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Optimized PCV2d ORF2 gene and preparation method of virus-like particles

A porcine circovirus and plasmid technology, applied in the field of molecular biology, can solve the problems of poor Cap protein solubility, low Cap protein yield, and low VLPs formation rate, and achieve the effect of shortening the experimental period, mature technology and low cost.

Pending Publication Date: 2020-09-11
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current eukaryotic expression system produces Cap protein with low yield and high cost; although the E. coli expression system is simple to operate, high in expression and low in cost, the obtained Cap protein is usually poorly soluble, difficult to purify, and low in VLPs formation rate

Method used

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  • Optimized PCV2d ORF2 gene and preparation method of virus-like particles
  • Optimized PCV2d ORF2 gene and preparation method of virus-like particles
  • Optimized PCV2d ORF2 gene and preparation method of virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] According to the ORF2 gene in the genome of PCV2d HB-MC1 strain, and optimized according to the codon preference of Escherichia coli, an NdeI restriction site was introduced at its 5' end, a stop codon and a Hind III restriction site were introduced at the 3' end, After the gene is synthesized, it is cloned into the pET30a(+) expression vector by double enzyme digestion to obtain the recombinant expression plasmid carrying the target fragment. The CAI (Codon Adaptation Index) of the original sequence was only 0.63, which was increased to 0.85 after optimization, which greatly increased the expression level and the probability of soluble expression.

Embodiment 2

[0071] The positive recombinant expression plasmid pET30a-ORF2 was transformed into E.coli Rosetta (DE3) host expression bacteria, positive colonies were picked on the LB plate containing kanamycin resistance for sequencing and identification, and the sequences without mutations and frameshifts were obtained. Positive recombinant expression strain Rosetta-pET30a-ORF2.

Embodiment 3

[0073] Pick a single positive recombinant colony, shake and expand in LB medium containing 20 μg / mL kanamycin at 37°C at a shaking speed of 200 r / min for 12 hours, then mix the overnight culture with a ratio of 1:100 Re-inoculate in fresh LB medium containing 20 μg / mL kanamycin, shake at 200 r / min at 37°C until OD 600 After about 0.7, different induction conditions were used to induce the expression of the target protein.

[0074] Four induction temperatures of 27°C, 32°C, 37°C and 42°C were used to induce the expression of the engineered bacteria for the same time, and the maximum amount of soluble target protein was obtained at the induction temperature of 37°C.

[0075] 1. Under the induction temperature of 37°C, the engineered bacteria were induced and expressed for 1h, 2h, 3h, 4h, 5h, 6h, 7h and 8h, and the maximum amount of soluble target protein was obtained after the induction time of 6h.

[0076] 2. At an induction temperature of 37°C, add IPTG with a final concentra...

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Abstract

The invention belongs to the molecular biology field, and discloses an optimized PCV2d ORF2 gene and a preparation method of virus-like particles of the optimized PCV2d ORF2 gene. After a coding genesequence for coding porcine circovirus 2d subtype Cap proteins is optimized, an expression engineering bacteria expression system is constructed and is used for expressing the Cap proteins. Expressionbacteria is capable of expressing a large number of dissoluble and active porcine circovirus 2d subtype Cap proteins under a proper expression condition, the expressed proteins are purified by virtueof a monoclonal antibody column, and then the high-purity and high-concentration Cap proteins are obtained and are capable of forming the virus-like particles. According to the preparation method, efficient soluble expression of the porcine circovirus 2d subtype full-length Cap proteins in a prokaryotic expression system is realized, the operation is simple, the cost is low, the prepared monoclonal antibody column can be repeatedly used, and the Cap proteins obtained through purification are high in concentration and purity, are capable of spontaneously forming the virus-like particles, havegood immunogenicity and are suitable for industrial application.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a preparation method for obtaining soluble PCV2d Cap protein and virus-like particles by using optimized PCV2d ORF2 gene and monoclonal antibody column purification. Background technique [0002] Porcine circovirus type 2 (PCV2) is the main pathogen that causes porcine circovirus-related diseases such as multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, and proliferative necrotizing pneumonia in weaned piglets. At present, PCV2 is ubiquitous in pig herds in China and even in the world, causing severe economic losses to the pig industry. Once pigs are infected with circovirus disease, there is no effective drug treatment, so vaccination is the main way to prevent the disease. PCV2 has 11 open reading frames (ORFs), among which ORF1 and ORF2 are the two main reading frames. Studies have shown that ORF2 encodes the main structural protein and capsi...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/70C07K14/01C07K16/08
CPCC07K14/005C07K16/081C12N15/70C12N2750/10022C12N2750/10023
Inventor 缪德年夏叶李春华郑琳琳王宏华黄冬薄宗义廖学文
Owner SHANGHAI ACAD OF AGRI SCI
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