表达O型口蹄疫病毒保护性抗原表位重组PRRSV的构建及其鉴定

Construction and identification of a recombinant PRRSV expressing protective antigens of type O foot-and-mouth disease virus

摘要:

以高致病性猪繁殖与呼吸综合征弱毒疫苗的感染性分子克隆 (rHuN4-F112) 作为载体，将O型口蹄疫病毒 (FMDV) VP1基因的421~480nt (141~160aa) 和598~639nt (200~213aa) 两优势保护性抗原表位串联成的目的基因，通过突变PCR的方法插入Nsp2中的508~532位缺失区域，经体外转录后转染至BHK-21细胞中培养36 h，将上清接种至MARC-145细胞中培养，并在MARC-145细胞中连续传代，拯救重组病毒。经RT-PCR扩增，MluⅠ酶切及测序验证，结果表明插入的外源基因及人为突变的MluⅠ分子标记都正确，说明重组病毒拯救成功，且该重组病毒能够在MARC-145细胞中稳定传代，将此重组病毒命名为rPRRSV-F112-O/VP1ep。rPRRSV-F112-O/VP1ep能够在MARC-145细胞上引起明显的细胞病变，间接免疫荧光检测表明外源基因在该病毒中成功获得了表达。经过生物学特性分析，该病毒的TCID50=-log10-6.75/0.1 mL, 且在MARC-145细胞中整体生长速度与其亲本病毒rHuN4-F112(△508-532) 相似，但明显高于rHuN4-F112病毒。

Abstract:

Using mutation PCR, we cloned the target gene containing 421?480nt (141?160aa) and 598-639nt (200?213aa) of VP1 gene of foot and mouth disease virus (FMDV) into the deleted region (508?532aa) of Nsp2 gene of a highly pathogenic porcine reproductive and respiratory syndrome virus derived vaccine strain (HuN4-F112) that was used as vector. The recombinant cDNA was in vitro transcribed followed by trancfection of BHK-21 cells for 36 h. Then, the supernatant of the cell culture was continuously seeded to monolayer of MARC-145 cells for recovery of the recombinant virus. CPE was obviously visible after a couple of passages in the seeded MARC-145, and the rescued virus (designated as rPRRSV-F112-O/VP1ep) was identified by Mlu I digestion, sequencing and immunofluorescence assay. Meanwhile, expression of inserted FMDV epitopes was also detected by indirect immunofluorescence assay with polyclonal antibodies against VP1 protein of FMDV. The analysis of biological characteristics shows that the titer of the rescued recombinant PRRSV (TCID50=?log10?6.75/0.1 mL) was similar to its direct parental virus rHuN4-F112-△508-532, but higher than rHuN4-F112.