本文選題：豬圓環(huán)病毒　切入點(diǎn)：MPRCA　出處：《湖南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：豬圓環(huán)病毒(Porcine circo virus,PCV)是圓環(huán)病毒科(Circoviridae)圓環(huán)病毒屬(Circo virus)的成員,是目前發(fā)現(xiàn)的最小的哺乳動(dòng)物病毒。PCV2主要有兩種基因型(亞型)：PCV2a和PCV2b。當(dāng)前研究表明PCV2的變異速度加快,其中PCV2b逐漸成為優(yōu)勢(shì)毒株。臨床上常見(jiàn)多種PCV2亞型共感染和PCV2與多病原混合感染現(xiàn)象。PCV2呈現(xiàn)全球性分布,能損傷豬免疫系統(tǒng),引起豬嚴(yán)重的免疫抑制,給養(yǎng)豬業(yè)造成重大經(jīng)濟(jì)損失。本研究首先采用常規(guī)PCR法對(duì)31份湖南省某地區(qū)豬病料進(jìn)行PCV2檢測(cè)和序列分析,選取PCV2 capsid蛋白氨基酸序列突變株HNLYYA1用于多引物滾環(huán)復(fù)制擴(kuò)增(Multiply-primed rolling-circle amplification, MPRCA)技術(shù)體系的建立。利用特異性引物和隨機(jī)引物的組合對(duì)PCV2突變株HNLYYA1的總DNA進(jìn)行MPRCA反應(yīng),最后利用限制性?xún)?nèi)切酶(EcoR Ⅰ和Nco Ⅰ)酶切、PCR和測(cè)序反應(yīng)來(lái)鑒定MPRCA產(chǎn)物。結(jié)果顯示,酶切后的MPRCA產(chǎn)物條帶與PCV2基因組或片段的大小一致。將回收的目的條帶與pSP72載體連接,轉(zhuǎn)化DH5a菌,最后對(duì)提取的重組質(zhì)粒進(jìn)行測(cè)序和序列比對(duì)分析,并將PCV2突變株HNLYYA1提交于GenBank(登錄號(hào)KJ867555),其全基因組大小為1767 bp,基因型為PCV2b-1C。該株P(guān)CV2 Cap蛋白與其他毒株進(jìn)行比對(duì),發(fā)現(xiàn)其具有獨(dú)特氨基酸突變。其中NLS區(qū)34位組氨酸(H)突變?yōu)槔野彼?Y)、LOOP GH區(qū)169位精氨酸(R)突變?yōu)楦拾彼?G)、LOOP HI區(qū)206位賴(lài)氨酸(K)突變?yōu)楫惲涟彼?I)。后續(xù)將PCV2突變株HNLYYA1的MPRCA產(chǎn)物EcoR Ⅰ單酶切和重新連接環(huán)化,獲得該株P(guān)CV2的全長(zhǎng)環(huán)化基因組,并將其體外轉(zhuǎn)染PK-15細(xì)胞,經(jīng)過(guò)細(xì)胞傳代培養(yǎng)完成PCV2突變株HNLYYA1感染性克隆的病毒拯救。本研究成功建立了PCV2 MPRCA的技術(shù)體系,并揭示了湖南PCV2分離株HNLYYA1的全基因組特征,利用細(xì)胞轉(zhuǎn)染法構(gòu)建了PCV2感染性克隆。所形成的研究結(jié)果為PCV2毒株突變進(jìn)化提供序列信息基礎(chǔ),也為PCV2感染性克隆構(gòu)建、PCV2毒株的復(fù)制和致病機(jī)理的研究奠定實(shí)驗(yàn)基礎(chǔ)。PCV2 MPRCA技術(shù)體系的建立為其他環(huán)狀病毒的全基因組分離和感染性克隆的構(gòu)建研究提供新的技術(shù)途徑。
[Abstract]:Porcine circo virus.PCV2 is a member of the genus Circoviridae.PCV2, the smallest mammalian virus found at present, has two main genotypes (subtypes: PCV2a and PCV2b.current studies have shown that the mutation rate of PCV2 is increasing. Among them, PCV2b gradually became the dominant virus strain. In clinic, many kinds of PCV2 subtype co-infection and mixed infection phenomenon of PCV2 and multi-pathogen. PCV2 showed a global distribution, which can damage the immune system of pigs and cause serious immunosuppression in pigs. In this study, 31 samples of pig disease in Hunan Province were detected by PCV2 and sequenced by routine PCR method. PCV2 capsid amino acid sequence mutant (HNLYYA1) was selected for the establishment of multiply-primed rolling-circle amplification (MPRCA) technique system. The total DNA of PCV2 mutant HNLYYA1 was reacted with specific primers and random primers. Finally, the restriction endonuclease Ecor 鈪，

本文編號(hào)：1616504