【摘要】：輸血傳播病毒(Transfusion transmitted virus,TTV)又稱細(xì)環(huán)病毒(Torque teno virus)。豬細(xì)環(huán)病毒即豬源TTV病毒(Torque teno sus virus,TTSu V),屬于細(xì)環(huán)病毒科(Anelloviridae),壬型細(xì)環(huán)病毒屬(Iotatorquevirus)和Kappatorquevirus病毒屬。TTSu V由美國(guó)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(bào)道。目前對(duì)于TTSuV的直接致病性和復(fù)制機(jī)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(yàn)針對(duì)TTSu V2建立了其相應(yīng)的ELISA和LAMP檢測(cè)方法。1.TTSu V2 ORF1基因序列分析比對(duì)GenBank中TTSu V2 ORF1基因序列,設(shè)計(jì)一對(duì)特異性引物,利用PCR方法擴(kuò)增獲得兩條TTSuV2 ORF1序列。測(cè)序結(jié)果顯示兩條序列均包含了整個(gè)TTSuV2ORF1區(qū)域,與TTV2Hn93株(JQ664305.1)同源性分別為97,2%和97.7%。運(yùn)用MEGA6.0和DNAstar將獲得的兩條序列與GenBank中TTSuV2 ORF1基因序列進(jìn)行遺傳進(jìn)化和同源性分析。從遺傳進(jìn)化關(guān)系來(lái)看,TTSu V2可分為四個(gè)分支,各分支間同源性在53.6~82.2%之間,我國(guó)范圍內(nèi)存在四個(gè)分支,表明我國(guó)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(dá)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(jìn)行PCR擴(kuò)增,將擴(kuò)增片段與表達(dá)載體pcoldⅠ連接,構(gòu)建了重組表達(dá)質(zhì)粒pcold-TTSu V2-ORF1,轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞后經(jīng)0.5mM IPTG誘導(dǎo)24 h后表達(dá)了大小約為34 KD的帶有His標(biāo)簽的重組蛋白,蛋白形式主要呈可溶性。Western Blot試驗(yàn)顯示純化的蛋白反應(yīng)原性良好,可作為下一步ELISA試驗(yàn)抗原基礎(chǔ)。3.TTSu V2間接ELISA檢測(cè)方法的建立以純化的重組蛋白作為包被抗原,分別對(duì)血清稀釋度及反應(yīng)時(shí)間、封閉液類型及封閉時(shí)間、酶標(biāo)二抗(HRP標(biāo)記的兔抗豬IgG)稀釋度及反應(yīng)時(shí)間等梯度進(jìn)行優(yōu)化建立了TTSu V2間接ELISA檢測(cè)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(biāo)二抗稀釋度為1:4000,反應(yīng)為1 h。對(duì)陰性血清反應(yīng)結(jié)果通過(guò)統(tǒng)計(jì)學(xué)計(jì)算得出陰陽(yáng)性血清臨界值為0.182。特異性試驗(yàn)說(shuō)明該方法特異性強(qiáng)。批內(nèi)批間重復(fù)性試驗(yàn)表明該方法重復(fù)性良好。運(yùn)用該方法對(duì)采自江西3家規(guī)�；i場(chǎng)的84份血清樣品進(jìn)行檢測(cè),陽(yáng)性檢出率分別為72%(13/18)、57.5%(19/33)、36.3%(12/33)。本試驗(yàn)建立的間接ELISA方法可運(yùn)用于臨床上豬血清中TTSuV2抗體的檢測(cè)。4.TTSu V2環(huán)介導(dǎo)等溫?cái)U(kuò)增(LAMP)檢測(cè)方法的建立本試驗(yàn)選取TTSu V2 UTR和ORF1前端區(qū)域?yàn)榘行蛄?利用生物軟件網(wǎng)站設(shè)計(jì)了兩對(duì)LAMP引物。對(duì)反應(yīng)參數(shù)和反應(yīng)條件摸索后,建立了TTSu V2 LAMP檢測(cè)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(cè)方法具有強(qiáng)特異性,高靈敏度等優(yōu)勢(shì),有望成為快速檢測(cè)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(hào)：2494906