本文選題：新孢子蟲 + Nc-43-p��； 參考：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：新孢子蟲病(Neosporosis)由犬新孢子蟲(Neospora caninum)感染引起的一種原蟲病[1]。新孢子蟲病是嚴(yán)重危害奶牛和犬的一種原蟲病,臨床報(bào)道發(fā)生新孢子蟲病的動(dòng)物包括綿羊、山羊、鹿、犀牛和馬,除此之外,在水牛、紅狐、灰狐、狼、駱駝以及貓科動(dòng)物中也檢測(cè)到了新孢子蟲抗體[2]。甚至在人的血清中也檢測(cè)到新孢子蟲的抗體,目前還沒有在人體內(nèi)發(fā)現(xiàn)新孢子蟲體的報(bào)導(dǎo),但是新孢子蟲可能存在人獸共患的風(fēng)險(xiǎn)。新孢子蟲可感染兔,為了減少新孢子蟲病給養(yǎng)兔業(yè)帶來(lái)的經(jīng)濟(jì)損失,做好該病的診斷與防控非常重要。目前沒有針對(duì)兔感染新孢子蟲的診斷方法。因此,本研究選取新孢子蟲的主要表面抗原基因Np-43基因,克隆到pET-32a,構(gòu)建pET-Nc-p43重組質(zhì)粒,在BL21中進(jìn)行Ncp43P重組蛋白表達(dá),利用鎳柱進(jìn)行表達(dá)蛋白的純化,作為包被抗原,建立檢測(cè)兔感染新孢子蟲的間接ELISA抗體檢測(cè)方法。利用表達(dá)的Ncp43P蛋白建立檢測(cè)兔血清中新孢子蟲抗體的間接ELISA方法。包被抗原濃度為5μg/mL。分別選擇了小鼠血清、胎牛血清、明膠作為封閉劑,并對(duì)一抗、二抗的稀釋濃度進(jìn)行了優(yōu)化,結(jié)果確定采用10%的小鼠血清作為封閉劑,血清按照1:100稀釋,酶標(biāo)二抗按照1:1000稀釋效果最好。建立的方法通過同一板內(nèi)檢測(cè)不同的血清,和同一樣品在不同板間進(jìn)行檢測(cè),分析建立的方法批間、批內(nèi)穩(wěn)定性。通過實(shí)驗(yàn)測(cè)得批間差為1.6%-5.0%,批內(nèi)差為0.4%-2.3%,均小于10%,說(shuō)明建立的方法具有很好的穩(wěn)定性。因?yàn)樾骆咦酉x與弓形蟲最為接近,所以利用該方法對(duì)弓形蟲抗體陽(yáng)性血清進(jìn)行檢測(cè),結(jié)果顯示該方法與弓形蟲陽(yáng)性血清無(wú)交叉反應(yīng),具有較好的特異性。Cut off值的確定,根據(jù)Cut off值=陰性血清OD值平均值+3SD,計(jì)算得Cut off值為0.37。為減少假陽(yáng)性與假陰性,在Cut off值的基礎(chǔ)上加減一個(gè)標(biāo)準(zhǔn)差作為判斷陰陽(yáng)性血清的標(biāo)準(zhǔn),即OD值0.4為陽(yáng)性,OD值0.3為陰性,OD值在兩者之間為疑似。該方法與VMRD的ELISA試劑盒進(jìn)行比較符合率達(dá)97.5%。該方法可用于兔血清中新孢子蟲抗體的檢測(cè),利用該方法對(duì)吉林地區(qū)采集的468份兔血清樣品進(jìn)行檢測(cè),結(jié)果檢測(cè)為陽(yáng)性血清為149份,陽(yáng)性率達(dá)31.8%。
[Abstract]:Neosporosis A protozoa disease caused by Neospora caninum infection. Neosporidiosis is a protozoan disease that seriously harms cows and dogs. Clinical reports of neosporidiosis include sheep, goats, deer, rhinoceros and horses, in addition to buffalo, red fox, gray fox, wolf, etc. Antibodies to neosporidium were also detected in camels and cats [2]. Antibodies to neosporidium have been detected even in human serum. There are no reports of neosporidium in human body, but neosporidium may be at risk of zoonosis. Neosporidium can infect rabbits. In order to reduce the economic loss caused by neosporidiosis, it is very important to make a good diagnosis, prevention and control of the disease. There is no diagnostic method for neosporidium infection in rabbits. Therefore, the main surface antigen gene of neosporidium, Np-43 gene, was cloned into pET-32a, and the recombinant plasmid pET-Nc-p43 was constructed. Ncp43P recombinant protein was expressed in BL21 and purified by nickel column. An indirect Elisa method for detection of neosporidium infection in rabbits was established. An indirect Elisa method for detection of neosporidium antibodies in rabbit serum was established by using the expressed Ncp43P protein. The concentration of coated antigen was 5 渭 g / mL. Mouse serum, fetal bovine serum and gelatin were selected as sealants, and the dilution concentration of first antibody and second antibody was optimized. The results showed that 10% mouse serum was used as sealant and the serum was diluted according to 1: 100. The enzyme labeled second antibody was best diluted according to 1: 1000. The established method was used to detect different serum in the same plate, and the same sample was detected in different plates. The stability of the method between batches and batches was analyzed. The results showed that the difference between batches was 1.6-5.0, and the difference within batches was 0.4-2.3, which was less than 100.The results showed that the established method had good stability. Because neosporidium and Toxoplasma gondii are the closest, the method is used to detect Toxoplasma antibody positive serum. The results show that this method has no cross reaction with Toxoplasma gondii positive serum, and has good specificity. According to the mean value of cut off = negative serum OD value 3 SD, the cut off value was calculated to be 0. 37. In order to reduce false positive and false negative, a standard deviation was added and subtracted on the basis of cut off value as the criterion for judging yin-yang serum, that is, OD value 0.4 was positive, OD value 0.3 was negative, OD value was suspected between them. Compared with the Elisa kit of VMRD, the coincidence rate of this method was 97.5. The method can be used for the detection of neosporidium antibody in rabbit serum. 468 rabbit serum samples collected in Jilin area were detected by this method. 149 positive sera were detected, and the positive rate was 31.8%.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.291

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