利用猪瘟病毒Npro蛋白水解酶活性融合表达标记疫苗marker的研究

Two main problems to develop CSF marker vaccines using reverse-genetics techniques are the locations where the marker is inserted into classical swine fever virus (CSFV) genome and the ability of inducing a response differentiable between infected and vaccinated animals by the vaccines. Here we describe a expression strategy of vaccine's marker using the autoproteolytic function of Npro from CSFV. FLAG as fusions with wild-type CSFV Npro is acquired and cleavaged from the polyprotein of CSFV by ligating of Npro sequence to the 3' terminal sequences of FLAG. The presence of an N-terminal secretion signal peptide validates FLAG-Npro being secreted to the extracellular space. Specifically, the "signal peptide-FLAG-Npro" sequence is inserted into the site between 5' terminal sequences and Npro of the full-length cDNA clone pACYC-SM of CSFV strain Shimen. RNA transcribed in vitro from the resulting plasmid was transfected into PK-15 porcine kidney cells and infectious progeny virus recovered from transfected cells. The genetic stability of the recombinant virus and the predicted fusion protein, FLAG-Npro will be detected. Further, essential residues in Npro for proteolysis will be identi?ed by constructing of deletion mutants of Npro. These results will provide important strategies for successful developing of a live marker vaccine against CSFV.

利用猪瘟反向遗传技术研制标记疫苗的关键问题一是确定marker序列插入病毒基因中的位置及marker表达策略；二是解决鉴别诊断的问题。本项目拟对问题一进行研究，提出利用猪瘟病毒Npro蛋白水解酶活性融合表达标记疫苗marker的科学假想，通过在FLAG标签序列后连接Npro蛋白达到FLAG-Npro融合表达并依靠Npro自体蛋白酶活性从多聚蛋白上分离；通过FLAG序列前加信号肽达到FLAG-Npro表达后分泌到细胞外的效果。具体内容是利用建立的石门毒株反向遗传平台（pACYC-SM），将"信号肽-FLAG-Npro"序列插入到pACYC-SM中猪瘟病毒全长cDNA的5'-NTR与Npro之间，最终拯救出重组病毒。检测重组病毒遗传稳定性和分泌表达FLAG效果，再通过截短表达技术确定重组病毒中Npro发挥自体蛋白酶活性的关键氨基酸序列，为后续成功研制猪瘟标记疫苗提供重要策略。

猪瘟标记疫苗免疫猪能产生与野毒感染相鉴别的抗体，对剔除带毒猪净化猪群具有重要实践意义，目前国内外尚无商品化猪瘟标记疫苗。利用猪瘟反向遗传技术研制标记疫苗的关键问题一是确定marker序列插入病毒基因中的位置及marker表达策略；二是解决鉴别诊断的问题。本项目首先通过分段扩增和拼接CSFV石门株全基因组，成功构建了的CSFV 石门毒株全长感染性克隆，在此基础之上，将“信号肽-FLAG-Npro”序列插入到pACYC-SM中猪瘟病毒全长cDNA的5’-NTR与Npro之间，成功构建了含FLAG标签的CSFV 石门毒株全长的克隆质粒，然而转染细胞，最终没有拯救出重组病毒。分析原因可能是由于外源基因的插入，造成CSFV石门毒株基因组克隆质粒不稳定，具体原因不明，有待进一步研究。在项目执行期间，发表学术论文3篇，其中SCI论文2篇。