ORF9基因缺失突变PCV2的构建与部分生物学特性

设计1对针对猪圆环病毒2型（PCV2）全基因组的特异性引物,从疑似断乳仔猪多系统衰竭综合征（PMWS）的病料中经PCR直接扩增出PCV2全基因组,再与载体pMD18-T Simple连接后构建重组质粒pMD18-T Simple-PCV2（命名为P-S-PCV2）。用ORF9特异的限制性内切酶Bpu10Ⅰ对P-S-PCV2进行酶切、补平连接反应,构建了ORF9基因缺失突变的重组质粒（命名为P-S-PCV2-J）。用SacⅡ对基因缺失突变的重组质粒进行酶切,获得的线性化基因突变PCV2基因组在体外进行自身环化,形成了相应缺失基因DNA（命名为PCV2-J）。用PCV2-J缺失突变株进行细胞转染、动物致病性和免疫原性、T淋巴细胞亚群的动态变化等部分生物学特性的研究。结果显示：PCV2-J转染IBRS-2细胞后,电镜观察可见病毒颗粒,PCR-RFLP检测有突变株生长;PCV2-J接种仔猪后无临床典型大体病变,PCR-RFLP检测淋巴结中有PCV2-J突变病毒的感染;PCV2-J免疫仔猪后的抗体水平在第2周开始上升,与对照组相比差异显著,CD3＋下降,与对照组无差异,CD4＋下降,第1周与对照组差异显著,CD8＋与对照组差异不显著。结果表明,ORF9基因缺失突变的PCV2仍具有复制感染能力,但免疫原性减弱。

Construction of PCV2 mutated in ORF9 gene and its biologic characteristics

After designed a pair of specific primer,the porcine circovirus type 2（PCV2） complete genome was amplified directly by PCR from the pigs with doubtful PMWS and cloned into pMD18-T Simple vector to construct the recombinant plasmid pMD18-T Simple-PCV2（named P-S-PCV2） in this study.To construct recombinant plasmid P-S-PCV2 mutated in ORF9 gene（named P-S-PCV2-J）,the P-S-PCV2 was digested and connected with restriction enzyme Bpu10Ⅰ.Then,the P-S-PCV2-J was digested with SacⅡ to form linear PCV2 genome which became the DNA mutated in ORF9 gene（named PCV2-J） after circling by self-ligation.The mutated PCV2-J virus was transfected into IBRS-2 cells,infected pigs and determined its ability of infection and immunity and the dynamic variety of T lymphocyte sub-group to study its biologic characteristics.As a result,the virus antigen and nucleic acid were discovered in the IBRS-2 cells and tissues.Compared with the control group,the antibody began to increase in pigs inoculated PCV2-J at the 2nd week,the CD3＋ and CD4＋ decreased,the CD8＋ showed no marked difference.The results proved that the PCV2 mutated in ORF9 still has the ability of replication and infection,but its ability of immunity is weaker than the original strain.