嵌合型猪圆环病毒1-2型感染性DNA克隆的构建及其对小鼠的免疫原性  

Construction of infectious DNA clone of chimeric porcine circovirus type 1-2 and its immunogenicity in mice

作　　者：宋益[1] 朱丽娜[1] 高崧[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点开放实验室,扬州225009

出　　处：《微生物学报》2008年第9期1234-1240,共7页Acta Microbiologica Sinica

基　　金：高等学校博士学科点专项科研基金(20041117005);江苏省农业三项工程项目[SX(2007)080]~~

摘　　要：【目的】本研究旨在构建嵌合型猪圆环病毒1-2型感染性DNA克隆。【方法】利用PCR技术扩增PCV2 ORF2,克隆入缺失PCV1 ORF2的pSK-PCV1△ORF2中,得到pSK-sPCV1-2。该重组质粒中包含嵌合型PCV1-2全基因组,通过不完全酶切的方法,将嵌合型PCV1-2全基因组串联入pSK载体中,得到含串联双拷贝的嵌合型PCV1-2感染性DNA克隆。【结果】经序列测定,获得含串联双拷贝的嵌合型PCV1-2感染性DNA克隆。PCV1-2嵌合病毒接种BALB/c小鼠,用间接ELISA方法对接种后7、14、21、28、35、42d的小鼠进行血清抗体检测。结果显示从接种后14d开始,即有部分小鼠产生了针对PCV2Cap蛋白的特异性抗体;至接种后42d,几乎全部接种小鼠的血清均呈阳性。【结论】构建了PCV1-2型感染性DNA克隆,该嵌合病毒能够激发机体产生体液免疫应答。[Objective] To construct infectious DNA clone of chimeric porcine circovirus type 1-2. [Methods] Recombinant plasmid pSK-PCV1AORF2-PCV2-ORF2 （pSK-sPCV1-2） was constructed by replacing the PCV1 capsid gene with that of PCV2 in the backbone recombinant containing the complete genomic DNA of PCV1. The full-length chimeric fragment PCV1-2 was excised from pSK-sPCV1-2 and cloned into the same recombinant to form the dimerized tandem DNA clone pSK-dPCV1-2. [Results] The sequence of PCV1-2 infectious clone was confirmed by sequencing. PCV1-2 virus was observed to be infectious after transfecting into both PK-15 and Dulac cells. BALB/c mice were immunized with recombinant virus PCV1-2 and sera samples collected from all control and vaccinated animals at -1, 7, 14, 21, 28, 35, 42 day post-inoculation （dpi） were assayed for anti-PCV2 capsid antibodies by ELISA. The results indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induced specific antibodies against the pathogenic PCV2 capsid antigen in almost all mice at 42 dpi. [Conclusion] PCV 1-2 infectious clone was constructed, and the recombinant virus strain of PCV1-2 was of some immunogenicity.

关 键 词：PCV1 PCV2 ORF2基因 嵌合型DNA克隆 

分 类 号：S852.5[农业科学—基础兽医学]