Abstract
Porcine reproductive and respiratory syndrome (PRRS) is an economically important swine disease that has devastated the swine industry worldwide. Vaccination with live attenuated vaccine or inactivated vaccine is the main treatment to control PRRS. However, the disadvantages such as virulence resumption of the attenuated vaccine and low immunogenicity of the inactivated vaccine call for a more efficient and safer genetically engineered vaccine. In this study, the structural protein GP5 of the PRRS virus (PRRSV), one of the major protective antigens which stimulates a protective immune response was selected to develop a genetically engineered subunit vaccine. In order to promote the immune reaction of the host to GP5, heat shock protein 70 (Hsp70) was selected as immuno-adjuvant to enhance PRRSV GP5 immunogenicity. The Hsp70 gene was amplified by PCR from attenuated Mycobacterium bovis, and the PRRSV GP5 gene was amplified by RT-PCR from the total RNA of PRRSV SCQ strain which was isolated, identified and maintained by the Animal Biotechnological Center, Sichuan Agricultural University, China. The fusion expressing plasmid pET32-GP5-Hsp70 was constructed and expressed in Escherichia coli BL21. Ni2+-chelating resin was used to purify the His-tagged fusion protein expressed under optimized expressing conditions. The rabbit anti-GP5-Hsp70 fusion protein antibody was made, and Western blot assay verified the successful expression of the fusion protein, making it possible for further investigation whether Hsp70 could improve the immunogenicity of the PRRSV GP5 subunit vaccine, or evaluating the immunogenicity of the GP5-Hsp70 subunit vaccine. Key words: Porcine reproductive and respiratory syndrome virus (PRRSV) GP5 gene, Mycobacterium bovis Hsp70 gene, cloning, prokaryotic expression, identification.
Highlights
Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), a positive-sense single stranded enveloped RNA virus of the family Arteriviridae and the genus Arterivirus (Meulenberg 2000)
In order to promote the immune reaction of the host to GP5, heat shock protein 70 (Hsp70) was selected as immuno-adjuvant to enhance PRRSV GP5 immunogenicity
The Hsp70 gene was amplified by PCR from attenuated Mycobacterium bovis, and the PRRSV GP5 gene was amplified by Reverse transcription (RT)-PCR from the total RNA of PRRSV SCQ strain which was isolated, identified and maintained by the Animal Biotechnological Center, Sichuan Agricultural University, China
Summary
Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), a positive-sense single stranded enveloped RNA virus of the family Arteriviridae and the genus Arterivirus (Meulenberg 2000). PRRS has caused enormous financial losses to swine industry worldwide with clinical signs such as respiratory disease in growing pigs and reproductive failure in breeding swine (Tong et al, 2007). From 2006 till a highly pathogenic PRRSV spread widely in China and has given a serious problem to Chinese swine industry (Li et al, 2009). PRRSV is subject to heterogenesis and always coinfects with other pathogens, such as Steptococcus suis, Haemophilus parasuis (H. parasuis), Mycoplasma hyopneumoniae, Actinobaccillus pleuropneumoniae, Salmonella spp., Pasteurella multocida, and swine influenza virus, which makes it more difficult to prevent and control (Groschup et al, 1993; Zeman et al, 1993). Gene Primer PRRSV GP5 (upper) P1 5'-AATGGATCCCTCGCCAGCAGCGAC-3' PRRSV GP5 (lower) P2 5'-CCGAAGCTT AGGACGACCCCATTG -3' Hsp (upper) P3 5'-AATAAGCTTACCATGGCTC GTGCG-3' Hsp (lower) P4 5'-AATCTCGAGTGCCATCCGGCTTTT-3'
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