Method of preparing coexpression PRRSV ORF5 and ORF6 double-gene nucleic acid vaccine

Abstract

The invention provides a co-expression porcine reproduction and a method for preparing a dual-gene nucleic acid vaccine of respiratory syndrome virus (PRRSV) ORF5 and ORF6. The method comprises the following steps: amplifying the whole gene sequences of the PRRSV ORF5 and the ORF6 respectively by an RT-PCR method, and inserting two genes into two multiple cloning sites of a mammalian eukaryotic expression vector pIRES to obtain recombinant plasmids pIRES-ORF5 and ORF6; transfecting the recombinant plasmids with CHO cells to establish a stable expression cell line; and detecting the transcription of the genes PRRSV ORF5 and ORF6 by an RT-PCR method, and detecting the expressions and the immune activities of GP5 and M proteins through the SDS-PAGE and the Western blot. The nucleic acid vaccine can generate GP5 proteins and M proteins in animals continuously so that organisms generate antibodies against both of the proteins continuously, thus the nucleic acid vaccine provides long-lasting immune protection for pig bodies, and has wide application prospect in PRRS prevention and control.

Description

1. Technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a nucleic acid vaccine capable of effectively controlling porcine reproductive and respiratory syndrome and co-expressing PRRSV ORF5 and ORF6 double genes, which is used to provide porcine reproductive and respiratory syndrome virus to pigs immune protection. 2. Background technology [0002] Porcine reproductive and respiratory syndrome can cause reproductive disorders such as abortion and stillbirth in pregnant sows and respiratory symptoms in piglets. Since the beginning of June 2006, "unknown hyperthermia syndrome" in pigs with highly pathogenic PRRSV as the main pathogen has appeared in more than a dozen provinces such as Hubei, Hunan, Jiangxi, Anhui, etc., which has caused serious damage to my country's pig industry. Economic losses. Porcine reproductive and respiratory syndrome virus is a single-stranded positive-sense RNA virus with a length of about 15Kb. ORF...

AI Technical Summary

Problems solved by technology

At present, attenuated vaccines and inactivated vaccines used to prevent PRRS have been widely used in my country, but porcine reproductive and respiratory syndrome has not been effectively controlled

Nucleic acid vaccines can introduce the genes of virus protective antigens into animals in the form of plasmids, and the expressed proteins can stimulate both humoral immunity and cellular immunity. In the present invention, PRRSV ORF5 and ORF6 genes are simultaneously inserted into eukaryotic recombinant expression vectors to construct Nucleic acid vaccines have not been reported yet

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