected by I TRAQ method with the adult of Echinococcus granulosus, six hook and Echinococcus, and the differential expression protein of six Echinococcus could be used as a preliminary study on the early diagnosis of antigen. In this study, the effective and protective paromyosin (M26) was screened in these differentially expressed proteins. The gene was added and the cloning vector and expression vector were successfully constructed. The recombinant protein of 41K Da was obtained by prokaryotic expression. The protein concentration was 0.5 mg/mL and the purity of 85%.2.M26 polyclonal antibody was prepared and purified. The New Zealand white rabbit was immunized with M26 as antigen, and the M26 polyclonal antibody was successfully prepared. The antibody titer was 1:12800. through Western B. Lot verification, with only one band, shows that the antibody can specifically identify M26. by indirect ELISA method using polyclonal antibody to detect the paromyosin in the serum of artificial infected sheep hydatid disease (5-10h, 24 h and 48 h). The result shows that when the dilution multiple of polyclonal antibody is in 1:3200-1:12800, the sample holes and negative of each time point are negative. The ratio of the data of the holes was greater than or equal to 2.1, that is, the positive, indicating that the protein.3.M26 positive hybridoma cells were screened at all time points: Balb/c mice were immunized with M26 as antigen, and 2 positive hybridoma cells were screened by cell fusion, hybridoma screening and ELISA method, and the artificial infected sheep were detected by the indirect ELISA method. In the early stages of Echinococcus granulosus (5-10 h, 24 h and 48 h), the para myosin was detected in the serum of 10 h and 24 h. The results showed that this detection method was successfully established. It can be shown that the use of this protein as an antigen by indirect ELISA can effectively diagnose early echinococcosis in sheep.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.734

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