【摘要】：豬圓環(huán)病毒感染是由豬圓環(huán)病毒2型(Porcine Circovirus type 2,PCV2)引起的豬傳染病。臨床上以消瘦、體質(zhì)下降、呼吸困難為主要特征給我國的養(yǎng)豬業(yè)造成了巨大的損失,豬圓環(huán)病毒衣殼蛋白(Cap蛋白)是主要的免疫原性蛋白。豬痘(Swinepox)是由豬痘病毒(Swinepox Virus,SWPV)引起豬皮膚形成痘斑和痘痂為特征的豬傳染病,絕大部分豬能自行康復。因豬痘病毒基因組大(約146 kb),復制非必需區(qū)多,可攜帶多個外源片段且具有嚴格的宿主特異性,是一種理想的基因工程疫苗載體。本研究構建了8個含有P28-ORF2不同拷貝數(shù)的重組質(zhì)粒,利用同源重組技術,得到8株重組豬痘病毒。旨在探索不同拷貝數(shù)P28-ORF2重組豬痘病毒表達Cap蛋白含量的關系。1.豬圓環(huán)病毒2型單克隆抗體的制備本研究基于PCV2 JX-CZH株ORF2目的基因,以豬痘病毒為載體,采用同源重組技術得到表達PCV2衣殼蛋白(PCV2-Cap)的重組豬痘病毒,以重組豬痘病毒表達的PCV2-Cap為抗原免疫BALB/c鼠,應用雜交瘤技術,以原核表達的His-Cap蛋白為檢測抗原,篩選到4株PCV2單克隆抗體。Westernblotting、免疫熒光實驗結果表明:4株單克隆抗體不僅能應用于PCV2-Cap的Western-blotting檢測,也能與PCV2病毒粒子特異性結合,應用于PCV2的免疫熒光和免疫組化染色。本研究獲得的4株PCV2單克降抗體為今后的PCV2-Cap的定性和定量檢測方法的建立打下了基礎。2.表達PCV2-Cap蛋白的多拷貝P28-ORF2重組豬痘病毒構建為了解豬痘病毒載體表達PCV2-Cap時,插入豬痘病毒基因組中的P28-ORF2拷貝數(shù)與PCV2-Cap表達量的關系,以豬痘病毒為載體,采用分子生物學方法,構建了8株不同拷貝數(shù)(1-8拷貝)P28-ORF2的重組重組豬痘病毒,并測定了8株重組豬痘病毒的部分微生物學特性。(1)重組病毒的熒光斑大小(330μm左右)、TCID50(約10-12/0.1mL)測定結果表明,豬痘病毒基因組中插入不同拷貝數(shù)P28-ORF2后,對重組豬痘病毒在PK15細胞內(nèi)的增殖無影響。(2)電鏡觀察結果顯示:不同拷貝數(shù)的重組豬痘病毒粒子形態(tài)與親本病毒一致。(3)Western-blotting結果:1拷貝的PCV2-Cap表達量最低;隨著P28-ORF2拷貝數(shù)的增加,PCV2-Cap表達量也隨之增加;至4拷貝P28-ORF2時,PCV2-Cap表達量接近峰值。本研究結果表明基于豬痘病毒載體表達外源蛋白時,單啟動子多拷貝重組豬痘病毒能明顯提高外源蛋白的表達量,4拷貝為較合適的拷貝數(shù)。
[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，

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