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Gene for encoding Cap protein of porcine circovirus 2 and application thereof

A porcine circovirus and protein technology, applied in the field of molecular biology, can solve the problems of large-scale production of Escherichia coli, which is difficult for Cap protein, and achieve the effects of low cost, soluble expression, and low expression level

Active Publication Date: 2017-11-10
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the current problems that the complete Cap protein is difficult to use Escherichia coli for large-scale production, the present invention provides a gene encoding porcine circovirus type 2 Cap protein that is easy to be soluble expressed

Method used

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  • Gene for encoding Cap protein of porcine circovirus 2 and application thereof
  • Gene for encoding Cap protein of porcine circovirus 2 and application thereof
  • Gene for encoding Cap protein of porcine circovirus 2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction and identification of recombinant expression vector pET30a-ORF2.

[0039] 1. ORF2 gene acquisition and codon optimization

[0040] PCV2 SD strain (CGMCC NO.5774, storage date: February 8, 2012) was used to inoculate PK-15 cells without PCV2 contamination, and the virus was harvested after 72 hours as a template, and the whole genome of porcine circovirus type 2 was amplified by PCR, and the primers The sequence is as follows:

[0041] The upstream primer sequences are as follows:

[0042] 5'-GAACCGCGGGCTGGCTGAACTTTTGAAAGT-3' (SEQ ID NO. 4);

[0043] The downstream primer sequences are as follows:

[0044] 5'-GCACCGCGGAAATTTCTGACAAACGTTACA-3' (SEQ ID NO.5);

[0045] The genome sequence of the PCV2 SD strain is shown in SEQ ID NO.6 (GenBank No.DQ 346683). The ORF2 gene of the virus shown in SEQ ID NO.7 is optimized and transformed into codons for Escherichia coli. The sequence is shown in SEQ ID Shown in NO.1. Add BamH I and Xho I restriction ...

Embodiment 2

[0062] Example 2 Construction and induced expression of recombinant expression strain BL21 / pET30a-ORF2.

[0063] 1. Transformation of competent Escherichia coli BL21 with recombinant plasmid

[0064] The correctly sequenced pET30a-ORF2 prokaryotic expression vector plasmid in Example 1 was transformed into Escherichia coli BL21 (NEB) to obtain the recombinant expression strain BL21 / pET30a-ORF2.

[0065] 2. Induced expression of recombinant expression strain BL21 / pET30a-ORF2

[0066] Pick a single colony of the recombinant bacteria, inoculate it into 10 mL of LB medium containing kanamycin, and culture it with shaking at 37°C until OD 600 When it reaches 0.6-1.0, add IPTG to a final concentration of 1 mmol / L, and continue culturing for 5 hours. Collect the bacteria by centrifugation, discard the supernatant, wash with PBS (0.015mol / L, pH7.2), add 3mL lysate, ultrasonically lyse for 3min, collect the supernatant after centrifugation, take an appropriate amount and add 5×SDS sa...

Embodiment 3

[0069] Example 3 Soluble expression of recombinant expression strain BL21 / pET30a-ORF2

[0070] Inoculate 2% of the recombinant strains into the liquid LB medium containing kanamycin according to the volume of the liquid LB medium containing kanamycin, and culture with shaking at 37°C for 2 hours to OD 600 When the concentration is 0.82, add IPTG with a final concentration of 0.8mmol / L to induce for 3 hours, centrifuge at 5000rpm at 4°C for 10min, wash the precipitate with PBS (0.015mol / L, pH7.2), then add 10mL of lysate, ultrasonically lyse for 20min, 4°C The supernatant was collected after centrifugation at 10,000 rpm for 15 minutes; the porcine circovirus type 2 Cap protein was obtained after purification and concentration.

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Abstract

The invention provides a gene for encoding Cap protein of porcine circovirus 2, a recombinant expression vector containing the gene and a recombinant bacterium containing the recombinant vector. The gene expresses easily in escherichia coli and comprises a sequence shown in SEQ ID NO.1. The invention further provides a method for inducing the recombinant bacterium to produce the Cap protein of the porcine circovirus 2 and application in the aspect of utilizing the protein to produce a vaccine of the porcine circovirus 2. Through the adoption of the gene for encoding Cap protein of the porcine circovirus 2 and the application thereof, the full-length Cap protein can efficiently express in escherichia coli, the operation is simple, the cost is cost, and the large-scale production is easy to achieve; the recombinant bacterium and the vaccine have wide application prospects in preventing and controlling diseases of the porcine circovirus 2.

Description

technical field [0001] The invention relates to a gene encoding porcine circovirus type 2 Cap protein and application thereof, belonging to the field of molecular biology. Background technique [0002] Porcine circovirus type 2 (Porcine circovirus 2, PCV-2) is one of the viruses that have been newly discovered in recent years and seriously endanger the pig industry. The virus is closely related to various disease syndromes of pigs, including post-weaning Multi-systemic wasting syndrome (Post-weaning multi-systemic wasting syndrome, PMWS) and sow reproductive disorders (Sowabortion and mortality syndrome, SAMS), etc. Since PMWS was first reported in Canada in 1991, the disease has now spread to all parts of the world. At the International Pig Veterinary Congress (IPVS) in 2010, porcine circovirus infection became the disease with the highest attention, and it is a globally recognized important disease that endangers the pig industry. one of infectious diseases. In recent ye...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01A61K39/12A61P31/20C12N1/21C12R1/19
CPCA61K39/12C07K14/005C12N2750/10022C12N2750/10034C12N2750/10051
Inventor 李俊吴晓燕时建立彭喆王金宝于江郑书轩张玲玲辛长勋王硕
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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