Construction and transfection experiment of a goose circovirus infectious clone.
- Author:
Ya-Ping XU
1
;
Jing TIAN
;
Hai-Xia YUAN
;
Jing GUO
;
Hong-Xia SUN
;
Wen-Wen LI
;
Wei-Hu CHEN
;
Xu-Ping YU
Author Information
1. Key Laboratory of Animal Virology of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Circovirus;
genetics;
Geese;
virology;
Real-Time Polymerase Chain Reaction;
Transfection
- From:
Chinese Journal of Virology
2012;28(1):29-34
- CountryChina
- Language:Chinese
-
Abstract:
A pair of primers with BamH I restriction site were designed to amplify the complete genome of goose circovirus. Two copies of the genome were ligated in tandem and cloned into pGEM-T Easy vector to construct an infectious clone named as pGEMT-2GoCV. The pGEMT-2GoCV linearized with EcoR I was transfected to negative embryos and gosling with Lipfectamine. PCR detection verified the proliferation of GoCV in geese. Some sera of the embryo transfected group were detected to be positive at 2 and 4 weeks after hatching and one bursa was detected to be positive at 4 weeks. Some sera of the gosling transfected group were also detected to be positive at 2 weeks after transfection. Furthermore, the mark in the PCR products were identified by BamH I digestion and the GoCV in positive tissue and sera were quantitated by Real-time PCR. The results showed that the virus load in positive bursa was 1.57 x 10(6) copies/mg, the virus load in positive sera were 3.52 x 10(4)-5.92 x 10(5) copies/microL. In conclusion, the infectious DNA clone constructed with two copies of full-length GoCV genome in tandem can transfect embryo and gosling and propagate the marked goose circovirus.
