Cloning, Sequencing and Expression of ORF7 Gene of Porcine Reproductive and Respiratory Syndrome Virus B_(13) Strain in a Baculovirus Expression System
ZHAO Yun1, LUO Chang-bao2, CHEN Ru2, LIN Zhi-xiong2, LI Shu-gen2, CHEN Bo-wen2, LIU Shang-gao3 (1. National Control Institute of Veterinary Bioproducts and Pharmaceuticals, Beijing 100081, China; 2. Guangzhou Animal and Plant Quarantine Bureau, Guang
The DNA fragments were amplified separately by one-tube RT-PCR, in which contained the complete ORF 7 gene of B 13 strain of PRRSV. The ORF 7 gene of B 13 strain of PRRSV was sequenced by automatic sequencing. The sequence of ORF 7 gene consisted of 384bp. The predicted polypeptide of ORF 7 gene consisted of 128 amino acids, and its molecular weight was about 15kD. After comparing the ORF 7 gene of B 13 strain with that of LV and VR-2332 which have been published previously, it was found that the nucleotide sequence of B 13 was 99.2% and 59.4% identical to LV and VR-2332 respectively, the predicted amino acid sequence was 98.4% and 54.7% identical with LV and VR-2332 respectively. The results showed that the B 13 strain of PRSV and the LV strain both may belong to a same subgroup, the European subgroup. Meanwhile, recombinant plasmid vector pAcGHLT-B-ORF 7 was constructed. After cotransfecting the cultured Sf9 cells with pAcGHLT-B-ORF 7 and the linearized genome DNA of AcNPV-SVI-G and TnNPV-SVI-G respectively, the recombinant virus AcNPV-OCC--GST-6×His-ORF 7 was obtained. In the Sf9 cells infected with the recombinant virus, the expressed ORF 7-GST fusion protein of B 13 strain with molecular weight about 46kD was successfully detected by Western-blot using anti-PRRSV B 13 polyclonal sera. The results of this study lay a foundation for developing new PRRS diagnostic antigen.
【CateGory Index】： S852.65