Wang Yuan, Yu Qian, Li Yi, Dong Yanming
College of Medical, Hubei University of Art and Science, Xiangyang 441053, Hubei, China.
School of Life Sciences, Jiangsu University, Zhenjiang 212013, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2020 Oct 25;36(10):2083-2091. doi: 10.13345/j.cjb.200111.
Rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV), are two pathogens that have harmful effect on rabbit breeding and population decline of European rabbits in their native range, causing rabbit haemorrhagic disease (rabbit fever) and myxomatosis, respectively. The capsid protein VP60 of the RHDV represents the major antigenic protein. To develop a recombinant bivalent vaccine candidate that can simultaneously prevent these two diseases, we used the nonessential gene TK (thymidine kinase) of MYXV as the insertion site to construct a recombinant shuttle vector p7.5-VP60-GFP expressing the RHDV major capsid protein (VP60) and the selectable marker GFP. Then the shuttle vector p7.5-VP60-GFP was transfected into rabbit kidney cell line RK13 which was previously infected with MYXV. After homologous recombination, the recombinant virus expressing GFP was screened under a fluorescence microscope and named as rMV-VP60-GFP. Finally, the specific gene-knock in and expression verification of the vp60 and gfp genes of the recombinant virus was confirmed by PCR and Western blotting. The results showed that these two genes were readily knocked into the MYXV genome and also successfully expressed, indicating that the recombinant MYXV expressing the vp60 of RHDV was generated. Protection against MYXV challenge showed that the recombinant virus induced detectable antibodies against MYXV which would shed light on development of the effective vaccine.
兔出血症病毒（RHDV）和黏液瘤病毒（MYXV）是两种对家兔养殖有有害影响并导致其在原生范围内数量下降的病原体，分别引起兔出血症（兔热病）和黏液瘤病。RHDV的衣壳蛋白VP60是主要的抗原蛋白。为了研发一种能够同时预防这两种疾病的重组二价候选疫苗，我们利用MYXV的非必需基因TK（胸苷激酶）作为插入位点，构建了一个表达RHDV主要衣壳蛋白（VP60）和选择标记绿色荧光蛋白（GFP）的重组穿梭载体p7.5-VP60-GFP。然后将穿梭载体p7.5-VP60-GFP转染到先前感染过MYXV的兔肾细胞系RK13中。经过同源重组后，在荧光显微镜下筛选出表达GFP的重组病毒，并命名为rMV-VP60-GFP。最后，通过PCR和蛋白质免疫印迹法对重组病毒的vp6基因和gfp基因进行了特异性基因敲入和表达验证。结果表明，这两个基因很容易敲入MYXV基因组并且成功表达，表明表达RHDV的vp60的重组MYXV构建成功。针对MYXV攻击的保护性研究表明，重组病毒诱导产生了可检测到的抗MYXV抗体，这将为有效疫苗的研发提供线索。
