表达猪源GM-CSF蛋白的重组PRRSV构建及鉴定

Construction and identification of a recombinant live attenuated PRRSV expressing porcine GM-CSF

为提高猪繁殖与呼吸障碍综合征(PRRS)弱毒疫苗的免疫效果,本研究以高致病性PRRS弱毒疫苗株HuN4-F112感染性分子克隆为基础,在NSP2区域删除了110个氨基酸,将猪源GM-CSF基因和PRRSV ORF6转录引导序列(TRS6)通过融合PCR方法插入HuN4-F112疫苗株的ORF1b和ORF2a之间,构建了全长重组质粒pHN4-△110-GM-CSF。将该重组质粒采用SwaⅠ酶切线性化,经体外转录病毒基因组RNA后转染BHK-21细胞,然后在Marc-145细胞中进行病毒拯救及传代。结果显示重组病毒感染Marc-145细胞可以引起明显的细胞病变;经RT-PCR、DNA测序、MluⅠ酶切鉴定和免疫荧光鉴定结果显示,救获的重组PRRSV含有GM-CSF基因并能够有效表达,将其命名为rHN4-△110-GM-CSF。生物学特性分析表明重组病毒与亲本病毒在Marc-145细胞中具有相似的生长特性,插入的外源基因具有良好的遗传稳定性。由于GM-CSF能够活化树突状细胞,是一种良好的免疫佐剂。该重组病毒的构建为动物体内免疫保护攻毒试验评价奠定了基础。

To improve protection efficacy of the attenuated porcine reproductive and respiratory syndrome （PRRS） vaccine, the porcine GM-CSF gene and the transcription regulatory sequence for ORF6 （TRS6） of PRRSV were joined together by overlap PCR, and inserted between the ORFlb and ORF2a of attenuated PRRSV HuN4-F112 vaccine strain with the deletion of a sequence encoding 110 amino acids in the region of Nsp2 to construct the full length viral cDNA recombinant plasmid of pHN4- A110-GM-CSF by reverse genetics techniques. To rescue the recombinant virus, capped RNAs of the viral genome were transcribed in vitro from the linearized pHN4-A110-GM-CSF digested with Swa Ⅰ . Then the recombinant virus of rHN4- △ 110-GM-CSF was successfully rescued by transfection of the viral genomic RNAs into BHK-21 cells and passaged on Marc-145 cells which shown obviously cytopathic effects. In addition, the GM-CSF gene insertion and expression in rescued rHN4- △ 110-GM-CSF were detected by RT-PCR, DNA sequencing and immunofluorescence assay. It was also demonstrated that the inserted GM-CSF gene was stably integrated in rHN4-△ 110-GM-CSF and no impact on the virus growth characters as the parental strain. The generated recombinant PRRSV vaccine strain expressing the GM-CSF as an immune-adjuvant provides a strong support to develop an efficient vaccine with a deletion gene maker.