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Preparation and application of a fusion protein and its vaccine composition

A vaccine composition and fusion protein technology, applied in the field of veterinary biological products, can solve problems such as poor immune effect

Active Publication Date: 2018-02-06
PULIKE BIOLOGICAL ENG INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the current subunit vaccine for Mycoplasma pneumoniae in swine has poor immune effect and cannot provide complete protection to pigs (Fagan PK, Djordievic SP. Chin J, et al. Oral immunization of mice with attenuated Salmonella typhimurium aroAexpressing a recombinant Mycoplasma hyopeumoniae antigen (NrdF). Infection and immunity, 1997, 65:2502-2507)

Method used

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  • Preparation and application of a fusion protein and its vaccine composition
  • Preparation and application of a fusion protein and its vaccine composition
  • Preparation and application of a fusion protein and its vaccine composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1 Expression, identification and purification of fusion protein PEA-NrdF-KDEL

[0092] According to NCBI (http: / / www.ncbi.nlm.nih.gov) reported Pseudomonas aeruginosa PAO1 (accession number: AE004091), Mycoplasma hyopneumoniae J strain (accession number: AE017243.1) The gene sequence (seeing SEQ ID No.3) in the NrdF protein in, amplifies Pseudomonas exotoxin A (PEA) structural domain I and II with PCR method, amplifies Mycoplasma hyopneumoniae NrdF protein with RT-PCR method and using continuous PCR to amplify the gene (SEQ ID NO.16) containing the KDEL polypeptide at the carboxy-terminal part. After the cloning is completed, they are connected in series by means of genetic engineering as a fusion protein.

[0093] 1.1 Construction and identification of PEA domain I and II, NrdF gene cloning vector

[0094] According to the selected PEA domain I and II gene (see SEQ ID No.1) sequence and NrdF gene (see SEQ ID NO.3) sequence, primers were designed using Prim...

Embodiment 2

[0118] Example 2 Expression, identification and purification of fusion protein PEA-P97R1-NrdF-KDEL

[0119] 2.1 Construction and identification of pMD18-T-PEA-P97R1-NrdF-KDEL cloning vector

[0120] According to the gene sequence in the P97R1 protein in PEA (accession number: AE004091), Mycoplasma hyopneumoniae J strain (accession number: AE017243.1) reported by NCBI (http: / / www.ncbi.nlm.nih.gov) (see SEQ ID No.7), use Primer Premier5.0 software to design primers, respectively amplify PEA gene, P97R1 gene, the primer sequences are as follows:

[0121] PEA upstream primer: 5'-GCCGAGGAAGCCTTCGAC-3',

[0122] PEA downstream primer: 5'-GCCGTCGCCGAGGAACT-3',

[0123] P97R1 upstream primer: 5'-GGTATATTACTCTCAGCCCCCA-3',

[0124] P97R1 downstream primer: 5'-TTCCGGTGTTTTTAGGCTTAA-3',

[0125] The amplified products of PEA gene and P97R1 gene were recovered and stored at -20°C.

[0126] The purified P97R1 gene and PEA gene were used as templates, and the two fragments were connect...

Embodiment 3

[0133] Example 3 Preparation of fusion proteins PEA-P102-KDEL, PEA-P97R1-KDEL, PEA-P97R2-KDEL, PEA-P97R2-NrdF-KDEL

[0134] 3.1 Preparation of fusion proteins PEA-P102-KDEL, PEA-P97R1-KDEL, PEA-P97R2-KDEL

[0135] According to the PEA (accession number: AE004091) reported by NCBI (http: / / www.ncbi.nlm.nih.gov), and referring to the gene sequences in the P102 protein, P97R1 protein, and P97R2 protein in the Mycoplasma hyopneumoniae J strain ( See SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.9 in sequence), construct the cloning vector and expression vector according to the method of Example 1 and perform double enzyme digestion, and name the fusion protein identified correctly after enzyme digestion as PEA-P102-KDEL, PEA-P97R1-KDEL, PEA-P97R2-KDEL.

[0136] The identified fusion proteins PEA-P102-KDEL, PEA-P97R1-KDEL, and PEA-P97R2-KDEL were purified, dialyzed and then concentrated. The concentrated protein content was 1 mg / mL and stored at -20°C.

[0137] 3.2 Preparation of fusion prote...

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Abstract

The invention provides a fusion protein and a composition and application of the mycoplasma pneumonia vaccine containing the fusion protein. The fusion protein includes Pseudomonas aeruginosa exotoxin A domain I and II, Mycoplasma hyopneumoniae antigenic protein and carboxyl terminal part, and the Mycoplasma hyopneumoniae antigenic protein includes NrdF protein, P102 protein, P97R1 protein, P97R2 protein, P97R1 protein Any protein in -NrdF protein, P97R2-NrdF protein. The invention also discloses a vaccine composition containing immune amount fusion protein and its application in the preparation of medicine for preventing and / or treating swine mycoplasma pneumonia. The vaccine composition can carry out a large amount of recombinant expression on the components of the vaccine composition by means of genetic engineering, which not only takes a short time, but also facilitates large-scale production; the vaccine composition can produce both humoral immunity and cellular immunity , can obtain a better immune effect.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to the preparation and application of a mycoplasma hyopneumoniae fusion protein and a vaccine composition containing the fusion protein. Background technique [0002] Mycoplasma pneumonia of swine (MPS) is also known as swine asthma (Li Yanming, Zhang Ying. Advances in biological research on Mycoplasma hyopneumoniae, Advances in Veterinary Medicine, 2003, 24(3): 25-27) or pig endemic Pneumonia is a contact chronic respiratory disease of pigs caused by Mycoplasma hyopneumoniae (Mhp). Manifested as cough and dyspnea, dissection showed fleshy or marbled lung tissue lesions. [0003] The Ross study at the University of Iowa found that after Mhp infection, the ability of lymphocytes to produce antibodies decreased, cellular immunity decreased, and the ability of alveolar macrophages to phagocytize and clear pathogens also decreased, and the activity of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/70A61K39/02A61K39/295A61K39/116A61P31/04A61P31/14A61P31/16A61P31/20A61P31/22
Inventor 张许科孙进忠王莹田克恭
Owner PULIKE BIOLOGICAL ENG INC
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