猪细环病毒1型感染性克隆构建及致病性分析

Torque teno sus virus 1 (TTSuV1) is a newly emergent virus, whether it is aetiological or not is unknown to day. In this project, the infectious DNA clone of TTSuV1 will be constructed based on the complete genome clone plasmid of the TTSuV1 strain identified by our lab, and transfection with the infectious TTSuV1 DNA clone in several cell lines will be performed to obtain the rescue virus TTSuV1. Then, the adaptability of the rescue TTSuV1 in different cell lines will be investigated preliminarily. Meanwhile, the indirect enzyme-linked immune absorbent assay (ELISA) to detect the antibody against TTSuV1 will be established. Therefore, the infectious clone and antibody detection method of TTSuV1 will be developed, which the technical and material foundation will be built for the further exploration of the culture in vitro and pathogenicity analysis of TTSuV1.

目前，猪细环病毒1型（TTSuV1）作为一种新近发现的病毒，其是否具有致病性尚不清楚。本项目拟以我们克隆鉴定的TTSuV1全基因组克隆质粒为基础，构建TTSuV1的感染性DNA克隆，转染适宜细胞获得拯救病毒，并初步探索拯救病毒的细胞适应性；同时建立检测TTSuV1抗体的间接ELISA方法，从而构建出TTSuV1感染性克隆及其抗体检测方法，为TTSuV1的体外培养体系及其致病性分析等进一步研究建立技术与材料基础。

目前，猪细环病毒1型（TTSuV1）作为一种新近发现的病毒，其是否具有致病性尚不清楚。用截短的TTSuV1 ORF1蛋白免疫小鼠，免疫小鼠血清能够与其发生阳性western blotting反应，说明制备出了抗TTSuV1 ORF1蛋白的抗体。并用截短的ORF1蛋白建立了检测TTSuV1抗体的间接ELISA方法。以克隆鉴定的TTSuV1全基因组克隆质粒为基础，构建了环状TTSuV1全基因组，用其转染PK-15、PIEC、MARC-145、ST、IPEC、Vero、HEK293等细胞，间接免疫荧光结果显示，PK-15、MARE-145、IPEC、HEK293细胞中均可见特异性绿色荧光，表明获得了TTsuV1拯救病毒，成功构建了猪细环病毒1型的感染性DNA克隆。初步探索了拯救病毒的细胞适应性，结果发现TTSuV1在这些细胞中的适应性差。研究结果表明，建立了检测TTSuV1抗体的间接ELISA方法，构建了TTSuV1的感染性DNA克隆，为进一步探索TTSuV1的细胞适应性和分析其致病性建立了较好的实验基础。