Method for efficiently expressing PCV2 Cap and PCV3 Cap fusion proteins
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A fusion protein and protein technology, applied in the field of molecular biology, can solve the problems of complex purification process and low expression of fusion protein, achieve high biological activity, high expression efficiency, and shorten the time for virus purification and identification
Active Publication Date: 2020-05-22
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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[0005] Aiming at the current problems of low expression of PCV2 Cap-PCV3 Cap fusion protein and complicated purification process, the present invention provides a new method for expressing PCV2 Cap and PCV3 Cap fusion protein. Modified, close to the natural virus-encoded protein, with high biological activity
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Embodiment 1
[0040] Example 1 Construction of Baculovirus
[0041] 1. Obtaining the target gene
[0042] Refer to the methods in "Establishment of real-time fluorescent quantitative PCR detection method for porcine circovirus type 2 SYBR Green I" and "Establishment of real-time fluorescent quantitative PCR detection method for porcine circovirus type 3 SYBR Green I" to construct type 2 and 3 The plasmids pEASY-Blunt-PCV2 and pEASY-Blunt-PCV3 of the whole genome of porcine circovirus were stored at -80°C for future use.
[0043] Table 1 Target gene PCR primer sequence
[0044] .
[0045] Synthesize primers according to Table 1, use pEASY-Blunt-PCV2 as a template, and use HBM-PCV2Cap F and Linker1-PCV2Cap R as primers to amplify the base sequence of PCV2 Cap, and recover the amplified product with a gel recovery kit , named 2Cap. Using pEASY-Blunt-PCV3 as a template and using Linker2-PCV3Cap-phF and PCV3Cap-phR as primers to amplify the base sequence of PCV3 Cap, the amplified product ...
Embodiment 2
[0080] Example 2 Expression of fusion protein
[0081] The P2 recombinant baculovirus was inoculated to a density of 2.5×10 at MOI=1, 0.5, and 0.1, respectively. 6 cells / mL sf9 cells. Collect 200 μL of cell supernatant every 24 hours, collect for 5 consecutive days, and centrifuge and purify the protein samples. The steps are as follows:
[0082] 1) Centrifuge the collected supernatant at 4°C, 5000×g for 30 min to remove cell debris and impurities, and collect the culture supernatant;
[0083] 2) After centrifugation, the cell supernatant was centrifuged at 30,000 rpm for 1 h, and the pellet was harvested, that is, the pellet was resuspended in sterile PBS;
[0084] 3) After resuspending the pellet in sterile PBS, add it to the upper layer of the 10%-30%-50% sucrose density gradient, centrifuge at 35 000 rpm for 1.5 h for purification, and carefully collect the white flocs between the 30%-50% sucrose layer resuspended in PBS, centrifuged at 30 000 rpm for 1.5 h to remove su...
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Abstract
The invention provides a method for efficiently expressing PCV2 Cap and PCV3 Cap fusion proteins. The method comprises the following steps: firstly, constructing a recombinant baculovirus for efficiently expressing PCV2Cap protein and PCV3Cap protein; connecting the nucleotide sequence of the PCV2Cap protein of which the nuclear localization signal is cut off with the nucleotide sequence of the PCV3Cap protein; connecting the middle by using a hydrophobic flexible protein linker sequence; and adding a bee element signal peptide sequence to the N ends of the PCV2Cap protein sequence and the PCV3Cap protein sequence to promote secretory expression. Proteins expressed by recombinant positive baculovirus infected sf9 insect cells are subjected to various post-translational modifications, and the modified proteins are close to natural virus coded proteins and have high biological activity. Moreover, the baculovirus has high species specificity, only infects the insect cells, has no infectivity to vertebrate cells, and expression products of baculovirus are safe and reliable and can be used for subsequent tests after simple treatment. Therefore, the method for expressing the PCV2 Cap andPCV3 Cap fusion proteins is safer and more effective than a traditional mode.
Description
technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for highly expressing fusion proteins of PCV2 Cap and PCV3 Cap. Background technique [0002] Porcine circovirus (PCV) mainly affects weaned piglets and fattening pigs, and can cause a variety of porcine circovirus-related diseases, posing a huge threat to the healthy development of the pig industry. According to the difference of antigenicity, pathogenicity and gene homology, PCV is divided into porcine circovirus type 1 (Porcine circovirus 1, PCV1), porcine circovirus type 2 (Porcine circovirus 2, PCV2) and porcine circovirus type Virus type 3 (Porcine circovirus 2, PCV2) three genotypes. It is known that PCV1 has no pathogenicity, but PCV2 has strong pathogenicity, and can cause a variety of porcine circovirus-associated diseases (PCVADs), the most important of which is multisystem weakness syndrome in weaned piglets. Postweaning multisystemic wasting syn...
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