本文選題：P1病毒 + RT-PCR��； 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：類(lèi)豬圓環(huán)病毒P1是從臨床疑似豬斷奶后多系統(tǒng)衰竭綜合征(Postweaning multisystemic wasting syndrome,PMWS)病豬的血清中分離到的,全長(zhǎng)648個(gè)核苷酸,是目前發(fā)現(xiàn)的基因組最小的動(dòng)物病毒,部分序列與豬圓環(huán)病毒2型(Porcinecircovirus type 2,PCV2)開(kāi)放閱讀框2(open reading frame2,ORF2)的序列高度同源。前期研究表明,P1病毒可引起轉(zhuǎn)染的PK-15細(xì)胞凋亡,也可導(dǎo)致感染豬出現(xiàn)類(lèi)似PMWS的臨床癥狀。本論文主要內(nèi)容如下:1.類(lèi)豬圓環(huán)病毒P1 ORF2和ORF3的鑒定P1病毒主要包括3個(gè)開(kāi)放閱讀框,為從轉(zhuǎn)錄水平和蛋白水平鑒定其ORF2和ORF3,用P1雙拷貝分子克隆(pSK-P1)轉(zhuǎn)染PK-15細(xì)胞,采用Trizol法提取轉(zhuǎn)染細(xì)胞的總RNA,純化后進(jìn)行RT-PCR擴(kuò)增,回收PCR產(chǎn)物并測(cè)序。利用cDNA末端快速擴(kuò)增法(Rapid-amp1ification of cDNAends,RACE)技術(shù)分別擴(kuò)增 ORF2 和 ORF3 的5'-與3'-末端。同時(shí),根據(jù)ORF2和ORF3編碼的氨基酸序列預(yù)測(cè)其B細(xì)胞抗原表位,采用逐步固相合成法合成多肽,與匙孔血藍(lán)蛋白(Keyhole limpet hemocyanin,KLH)偶聯(lián),免疫新西蘭大白兔制備多克隆抗體,常規(guī)間接ELISA法檢測(cè)血清效價(jià)。通過(guò)免疫組化方法檢測(cè)轉(zhuǎn)染pSK-P1的PK-15細(xì)胞與兩種多抗的免疫反應(yīng)性。結(jié)果表明:以P1病毒RNA為模板,用ORF2和ORF3特異引物進(jìn)行的RT-PCR均能擴(kuò)增出目的基因片段,分別與P1病毒ORF2和ORF3的序列同源性達(dá)100%;RACE技術(shù)分析,可知P1病毒ORF2的5'-和3'-末端分別在nt648和nt481,ORF3的5'-和3'-末端分別在nt364和nt70。合成肽的氨基酸序列分別為ORF2:CLSRLPQSERPPGRW和ORF3:CVYPKVRERRVLKMP;用ORF2和ORF3表位肽與KLH的偶聯(lián)物制備的多抗效價(jià)均在1:512000以上,免疫組化檢測(cè)結(jié)果顯示2種抗體均能夠與P1病毒發(fā)生特異性顯色反應(yīng)。綜上,通過(guò)轉(zhuǎn)錄分析及免疫組化法分別從轉(zhuǎn)錄水平和蛋白水平證實(shí)了類(lèi)豬圓環(huán)病毒P1 ORF2和ORF3的存在。2.類(lèi)豬圓環(huán)病毒P1三種主要蛋白在昆蟲(chóng)細(xì)胞中的表達(dá)分析為鑒定P1病毒3個(gè)主要開(kāi)放閱讀框所編碼的蛋白能否在體外自組裝形成病毒樣顆粒(Virus-likeparticles,VLPs),設(shè)計(jì)3對(duì)引物分別擴(kuò)增P1病毒的ORF1、ORF2和ORF3,連接到載體pFastBacTM1,構(gòu)建重組表達(dá)質(zhì)粒,脂質(zhì)體法轉(zhuǎn)染Sf9細(xì)胞獲得重組桿狀病毒(rBac-ORF1、rBac-ORF2 和 rBac-ORF3)。通過(guò) RT-PCR 及 Western blot分別從轉(zhuǎn)錄水平和蛋白水平鑒定表達(dá)情況,透射電鏡觀察是否形成VLPs。結(jié)果顯示,重組質(zhì)粒轉(zhuǎn)染Sf9細(xì)胞后,均可導(dǎo)致細(xì)胞病變;從接毒細(xì)胞中提取總RNA,除去基因組DNA后,利用RT-PCR能夠擴(kuò)增出相應(yīng)目的片段;Western blot結(jié)果顯示,rBac-ORF1表達(dá)產(chǎn)物能夠與抗P1VP1單抗和多抗發(fā)生特異反應(yīng);電鏡觀察發(fā)現(xiàn),3種重組蛋白均可在Sf9細(xì)胞中形成近橢圓形的包涵體。綜上,本研究利用桿狀病毒表達(dá)載體系統(tǒng)成功地在Sf9細(xì)胞中表達(dá)了 P1病毒ORF1、ORF2和ORF3基因,并證實(shí)在本試驗(yàn)條件下這3種重組蛋白均不能自組裝成VLPs。本研究為進(jìn)一步探究P1病毒蛋白的其他功能特性奠定了基礎(chǔ)。
[Abstract]:Porcine circovirus P1 was isolated from the serum of clinically suspected pigs with postweaning multisystemic wasting syndrome (PMWS) and has a total length of 648 nucleotides. Part of the sequence is highly homologous to the sequence of Porcine circovirus type 2 (PCV2) open reading box 2 (open reading frame2 ORF2. Previous studies have shown that PK-15 cells transfected with PK-15 can be induced to apoptosis by the virus, and it can also cause PMWS like clinical symptoms in infected pigs. The main contents of this thesis are as follows: 1. The identification of porcine circovirus P1 ORF2 and ORF3 mainly consists of three open reading frames. In order to identify ORF2 and ORF3 from transcription and protein levels, PK-15 cells were transfected with P1 double copy molecular clone (pSK-P1). The total RNAs of transfected cells were extracted by Trizol method, then purified and amplified by RT-PCR. PCR products were recovered and sequenced. Rapid-amp1ification of cDNAendsrace was used to amplify the 5- and 3N-terminal of ORF2 and ORF3, respectively. At the same time, the B cell epitopes of ORF2 and ORF3 were predicted according to their amino acid sequences. Polypeptides were synthesized by solid phase synthesis and conjugated with keyhole limpet hemocyanin (KLH) to immunize New Zealand white rabbits to prepare polyclonal antibodies. Serum titers were detected by routine indirect Elisa. The immunoreactivity of pSK-P1 transfected PK-15 cells with two kinds of polyantibodies was detected by immunohistochemical method. The results showed that the target gene fragments could be amplified by RT-PCR with ORF2 and ORF3 specific primers using P1 virus RNA as template, and the sequence homology with P1 virus ORF2 and ORF3 were 100% and 100% respectively. The results showed that the 5- and 3- ends of P1 virus ORF2 were at the nt364 and nt70 ends of nt648 and nt481 ORF3, respectively. The amino acid sequences of the synthetic peptides were ORF2: CLSRLPQSERPPGRW and ORF3: CVYPKVRERRVLKMP.The polyclonal titers of the conjugates of ORF2 and ORF3 epitope peptides with KLH were above 1: 512000, and the results of immunohistochemistry showed that the two antibodies could react with P1 virus specifically. In conclusion, the existence of P1 ORF2 and ORF3 of porcine circovirus was confirmed by transcriptional analysis and immunohistochemistry, respectively. Expression Analysis of three main proteins of Porcine circovirus P1 in insect cells to identify whether the proteins encoded by the three main open reading frames of P1 virus can self-assemble into Virus-like particles-like VLPs in vitro, three pairs of primers were designed to expand respectively. ORF1, ORF2 and ORF3 of P1 virus were ligated to the vector pFastBacTM1 to construct the recombinant expression plasmid. Recombinant baculovirus (rBac-ORF1, rBac-ORF2 and rBac-ORF3) was obtained by transfection of Sf9 cells with liposome. The expression of VLPs was identified by RT-PCR and Western blot at the transcriptional level and protein level, and the formation of VLPswas observed by transmission electron microscope (TEM). The results showed that the recombinant plasmid could cause cytopathic changes after transfection of Sf9 cells, total RNAs were extracted from inoculated cells, and genomic DNA was removed. The expression products of rBac-ORF1 could react specifically with anti-P1VP1 monoclonal antibody and polyclonal antibody by RT-PCR, and the three recombinant proteins could form oval inclusion bodies in Sf9 cells by electron microscope. In conclusion, we successfully expressed the ORF1ORF2 and ORF3 genes of P1 virus in Sf9 cells using baculovirus expression vector system, and confirmed that these three recombinant proteins could not self-assemble into VLPsunder this condition. This study laid a foundation for further exploring other functional characteristics of P1 virus protein.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S852.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王志軍;郭慧娟;李秀麗;董樹(shù)仁;鄢明華;;豬圓環(huán)病毒病原學(xué)研究進(jìn)展[J];天津農(nóng)業(yè)科學(xué);2014年08期

2 王鳳芝;溫立斌;姚火春;何孔旺;;從蛋白質(zhì)和轉(zhuǎn)錄水平鑒定類(lèi)豬圓環(huán)病毒P1存在ORF2和ORF3[J];中國(guó)農(nóng)業(yè)科學(xué);2014年14期

3 王鳳芝;溫立斌;姚火春;何孔旺;;類(lèi)圓環(huán)病毒P1 VP1蛋白單克隆抗體的制備及應(yīng)用[J];華北農(nóng)學(xué)報(bào);2014年03期

4 溫立斌;何孔旺;解建平;楊浩;周建新;金全勝;王玉然;;類(lèi)豬圓環(huán)病毒P1的免疫電鏡觀察[J];華北農(nóng)學(xué)報(bào);2013年S1期

5 溫立斌;何孔旺;解建平;楊浩;周建新;金全勝;王玉然;;免疫親和層析純化類(lèi)豬圓環(huán)病毒P1[J];華北農(nóng)學(xué)報(bào);2013年S1期

6 Libin WEN;Fengzhi WANG;Bin LI;Yang YU;Zhengyu YU;Aihua MAO;Jianping XIE;Kong-wang HE;;Genetic Diversity of Intragenomic Rearrangement of Porcine Circovirus Type 2 in vitro and in vivo[J];Agricultural Science & Technology;2013年12期

7 王小敏;何孔旺;王東田;周忠濤;茅愛(ài)華;俞正玉;汪偉;倪艷秀;溫立斌;;PCV2、P1和TTV混合感染的流行病學(xué)調(diào)查[J];福建農(nóng)業(yè)學(xué)報(bào);2013年09期

8 溫立斌;何孔旺;王鳳芝;俞正玉;茅愛(ài)華;解建平;;類(lèi)豬圓環(huán)病毒P1的感染及其分子特征[J];華北農(nóng)學(xué)報(bào);2013年05期

9 溫立斌;何孔旺;俞正玉;茅愛(ài)華;高曉靜;倪艷秀;張雪寒;郭容利;李彬;王小敏;周俊明;呂立新;;類(lèi)豬圓環(huán)病毒因子P1在自然感染仔豬體內(nèi)的組織分布[J];中國(guó)獸醫(yī)學(xué)報(bào);2013年01期

10 溫立斌;何孔旺;倪艷秀;張雪寒;李彬;王小敏;郭容利;周俊明;俞正玉;茅愛(ài)華;呂立新;;類(lèi)豬圓環(huán)病毒因子P1核酸鏈型和極性的研究[J];華北農(nóng)學(xué)報(bào);2012年03期

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