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Method for expressing ORF2 protein of porcine circovirus 3 by using Escherichia coli and application thereof

A porcine circovirus, PVC3-ORF2 technology, applied in the biological field, can solve the problems of high cost and difficult protein purification, and achieve the effects of low cost, high immunogenicity and low production cost

Inactive Publication Date: 2019-04-12
镇江瑞华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Porcine circovirus type 3 ORF2 protein subunit vaccine has been reported on the use of prokaryotic expression of ORF2 truncated protein, but prokaryotic expression often exists in the form of inclusion bodies, which involves cumbersome steps such as denaturation and renaturation and high costs. However, baculovirus has the disadvantages of high expression cost and difficult protein purification.

Method used

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  • Method for expressing ORF2 protein of porcine circovirus 3 by using Escherichia coli and application thereof
  • Method for expressing ORF2 protein of porcine circovirus 3 by using Escherichia coli and application thereof
  • Method for expressing ORF2 protein of porcine circovirus 3 by using Escherichia coli and application thereof

Examples

Experimental program
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Embodiment 1

[0034] Example 1 Synthesis of porcine circovirus type 3 ORF2 protein

[0035] 1) Synthesis of porcine circovirus type 3 ORF2 protein gene (PVC3-ORF2 gene)

[0036] Based on the amino acid sequence of the Capsid protein protein of Porcine circovirus 3 (GenBank: AOO87130.1), such as the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, it is reverse-compiled into nucleotides by Escherichia coli codon optimization Sequence, and enzyme cutting sites were added at both ends of the entire fragment to make it suitable for expression in Escherichia coli (commissioned by General Biosystems (Anhui) Co., Ltd. to synthesize), the final gene sequence is as follows: SEQ ID NO: 2, named PCV3 -ORF2, the specific construction method:

[0037] PCR primers were designed using OLigo6.0 primer design software (P PVC3-ORF2 F, P PVC3-ORF2 R), the primer sequences are as follows:

[0038] P PVC3-ORF2 F: 5'-ATGCGCCATCGCGCGATTTTT-3'

[0039] P PVC3-ORF2 R: 5'-ATCCTCGAGTTACAGCACG...

Embodiment 2

[0084] Example 2 Porcine Circovirus Type 3 ORF2 Protein Subunit Vaccine Preparation and Immunization Test

[0085] 1) Preparation of porcine circovirus type 3 ORF2 protein subunit vaccine

[0086] Measure the purified porcine circovirus type 3 ORF2 protein stock solution concentration prepared by Example 1 with Bradford protein quantification reagent, adjust the protein concentration to a final concentration of 0.01mg / mL, 0.05mg / mL, 0.1mg / mL, 0.2mg / mL four concentrations, and then mixed with French SEPPIC adjuvant GEL01, the volume of adjuvant added accounted for 10% of the total volume, after mixing evenly and stirring, according to the requirements of the appendix of the current version of the Chinese Veterinary Pharmacopoeia, sterility testing, viscosity measurement, stability After the determination is qualified, place it at 2-8°C for later use.

[0087] 2) Porcine circovirus type 3 ORF2 protein subunit vaccine immunization test

[0088] Prepared 0.01mg / mL, 0.05mg / mL, 0...

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Abstract

The invention discloses a method for expressing an ORF2 protein of a porcine circovirus 3 (PCV3) by using Escherichia coli and application thereof. The method is as below: 1) cloning a solubility-enhancing tag GST gene, a solubility-enhancing tag His6-SUMO gene and a PVC3-ORF2 gene respectively; 2) ligating the obtained three genes to obtain a fusion gene; 3) ligating the obtained fusion gene to an E. coli expression vector to obtain a recombinant expression vector containing the fusion gene; 4) transforming the recombinant expression vector into E. coli expressing strain competent cells to obtain recombinant E. coli expressing strains containing the fusion gene; 5) culturing the obtained recombinant expression strains, and inducing expression of the fusion protein by IPTG; and 6) recovering the recombinant expression bacteria obtained by culture, breaking and purifying the recombinant expression bacteria to obtain the ORF2 protein of the porcine circovirus 3. The method not only obtains soluble protein with high expression, but also achieves high immunoreduction.

Description

technical field [0001] The invention relates to a method for expressing porcine circovirus type 3 ORF2 protein by using Escherichia coli and an application thereof, belonging to the field of biotechnology. Background technique [0002] Porcine circovirus (Porcine circovirus, PCV) is a representative species of the genus Circovirus, a covalently closed, single-stranded circular DNA virus, and the smallest animal virus discovered so far. Before 2015, only two types of porcine circovirus type 1 (PCV1) and porcine circovirus type 2 (PCV2) were found worldwide. In June 2015, a PDNS outbreak broke out in a commercial pig farm in North Carolina, USA. Compared with the past, the outbreak was more serious. PaLinski and others identified a new type of virus for the first time from sick sows and aborted fetuses with the help of next-generation sequencing technology (NGS). Type 3 (PCV3). [0003] PCV3, like PCV1 and PCV2, has an icosahedral structure, and its genome is a single-stran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N1/21C12P21/06C07K14/01C07K1/22A61K39/12A61P31/20C12R1/19
CPCC12N15/70A61K39/12A61K2039/552A61P31/20C07K14/005C07K2319/21C07K2319/23C07K2319/50C12N15/62C12N2750/10022C12N2750/10034C12N2750/10051C12P21/06
Inventor 李少辉陶家权单玉飞张开岳陶慧丽
Owner 镇江瑞华生物科技有限公司
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