【摘要】：輸血傳播病毒(Transfusion transmitted virus,TTV)又稱細環(huán)病毒(Torque teno virus)。豬細環(huán)病毒即豬源TTV病毒(Torque teno sus virus,TTSu V),屬于細環(huán)病毒科(Anelloviridae),壬型細環(huán)病毒屬(Iotatorquevirus)和Kappatorquevirus病毒屬。TTSu V由美國學(xué)者于1999年首次從豬群中發(fā)現(xiàn),之后世界范圍內(nèi)陸續(xù)有病例的報道。目前對于TTSuV的直接致病性和復(fù)制機理尚不清楚,只了解其與某些豬病發(fā)生可能存在某種協(xié)同作用。鑒于TTSu V在豬群中廣泛流行性及潛在威脅,本試驗針對TTSu V2建立了其相應(yīng)的ELISA和LAMP檢測方法。1.TTSu V2 ORF1基因序列分析比對GenBank中TTSu V2 ORF1基因序列,設(shè)計一對特異性引物,利用PCR方法擴增獲得兩條TTSuV2 ORF1序列。測序結(jié)果顯示兩條序列均包含了整個TTSuV2ORF1區(qū)域,與TTV2Hn93株(JQ664305.1)同源性分別為97,2%和97.7%。運用MEGA6.0和DNAstar將獲得的兩條序列與GenBank中TTSuV2 ORF1基因序列進行遺傳進化和同源性分析。從遺傳進化關(guān)系來看,TTSu V2可分為四個分支,各分支間同源性在53.6~82.2%之間,我國范圍內(nèi)存在四個分支,表明我國可能呈現(xiàn)TTSu V2多亞群的流行。2.TTSu V2 ORF1截短基因原核的表達在TTSu V2 ORF1基因序列分析基礎(chǔ)上,選擇兩端截短的ORF1基因作為靶序列進行PCR擴增,將擴增片段與表達載體pcoldⅠ連接,構(gòu)建了重組表達質(zhì)粒pcold-TTSu V2-ORF1,轉(zhuǎn)入BL21(DE3)感受態(tài)細胞后經(jīng)0.5mM IPTG誘導(dǎo)24 h后表達了大小約為34 KD的帶有His標簽的重組蛋白,蛋白形式主要呈可溶性。Western Blot試驗顯示純化的蛋白反應(yīng)原性良好,可作為下一步ELISA試驗抗原基礎(chǔ)。3.TTSu V2間接ELISA檢測方法的建立以純化的重組蛋白作為包被抗原,分別對血清稀釋度及反應(yīng)時間、封閉液類型及封閉時間、酶標二抗(HRP標記的兔抗豬IgG)稀釋度及反應(yīng)時間等梯度進行優(yōu)化建立了TTSu V2間接ELISA檢測方法。其中封閉條件為3%犢牛血清封閉2 h,血清稀釋度為1:400,反應(yīng)為45 min,酶標二抗稀釋度為1:4000,反應(yīng)為1 h。對陰性血清反應(yīng)結(jié)果通過統(tǒng)計學(xué)計算得出陰陽性血清臨界值為0.182。特異性試驗說明該方法特異性強。批內(nèi)批間重復(fù)性試驗表明該方法重復(fù)性良好。運用該方法對采自江西3家規(guī)�；i場的84份血清樣品進行檢測,陽性檢出率分別為72%(13/18)、57.5%(19/33)、36.3%(12/33)。本試驗建立的間接ELISA方法可運用于臨床上豬血清中TTSuV2抗體的檢測。4.TTSu V2環(huán)介導(dǎo)等溫擴增(LAMP)檢測方法的建立本試驗選取TTSu V2 UTR和ORF1前端區(qū)域為靶序列,利用生物軟件網(wǎng)站設(shè)計了兩對LAMP引物。對反應(yīng)參數(shù)和反應(yīng)條件摸索后,建立了TTSu V2 LAMP檢測方法。TTSu V2 LAMP方法最佳反應(yīng)條件為64℃恒溫90 min,最低病毒檢出限為100 copies/?l,特異性良好,與相關(guān)豬源病毒不存在交叉反應(yīng)。該LAMP檢測方法具有強特異性,高靈敏度等優(yōu)勢,有望成為快速檢測TTSu V2的主要手段之一。
[Abstract]:Transfusion transmitted virus (Transfusion transmitted virus,TTV) also known as microcyclic virus (Torque teno virus). Porcine TTV virus (Torque teno sus virus,TTSu V),) belongs to (Iotatorquevirus) and Kappatorquevirus viruses of (Anelloviridae), nontype microcircovirus family. TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. the TPTSu V was first discovered in pigs by American scholars in 1999. Since then, there have been reports of cases around the world. At present, the direct pathogenicity and replication mechanism of TTSuV are not clear, only that it may have some synergistic effect with the occurrence of some pig diseases. In view of the widespread epidemic and potential threat of TTSu V in pig populations, the corresponding ELISA and LAMP detection methods for TTSu V 2 were established in this experiment. 1. TTSu V 2 ORF1 gene sequence was compared with TTSu V 2 ORF1 gene sequence in GenBank, and a pair of specific primers were designed. Two TTSuV2 ORF1 sequences were amplified by PCR. The sequencing results showed that the two sequences contained the whole TTSuV2ORF1 region, and the homology with TTV2Hn93 strain (JQ664305.1) was 97%, 2% and 97.7%, respectively. MEGA6.0 and DNAstar were used to analyze the genetic evolution and homology of the two sequences with TTSuV2 ORF1 gene sequences in GenBank. According to the genetic evolution relationship, TTSu V 2 can be divided into four branches, the homology among them is 53.6 鈮，

本文編號：2494906