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Recombinant protein of Mycoplasma pneumonia and porcine circovirus type 2 and bivalent vaccine prepared therewith

A recombinant protein, vaccine technology, applied in the field of vaccines, can solve problems such as increasing mortality, increasing disease severity and potential persistence, and economic losses

Active Publication Date: 2019-01-15
浙江洪晟生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mycoplasma hyopneumoniae often associates with Pasteurella multocida ( P. multocida ), porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) and porcine circovirus (Porcinecircovirus, PCV) and other pathogens co-infected, increasing the severity and potential persistence of related diseases, and exacerbating respiratory diseases , and then cause respiratory disease syndrome (Porcine Respiratory Disease Complex, PRDC), eventually leading to an increase in mortality, bringing huge economic losses to the world pig industry

Method used

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  • Recombinant protein of Mycoplasma pneumonia and porcine circovirus type 2 and bivalent vaccine prepared therewith
  • Recombinant protein of Mycoplasma pneumonia and porcine circovirus type 2 and bivalent vaccine prepared therewith
  • Recombinant protein of Mycoplasma pneumonia and porcine circovirus type 2 and bivalent vaccine prepared therewith

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Experimental program
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Effect test

Embodiment 1

[0052] Acquisition of target gene and construction of expression vector

[0053] Search the Shandong strain PCV2 Cap gene sequence (accession number: KY656098.1) in the NCBI database to remove the nuclear localization signal peptide, and optimize the codons of the prokaryotic expression system for the remaining nucleic acid sequence. The same method was used to search for the P97R1 (MHP168_110), P46 (MHP168_522) and P42 (MHP168_069) gene sequences of the Mhp168 strain (accession number: CP002274.1), and optimize the codons. Combined with the pET32a expression vector multiple cloning site and gene sequence to select the appropriate restriction site and connecting peptide sequence, the following two sequences were chemically synthesized: ①NcoI-P97R1-GGSG-P46-GGSG-P42-XhoI; ②KpnI-Cap-XhoI.

[0054] Using molecular biology methods, the two sequences synthesized above were double-digested and ligated into the pET32a vector, and then transformed into TG1 cloning bacteria. The recomb...

Embodiment 2

[0057] Expression and purification of target protein

[0058] The pET32a-P97R1-P46-P42 and pET32a-Cap were transformed into BL21 DE3 expression bacteria, respectively, for induced expression and purification. The methods of obtaining and operating the two proteins are the same, specifically as follows:

[0059] ①Protein expression: insert recombinant expression bacteria into LB medium at a ratio of 1:100, add antibiotics with a final concentration of 1 mM Amp, shake the bacteria at 37°C and 220 rpm for 3-4 h, and then add a final concentration of 1 mM IPTG to induce , 16°C, 220 rpm to induce overnight culture.

[0060] ② Bacterial cell crushing: collect the cultured bacterial liquid induced overnight, and centrifuge at 4000 rpm for 40 min to obtain the bacterial cell; resuspend and wash with an appropriate amount of sterile PBS solution, centrifuge at 12000 rpm, 4°C for 10 min, and discard the supernatant; use a small amount of pre-cooled The bacterial cells were resuspended...

Embodiment 3

[0063] Identification, concentration and quantitative analysis of target protein

[0064] After the purified P97-P46-P42 and Cap were prepared separately, they were electrophoresed on 12% SDS-PAGE gel, stained with Coomassie brilliant blue, and decolorized. The results are as follows image 3 with Figure 4 . Using His antibody for Western Blot identification, the results are as follows Figure 5 with Image 6 .

[0065] On the basis of the above identification, the two proteins were concentrated and desalted using 10 kDa ultrafiltration tubes, and the two proteins were quantified by BCA method, and the protein concentration was finally adjusted to 1 mg / mL.

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Abstract

The invention relates to the technical field of vaccines, in particular to a recombinant protein of mycoplasma suis pneumonia and porcine circovirus type 2, a bivalent vaccine prepared therewith and application thereof. A Mycoplasma pneumonia and porcine circovirus type 2 bivalent genetically engineered vaccine is characterized in that the vaccine includes Mhp P97R1-P46-P42 recombinant protein andPCV2 Cap recombinant protein. The encoding gene sequence of the Mhp P97R1-P46-P42 recombinant protein is shown in SEQ ID NO. 1; The coding gene sequence of the PCV2 Cap recombinant protein is shown in SEQ ID NO. 2. The bivalent genetically engineered vaccine can effectively prevent and control Mycoplasma pneumonia and porcine circovirus type 2, and has good application prospect.

Description

technical field [0001] The invention relates to the technical field of vaccines, in particular to a recombinant protein of mycoplasma swine pneumonia and porcine circovirus type 2, a double genetic engineering vaccine prepared therefrom and application thereof. Background technique [0002] swine mycoplasma pneumonia ( Mycoplasmal pneumoniae of swine, MPS) is due to infection with Mycoplasma hyopneumoniae ( Mycoplasma hyopneumonia , Mhp), also known as "porcine asthma" or "porcine enzootic pneumonia (PEP)". Piglets are often infected, clinically characterized by asthma, paroxysmal spasmodic cough, loss of appetite and pulmonary edema, eventually leading to death by suffocation. The disease has the characteristics of high infection rate and high infectivity. Mycoplasma hyopneumoniae often associates with Pasteurella multocida ( P. multocida ), porcine reproductive and respiratory syndrome virus (Porcine Reproductive and Respiratory Syndrome Virus, PRRSV) and porcine ci...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/34C12N15/70A61K39/295A61K39/12A61K39/02A61P31/20A61P31/04
CPCA61K39/0241A61K39/12A61K2039/552A61K2039/70A61P31/04A61P31/20C07K14/005C07K14/30C07K2319/00C12N15/62C12N15/70C12N2750/10022C12N2750/10034
Inventor 何玉龙陶宇舒建洪陈健吴月红杨芳
Owner 浙江洪晟生物科技股份有限公司
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