糖基化位点突变对猪圆环病毒2型增殖的影响

Effect of glycosylation site mutation on proliferation of porcine circovirus type 2

为提高猪圆环病毒（PCV）的滴度，以本实验室已成功分离的1株PCV2d流行毒株PCV2 RC160307为模板，通过融合PCR突变其Rep蛋白上的1个N-糖基化位点，并成功构建自身环化感染性克隆HT—PCV2，转染ST细胞，盲传至F8代后，通过PCR、间接免疫荧光（IFA）及过氧化物酶单层试验（IMPA）进行验证，并通过IMPA测定TCID50发现，拯救成功的HT-PCV2病毒滴度为10^5.2×TCID50／0.1mL，较分离的流行毒株RC160307及未糖基化位点突变的感染性克隆HPCV2的病毒滴度有所提高。此种方法为PCV2复制机制的进一步研究疫苗的研制及其疾病的预防奠定基础。

In order to improve PCV2 virus titer, infectious clone was successfully constructed and one N-glycosylation site of Rep protein were mutated by means of fusion PCR with PCV2d epidemic isolates PCV2 RC160307 as template. Then ST cells was transfected,verification was build according to PCR,IFA and IMPA for the recombinant virus F8 and TCID50 was measured by IMPA. The virus titer of the recombinant virus F8 increased by 10^5.2×TCID50/0.1mL compared with the epidemic isolates PCV2 RC160307 and the infectious clone with no N- glycosylation sites mutation. The way provided theroy basis for PCV2 replication mechanism and layed a fundation for PCV2 vaccine development and prevention.