Mycoplasma hyopneumoniae fusion gene and application

A technology of mycoplasma hyopneumoniae and fusion gene, which is applied in the field of animal infectious diseases, can solve the problems of high price and difficult cultivation of mycoplasma, and achieve the effects of high expression, easy purification and preparation, and reduced preparation cost

Active Publication Date: 2015-01-21
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the difficulty in cultivating mycoplasma, the above-mentioned commercial vaccines are all quite expensive

Method used

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  • Mycoplasma hyopneumoniae fusion gene and application
  • Mycoplasma hyopneumoniae fusion gene and application
  • Mycoplasma hyopneumoniae fusion gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Construction of P97R1-P36-P46 fusion gene

[0027] 1. Design of primers (shown in Table 1)

[0028] Table 1 PCR primers

[0029]

[0030]

[0031] The underlined part in the primer sequence in Table 1 is the corresponding restriction site. The sequence in bold in the P36 primer and P46 primer is the nucleotide sequence of Linker. The primers in Table 1 were synthesized by Nanjing GenScript Biotechnology Co., Ltd. .

[0032] 2. Amplification of P46, P36 and P97R1 genes

[0033] Genomic DNA of 168 strains of Mycoplasma hyopneumoniae (Liu W, Feng Z X, Fang L R, et al. Complete genome sequence of Mycoplasma hyopneumoniae strain 168. Journal of Bacteriology. 2011, 193: 1016-1017) was used as a template to carry out P97R1( GenBank accession number ADQ90328.1), P36 (GenBank accession number ADQ90382.1) and P46 genes (GenBank accession number ADQ90718.1) were amplified.

[0034] The PCR amplification system is described in Table 2:

[0035] Table 2 PCR ...

Embodiment 2

[0048] Example 2: Prokaryotic expression of recombinant plasmid pET30a-P97R1-Linker-P36-Linker-P46

[0049] 1. Plasmid transformation

[0050] The expression plasmid pET30a-P97R1-Linker-P36-Linker-P46 (Kana resistance) containing the fusion gene of Mycoplasma hyopneumoniae P97R1-P36-P46 was transformed into Escherichia coli BL21 competent cells.

[0051] The specific operation is as follows:

[0052] 1) Transfer 100 μL E. coli BL21 competent cell suspension to a sterile 1.5ml EP tube, add 3 μL ligation product, swirl gently to mix the contents, and place on ice for 30 minutes.

[0053] 2) Put the centrifuge tube into a circulating water bath preheated to 42° C. and heat shock for 90 seconds.

[0054] 3) Quickly transfer the centrifuge tube to an ice bath to cool the cells for 1-2 minutes.

[0055] 4) Add 400 μL LB medium to each tube. Warm the medium to 37°C with a water bath, then transfer the centrifuge tube to a shaker at 37°C, and incubate for 45 minutes to recover the...

Embodiment 3

[0060] Example 3: Purification of pET30a-P97R1-Linker-P36-Linker-P46 fusion protein

[0061] The pET30a-P97R1-Linker-P36-Linker-P46 fusion protein expressed in soluble form was purified by AKTA FPLC (Amersham Biosciences UPC-900), and the purification steps were as follows:

[0062] (1) Centrifuge 200 mL of the bacterial solution after 5 hours of induction at 12,000 r / min for 1 minute, discard the supernatant, resuspend the precipitate with Binding Buffer (1 / 10), place it in a -80°C refrigerator, freeze and thaw three times, and use Ultra-high pressure wave crusher crushed it until clear, centrifuged at 12000r / min for 30min, took the supernatant, filtered the supernatant with a 0.22μm filter membrane, and put it on ice for later use.

[0063] (2) Turn on the analyzer, then turn on the computer, select UNICORNS.1.0, choose default, and click OK.

[0064](3) Select System control, put the two probes A and B into 20% absolute ethanol, and turn them on in turn: Manual→Pump→Pump W...

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Abstract

The invention belongs to the field of animal gene engineering, and in particular relates to a synthetic fusion gene for expressing mycoplasma hyopneumoniae and application. The fusion gene is characterized in that a mycoplasma hyopneumoniae P97R1 gene is connected in series with a P36 gene through a Linker, the termination codon of the P36 gene is deleted, the P46 gene with signal peptide removed is fused at the C end of the P36 gene through Linker, and then the fusion gene P97R1-P36-P46 is obtained, wherein the nucleotide sequence of the fusion gene is shown as SEQ ID NO: 1. The fusion gene is included in a prokaryotic expression plasmid, and escherichia coli BL21/pET30a-P97R1-Linker-P36-Linker-P46 containing the plasmid is collected in CCTCC with the collection number CCTCC NO: M2014269. The invention further discloses immune efficacy evaluation of the fusion gene and application of the fusion gene in a novel vaccine.

Description

technical field [0001] The invention belongs to the technical field of animal infectious diseases and is related to animal genetic engineering. It specifically relates to a fusion gene of mycoplasma hyopneumoniae, its preparation method and application. Background technique [0002] Mycoplasma pneumonia of swine (MPS) is caused by Mycoplasma hyopneumoniae (MHP). It is a chronic respiratory infectious disease with cough and wheezing as the main symptoms in pigs. It is chronic, contact, highly contagious, and highly Characterized by morbidity and low mortality. The disease is widely distributed all over the world. It is often called porcine enzootic pneumonia abroad, but in my country it is often called swine asthma (Maes D, Segales J, Meyns T, et al. Control of Mycoplasma hyopneumoniae infections in pigs. Vet Microbiol, 2008, 126:297-309). Mycoplasma hyopneumoniae infection leads to a 12%-16% decrease in the growth rate of pigs, a 13%-22% reduction in feed conversion rate, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/10C12N1/21A61K39/02A61P31/04C12R1/35C12R1/19
Inventor 肖少波方六荣刘威罗锐陈焕春
Owner HUAZHONG AGRI UNIV
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