猪圆环病毒2型感染性克隆构建

Construction of an Infectious Clone of Porcine Circovirus Type 2

关键词:  猪圆环病毒2型  感染性克隆  病毒拯救  遗传进化分析

Key words:  porcine circovirus type 2  infectious cloning  virus rescue  genetic evolution analysis

摘要:

基于PCV2滚环复制原理，在分析PCV2不同基因亚型代表毒株基因组结构和序列的基础上，设计2对引物用于PCR扩增PCV2基因组序列。以从收集到的疑似PCV2感染的10份猪血清样品中提取的DNA为模板，用设计的引物扩增目的片段，并进行测序分析，1株为PCV2a型，4株为PCV2b型，5株为PCV2d型。每种基因型毒株中各选取一个样本，利用上述2对引物进行PCR扩增，并将扩增产物克隆至pEASY-Blunt simple载体中，通过双酶切连接2个PCR片段，构建含有约1500 bp重叠序列的PCV2基因组的质粒。通过脂质体转染法将含PCV2全基因组的质粒转染至PK-15细胞进行病毒拯救，经PCR和IFA两种方法验证，证明成功拯救出3个基因型的PCV2毒株。通过对PCV2全基因组结构及序列分析，该方法同样适用于PCV2g、PCV2h基因型病毒的感染性克隆构建。本研究建立了一种基于反向遗传学技术的PCV2感染性克隆的构建方法，为开展PCV2的病原学研究提供了技术支持。

Abstract:

Based on the rolling loop replication principle of porcine circovirus type 2 (PCV2), two primers were designed for PCR amplification of the PCV2 genome sequence, taking into account the analysis of the genome structure and sequence of representative strains from different gene subtypes of PCV2. Using the DNA extracted from 10 pig serum samples suspected to be infected with PCV2 as templates, the target fragments were amplified with the designed primers and subjected to sequencing analysis. Among them, one strain was PCV2a type, four strains were of the PCV2b type, and five strains were of the PCV2d type. A sample of each genotype strain was selected and PCR amplified with the above two pairs of primers, and the amplification product was cloned into the pEASY-Blunt simple vector, and two PCR fragments were ligated by double digestion to construct a plasmid containing about 1500 bp overlapping sequences of PCV2 genome. To rescue the PCV2 virus, the plasmid containing the complete PCV2 genome was transfected into PK-15 cells using the liposome transfection method. The successful rescue of the PCV2 virus was confirmed through PCR and immunofluorescence assay (IFA), validating the rescue of three PCV2 genotypes. By analyzing the whole genome structure and sequence of PCV2, this method is also suitable for the construction of infectious clones of PCV2g and PCV2h genotypes. In this study, a method of constructing PCV2 infectious clone based on reverse genetics technology was established to provide technique support for the etiology research of PCV2.