PCV2单克隆抗体的制备及多拷贝P28-ORF2重组猪痘病毒的构建
发布时间：2019-06-22 07:51
【摘要】：猪圆环病毒感染是由猪圆环病毒2型(Porcine Circovirus type 2,PCV2)引起的猪传染病。临床上以消瘦、体质下降、呼吸困难为主要特征给我国的养猪业造成了巨大的损失,猪圆环病毒衣壳蛋白(Cap蛋白)是主要的免疫原性蛋白。猪痘(Swinepox)是由猪痘病毒(Swinepox Virus,SWPV)引起猪皮肤形成痘斑和痘痂为特征的猪传染病,绝大部分猪能自行康复。因猪痘病毒基因组大(约146 kb),复制非必需区多,可携带多个外源片段且具有严格的宿主特异性,是一种理想的基因工程疫苗载体。本研究构建了8个含有P28-ORF2不同拷贝数的重组质粒,利用同源重组技术,得到8株重组猪痘病毒。旨在探索不同拷贝数P28-ORF2重组猪痘病毒表达Cap蛋白含量的关系。1.猪圆环病毒2型单克隆抗体的制备本研究基于PCV2 JX-CZH株ORF2目的基因,以猪痘病毒为载体,采用同源重组技术得到表达PCV2衣壳蛋白(PCV2-Cap)的重组猪痘病毒,以重组猪痘病毒表达的PCV2-Cap为抗原免疫BALB/c鼠,应用杂交瘤技术,以原核表达的His-Cap蛋白为检测抗原,筛选到4株PCV2单克隆抗体。Westernblotting、免疫荧光实验结果表明:4株单克隆抗体不仅能应用于PCV2-Cap的Western-blotting检测,也能与PCV2病毒粒子特异性结合,应用于PCV2的免疫荧光和免疫组化染色。本研究获得的4株PCV2单克降抗体为今后的PCV2-Cap的定性和定量检测方法的建立打下了基础。2.表达PCV2-Cap蛋白的多拷贝P28-ORF2重组猪痘病毒构建为了解猪痘病毒载体表达PCV2-Cap时,插入猪痘病毒基因组中的P28-ORF2拷贝数与PCV2-Cap表达量的关系,以猪痘病毒为载体,采用分子生物学方法,构建了8株不同拷贝数(1-8拷贝)P28-ORF2的重组重组猪痘病毒,并测定了8株重组猪痘病毒的部分微生物学特性。(1)重组病毒的荧光斑大小(330μm左右)、TCID50(约10-12/0.1mL)测定结果表明,猪痘病毒基因组中插入不同拷贝数P28-ORF2后,对重组猪痘病毒在PK15细胞内的增殖无影响。(2)电镜观察结果显示:不同拷贝数的重组猪痘病毒粒子形态与亲本病毒一致。(3)Western-blotting结果:1拷贝的PCV2-Cap表达量最低;随着P28-ORF2拷贝数的增加,PCV2-Cap表达量也随之增加;至4拷贝P28-ORF2时,PCV2-Cap表达量接近峰值。本研究结果表明基于猪痘病毒载体表达外源蛋白时,单启动子多拷贝重组猪痘病毒能明显提高外源蛋白的表达量,4拷贝为较合适的拷贝数。
[Abstract]:Swine circovirus infection is a swine infectious disease caused by swine circovirus type 2 (Porcine Circovirus type 2, PC 2. The clinical characteristics of emaciation, physique decline and dyspnea have caused great losses to the pig industry in China. Pig circovirus capsid protein (Cap protein) is the main immunogenic protein. Swine pox (Swinepox) is a swine infectious disease characterized by the formation of acne spots and scab in pig skin caused by swine pox virus (Swinepox Virus,SWPV). Most pigs can recover by themselves. Because of the large genome of swine pox virus (about 146 kb), replication), which can carry many foreign fragments and has strict host specificity, it is an ideal genetic engineering vaccine vector. In this study, eight recombinant plasmids containing different copy numbers of P28-ORF2 were constructed. Eight strains of recombinant swine pox virus were obtained by homologous recombination technique. The purpose of this study was to explore the relationship between the expression of Cap protein in recombinant swine pox virus (P28-ORF2) with different copy numbers. Preparation of monoclonal antibody against swine circovirus type 2 based on ORF2 target gene of PCV2 JX-CZH strain, recombinant swine pox virus expressing PCV2 capsid protein (PCV2-Cap) was obtained by homologous recombination technique. PCV2-Cap expressed by recombinant swine pox virus was used as antigen to immunize BALB/c mice. Four strains of PCV2 monoclonal antibody were screened by hybridoma technique and prokaryotic expression His-Cap protein as detection antigen. The results of immunofluorescence assay showed that the four monoclonal antibodies could not only be used for the detection of Western-blotting in PCV2-Cap, but also specifically bind to PCV2 virus particles, and could be used in immunofluorescence and immunohistochemical staining of PCV2. The four strains of PCV2 monogram antibody obtained in this study laid a foundation for the establishment of qualitative and quantitative detection methods of PCV2-Cap in the future. 2. Multi-copy P28-ORF2 recombinant swine pox virus expressing PCV2-Cap protein was constructed to understand the relationship between the number of P28-ORF2 copies inserted into the genome of swine pox virus and the expression of PCV2-Cap when PCV2-Cap was expressed by swine pox virus vector. Eight recombinant swine pox viruses with different copy numbers (1 鈮，
本文编号：2504387
[Abstract]:Swine circovirus infection is a swine infectious disease caused by swine circovirus type 2 (Porcine Circovirus type 2, PC 2. The clinical characteristics of emaciation, physique decline and dyspnea have caused great losses to the pig industry in China. Pig circovirus capsid protein (Cap protein) is the main immunogenic protein. Swine pox (Swinepox) is a swine infectious disease characterized by the formation of acne spots and scab in pig skin caused by swine pox virus (Swinepox Virus,SWPV). Most pigs can recover by themselves. Because of the large genome of swine pox virus (about 146 kb), replication), which can carry many foreign fragments and has strict host specificity, it is an ideal genetic engineering vaccine vector. In this study, eight recombinant plasmids containing different copy numbers of P28-ORF2 were constructed. Eight strains of recombinant swine pox virus were obtained by homologous recombination technique. The purpose of this study was to explore the relationship between the expression of Cap protein in recombinant swine pox virus (P28-ORF2) with different copy numbers. Preparation of monoclonal antibody against swine circovirus type 2 based on ORF2 target gene of PCV2 JX-CZH strain, recombinant swine pox virus expressing PCV2 capsid protein (PCV2-Cap) was obtained by homologous recombination technique. PCV2-Cap expressed by recombinant swine pox virus was used as antigen to immunize BALB/c mice. Four strains of PCV2 monoclonal antibody were screened by hybridoma technique and prokaryotic expression His-Cap protein as detection antigen. The results of immunofluorescence assay showed that the four monoclonal antibodies could not only be used for the detection of Western-blotting in PCV2-Cap, but also specifically bind to PCV2 virus particles, and could be used in immunofluorescence and immunohistochemical staining of PCV2. The four strains of PCV2 monogram antibody obtained in this study laid a foundation for the establishment of qualitative and quantitative detection methods of PCV2-Cap in the future. 2. Multi-copy P28-ORF2 recombinant swine pox virus expressing PCV2-Cap protein was constructed to understand the relationship between the number of P28-ORF2 copies inserted into the genome of swine pox virus and the expression of PCV2-Cap when PCV2-Cap was expressed by swine pox virus vector. Eight recombinant swine pox viruses with different copy numbers (1 鈮，
本文编号：2504387
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