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Swine Mycoplasmal pneumonia live vaccine mucosal immune adjuvant, preparation method and application thereof

A technology of mycoplasma pneumonia and immune adjuvant, which is applied in vaccines, veterinary vaccines, chemical instruments and methods, etc., can solve the problem of the effect of adjuvant components, unclear action principle, lack of mucosal immune adjuvant, difficulty in adjuvant selection, etc. To improve the efficacy of mucosal immune protection, prolong the duration of immunity, and achieve the effect of less side effects

Active Publication Date: 2019-01-15
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reasons are mainly the following difficulties: First, the research on mucosal immune adjuvants is still very lacking, which is largely affected by the unclear research on mucosal immune response, and the effect of most adjuvant components on mucosal parts The mechanism of action is not clear, which brings considerable difficulties to the selection of adjuvants

Method used

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  • Swine Mycoplasmal pneumonia live vaccine mucosal immune adjuvant, preparation method and application thereof
  • Swine Mycoplasmal pneumonia live vaccine mucosal immune adjuvant, preparation method and application thereof
  • Swine Mycoplasmal pneumonia live vaccine mucosal immune adjuvant, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of recombinant engineering bacteria BL21-pET28a-CTB-P97R1

[0033]In order to screen the mucosal immune adjuvant of the live vaccine against Mycoplasma pneumoniae in swine, a variety of fusion proteins were designed. After testing, only the fusion protein CTB-P97R1 was found to have a better immune effect. The amino acid sequence of the fusion protein CTB-P97R1 is shown in SEQ ID NO:2, and the gene sequence is shown in SEQ ID NO:1.

[0034] This example describes the construction process of the recombinant genetically engineered bacteria BL21-pET28a-CTB-P97R1 used to prepare the fusion protein CTB-P97R1.

[0035] According to the gene sequence (AY307389.1) of cholera toxin B subunit (CTB) included in GenBank, the CTB gene (see SEQ ID NO: 3 for the specific sequence) was obtained by gene synthesis, and at both ends Add PagI and NheI restriction sites, respectively. The above synthetic sequence was double digested and purified, and the pET-28a(+...

Embodiment 2

[0037] Example 2: Expression and purification of fusion protein CTB-P97R1

[0038] The positive recombinant bacterium BL21-pET28a-CTB-P97R1 was cultured overnight at 37° C. with shaking in LB medium (kanamycin resistant). The next day, the bacterial solution was inoculated in LB (kanamycin-resistant) liquid medium at a ratio of 2%, and cultured with shaking at 37°C until OD 600 =0.4-0.6, adding isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 1.0mmol / L to induce expression, collect the bacterial liquid 4 hours after induction, centrifuge to get bacterial precipitate, wash with PBS 1 time. The cells were suspended in PBS buffer, ultrasonically lysed, centrifuged at 10,000 rpm for 30 min at 4°C, and the lysed supernatant was separated and purified by His-Tag affinity chromatography column, dialyzed for 2 days to remove salt, and freeze-dried. The purity of fusion protein CTB-P97R1 was analyzed by 15% SDS-PAGE electrophoresis. From figure 2 It can be ...

Embodiment 3

[0039] Example 3 In vitro biological activity detection of fusion protein CTB-P97R1

[0040] Methods: Ganglioside GM1-enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of fusion protein CTB-P97R1 and ganglioside GM1. Carbonate buffer containing 5 μg / ml GM1 (containing 35 mmol / L NaHCO 3 , 15mmol / L Na 2 CO 3 aqueous solution) at 4°C overnight, 100 μl per well; washed 3 times with PBST (PBS buffer containing 0.1% Tween-20), blocked with PBS solution containing 1% BSA at 37°C for 2 hours, washed 3 times with PBST, Add 100 μl of a solution containing 500 ng of CTB-P97R1 (purified) to each well, incubate at 37° C. for 2 hours, and replace it with CTB-P97R1 solution boiled at 100° C. for 10 minutes in the control. After washing with PBST, rabbit anti-CTB antibody (Bio-Rad, USA) diluted with PBS at a dilution ratio of 1:4000 was added, and incubated at 37°C for 1 h. After washing with PBST for 3 times, horseradish peroxidase-labeled goat anti-rabbit ...

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Abstract

The invention provides a swine Mycoplasmal pneumonia live vaccine mucosal immune adjuvant, a preparation method and application thereof, which relate to the field of veterinary vaccines. The mucosal adjuvant is a solution of a recombinant protein CTB-P97R1 containing fusion expression of cholera toxin B subunit and Mycoplasma hyopneumoniae P97R1. The invention also provides the preparation methodof the mucosal adjuvant, wherein preparing 10 mg / ml recombinant protein CTB-P97R1 mother liquor, then diluting the mother liquor with a solvent. The invention also provides the application of the mucosal immune adjuvant in preparing a live vaccine of Mycoplasmal pneumonia . The mucosal adjuvant of that invention is an aqueous compound adjuvant, The preparation method is simple, the adjuvant property is stable, the mycoplasma hyopneumoniae activity is not damaged, and the mixture is compatible with aerosol protectant, and the mixture is still harmless to mycoplasma hyopneumoniae activity, so itis suitable for aerosol immunization of live vaccine. Using the adjuvant can significantly improve the mucosal immune protection efficacy of vaccine, and prolong the immune duration.

Description

technical field [0001] The invention relates to the field of veterinary vaccines, in particular to a mucosal immune adjuvant for a live vaccine of mycoplasma pneumoniae in swine, a preparation method and an application thereof. Background technique [0002] Mycoplasma pneumonia is a chronic respiratory infectious disease of pigs caused by Mycoplasma hyopneumoniae (Mhp), the main symptoms of which are cough and wheezing. It mainly causes the feed conversion rate of pigs to decrease and the growth to be hindered, and the morbidity rate is high and the mortality rate is low. Hyopneumoniae infection destroys the cilia, predisposing to secondary infections with other respiratory bacteria and viruses, resulting in more severe outbreaks. The disease is widespread worldwide and has caused significant economic losses to the modern pig industry. [0003] Mycoplasma pneumonia in swine is a refractory chronic infectious disease. The effect of drug treatment is not ideal, and it is eas...

Claims

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Application Information

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IPC IPC(8): A61K39/39A61K39/02A61P31/04A61K9/72A61K47/40A61K47/10A61K47/02A61K47/18C07K19/00C12N15/70
CPCA61K39/0241A61K39/39A61K47/02A61K47/10A61K47/183A61K47/40A61P31/04A61K9/0078A61K9/12C12N15/62C12N15/70C07K14/30C07K2319/55A61K2039/57A61K2039/55561A61K2039/55577A61K2039/55544A61K2039/55516A61K2039/552A61K2039/541
Inventor 熊祺琰张珍珍邵国青冯志新韦艳娜王佳白昀甘源张磊
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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