类猪圆环病毒P1的转录分析及蛋白功能的初步鉴定

发布时间：2018-06-22 13:39

本文选题：P1病毒 + RT-PCR　；参考：《南京农业大学》2015年硕士论文

【摘要】：类猪圆环病毒P1是从临床疑似猪断奶后多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)病猪的血清中分离到的,全长648个核苷酸,是目前发现的基因组最小的动物病毒,部分序列与猪圆环病毒2型(Porcinecircovirus type 2,PCV2)开放阅读框2(open reading frame2,ORF2)的序列高度同源。前期研究表明,P1病毒可引起转染的PK-15细胞凋亡,也可导致感染猪出现类似PMWS的临床症状。本论文主要内容如下:1.类猪圆环病毒P1 ORF2和ORF3的鉴定P1病毒主要包括3个开放阅读框,为从转录水平和蛋白水平鉴定其ORF2和ORF3,用P1双拷贝分子克隆(pSK-P1)转染PK-15细胞,采用Trizol法提取转染细胞的总RNA,纯化后进行RT-PCR扩增,回收PCR产物并测序。利用cDNA末端快速扩增法(Rapid-amp1ification of cDNAends,RACE)技术分别扩增 ORF2 和 ORF3 的5'-与3'-末端。同时,根据ORF2和ORF3编码的氨基酸序列预测其B细胞抗原表位,采用逐步固相合成法合成多肽,与匙孔血蓝蛋白(Keyhole limpet hemocyanin,KLH)偶联,免疫新西兰大白兔制备多克隆抗体,常规间接ELISA法检测血清效价。通过免疫组化方法检测转染pSK-P1的PK-15细胞与两种多抗的免疫反应性。结果表明:以P1病毒RNA为模板,用ORF2和ORF3特异引物进行的RT-PCR均能扩增出目的基因片段,分别与P1病毒ORF2和ORF3的序列同源性达100%;RACE技术分析,可知P1病毒ORF2的5'-和3'-末端分别在nt648和nt481,ORF3的5'-和3'-末端分别在nt364和nt70。合成肽的氨基酸序列分别为ORF2:CLSRLPQSERPPGRW和ORF3:CVYPKVRERRVLKMP;用ORF2和ORF3表位肽与KLH的偶联物制备的多抗效价均在1:512000以上,免疫组化检测结果显示2种抗体均能够与P1病毒发生特异性显色反应。综上,通过转录分析及免疫组化法分别从转录水平和蛋白水平证实了类猪圆环病毒P1 ORF2和ORF3的存在。2.类猪圆环病毒P1三种主要蛋白在昆虫细胞中的表达分析为鉴定P1病毒3个主要开放阅读框所编码的蛋白能否在体外自组装形成病毒样颗粒(Virus-likeparticles,VLPs),设计3对引物分别扩增P1病毒的ORF1、ORF2和ORF3,连接到载体pFastBacTM1,构建重组表达质粒,脂质体法转染Sf9细胞获得重组杆状病毒(rBac-ORF1、rBac-ORF2 和 rBac-ORF3)。通过 RT-PCR 及 Western blot分别从转录水平和蛋白水平鉴定表达情况,透射电镜观察是否形成VLPs。结果显示,重组质粒转染Sf9细胞后,均可导致细胞病变;从接毒细胞中提取总RNA,除去基因组DNA后,利用RT-PCR能够扩增出相应目的片段;Western blot结果显示,rBac-ORF1表达产物能够与抗P1VP1单抗和多抗发生特异反应;电镜观察发现,3种重组蛋白均可在Sf9细胞中形成近椭圆形的包涵体。综上,本研究利用杆状病毒表达载体系统成功地在Sf9细胞中表达了 P1病毒ORF1、ORF2和ORF3基因,并证实在本试验条件下这3种重组蛋白均不能自组装成VLPs。本研究为进一步探究P1病毒蛋白的其他功能特性奠定了基础。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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