Method for preparing porcine circovirus 2 open reading frame 2 (ORF2) protein

A circovirus and ORF2 technology, which is applied in the field of preparation of porcine type II circovirus ORF2 protein, can solve the problems of high price, high expression cost, lack of production inspection methods and standards, etc., so as to reduce production costs and avoid protein denaturation. Effect

CN102994518AInactive Publication Date: 2013-03-27SHANDONG SINDER TECH
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Patent Information

Authority / Receiving Office
CN · China
Current Assignee / Owner
Publication Date
2013-03-27
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a method for preparing a porcine circovirus 2 open reading frame 2 (ORF2) protein. A Cap gene of an encoding porcine circovirus 2 (PCV2) nucleocapsid protein is connected to a pET-28a(+) plasmid to build an expression plasmid pET-28-Cap, and a BL21(DE3) competent cell is converted to obtain a genetic engineering strain pET-28-Cap / BL21. Induction expression and detection prove that the recombinant protein has good reactionogenicity; furthermore, the protein can be used for diagnosing antigen or preparing subunit vaccines; and the product can be expressed in Escherichia coli in a soluble manner, the expression quantity is large and a complicated process of albuminous degeneration repeatability is avoided, and the loss and production cost of the protein are lowered.
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Description

technical field

[0001] The invention relates to a method for preparing porcine type II circovirus ORF2 protein. Background technique

[0002] Porcine circovirus type II (PCV2) is one of the newly discovered animal viruses in recent years. It belongs to the Circoviridae family. , is the smallest animal virus found so far. The disease was first discovered in western Canada in 1991, and is divided into PCV-1 and PCV-2 according to the pathogenicity. Among them, PCV-2 has strong infectivity to pigs, and can infect pigs of different ages through the oral and respiratory routes, causing multisystemic wasting syndrome (PMWS) in weaned piglets. Infected pigs have progressive emaciation, sluggishness, dyspnea, jaundice, pale skin, and even death. Etiological and serological investigations have shown that the disease is widespread in many countries and has caused great losses to the world's pig industry.

[0003] The PCV-2 genome contains ORF1 and ORF2. ORF1 encodes the Rep protein...

Examples

Embodiment Construction

[0022] see figure 1 and figure 2 Shown, the preparation method of a kind of porcine type II circovirus ORF2 protein of the present invention comprises the following steps:

[0023] 1. Sequence Synthesis

[0024] Through sequence alignment, the nucleotide sequence is changed under the premise of ensuring that the encoded amino acids are the same, the rare codons in the sequence are replaced with E. coli preferred codons, and EcoR I and Not I restrictions are added to the two ends of the sequence respectively Restriction site, synthesize cap gene, connect to PES vector, digest according to the set restriction endonuclease, clone the target fragment into pMD18-T plasmid vector, transform competent DH5a cells, pick positive colonies , A small amount of plasmid was extracted with OMEGA kit, and the positive plasmid was identified by enzyme digestion.

[0025] 2. Construction and identification of prokaryotic expression vector

[0026] The plasmid pES-Cap was digested by EcoR ...