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Subunit vaccine for bovine fusobacterium necrophorum and preparation method of subunit vaccine

A subunit vaccine, Necroptosis bacillus technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of natural toxin side effects, difficulty in vaccine application, difficulty in bacterial culture, etc., and achieve high levels of specific antibodies, good cellular immunity and humoral Immunity, protection from attack effects

Active Publication Date: 2019-12-27
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is unavoidable that inactivated vaccines have disadvantages such as difficulty in cultivating a large number of bacteria, poor immune effect and serious side effects caused by natural toxins, which have brought difficulties to the wide application of the vaccine.

Method used

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  • Subunit vaccine for bovine fusobacterium necrophorum and preparation method of subunit vaccine
  • Subunit vaccine for bovine fusobacterium necrophorum and preparation method of subunit vaccine
  • Subunit vaccine for bovine fusobacterium necrophorum and preparation method of subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This example illustrates the cloning of 43kDa OMP, PL-4 and H2 genes and the construction of expression vectors.

[0048] According to the gene sequences of Necroptosis 43kDa OMP, leukotoxin lktA and hemolysin hly published on GenBank, specific primers were designed for PCR amplification using Primer5.0 software. The designed specific primers were all provided by Harbin Boshi Biotechnology Co., Ltd. synthesis. The primer sequences are as follows:

[0049] Table 1 Fusobacterium necroptosis specific amplification primers

[0050]

[0051] The genome of Necroptosis bacilli was extracted using Tiange Bacteria Genomic DNA Extraction Kit. The extracted genome was used as a template, and PCR amplification was performed according to conventional methods. The amplification system and reaction conditions are as follows:

[0052] Table 2 PCR reaction system and reaction conditions

[0053]

[0054] Continued Table 2 PCR reaction system and reaction conditions

[0055] ...

Embodiment 2

[0060] This example illustrates the induction of expression in recombinant E. coli.

[0061] Use pGEX-6p-1-PL-4-BL21, pET-32a-H2-BL21, pET-32a-43kDa OMP-BL21 positive bacteria and pET-32a, pGEX-6p-1 empty vector at a ratio of 1:1 000 Inoculated in LB (Amp + ) liquid medium, cultured on a constant temperature shaker at 37°C at 220r / min. When bacteria OD 600 When the value reaches 0.4-0.6, add IPTG to the bacterial solution to induce expression, the final concentration of IPTG is 0.5mmol / L, and the induction condition is 16°C, 160r / min for overnight culture. Collect the induced bacterial liquid, centrifuge and sonicate, as follows:

[0062] Bacterial precipitates were collected, and each induced bacterial solution was placed in a high-speed centrifuge for 10 min at 8 000 r / min, the supernatant was discarded to collect the precipitate, and the precipitate was washed with sterilized PBS buffer 3 times, each time at 5 000 r / min Centrifuge for 10 min, and finally suspend each in...

Embodiment 3

[0065] This example illustrates the purification of recombinant proteins.

[0066] 1. Purification of pET-32a-43kDa OMP recombinant protein

[0067] Gently invert the column to resuspend the Ni-NTA Agarose while tapping repeatedly. Add resin to the purification column, the resin sinks completely due to gravity, suck out the supernatant, then add sterilized distilled water, turn over and tap the purification column again and again to resuspend the resin. The resin sinks completely due to gravity, and the supernatant is aspirated. Add Denaturing binding buffer, gently flip the column up and down to resuspend the resin, then allow the resin to sink naturally by gravity, suck out the supernatant, and repeat 2-3 times to make protein purification under denaturing conditions. Put the bacterial lysate in the Ni-NTA column, and gently stir it with a magnetic stirrer at room temperature, so that the lysate and the resin fully interact for 20-30min. Precipitate the resin naturally ag...

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Abstract

The invention relates to a subunit vaccine for bovine fusobacterium necrophorum. The subunit vaccine comprises three proteins: an outer membrane protein 43kDa OMP of bovine fusobacterium necrophorum,a leukotoxin truncated protein PL-4 of fusobacterium necrophorum and a hemolysin truncated protein H2 of fusobacterium necrophorum, wherein the dosage ratio of the three proteins is 1:1:1. The invention also relates to a preparation method of the subunit vaccine for bovine fusobacterium necrophorum. The subunit vaccine for bovine fusobacterium necrophorum comprises the three proteins: the outer membrane protein 43kDa OMP of bovine fusobacterium necrophorum, the leukotoxin truncated protein PL-4 of fusobacterium necrophorum and the hemolysin truncated protein H2 of fusobacterium necrophorum that are used in combination to produce a high level of specific antibody after immunity, thereby provoking cellular immunity and humoral immunity well in mice and effectively protecting the mice from attack of the fusobacterium necrophorum. The subunit vaccine has a better immune protection effect comparable to that of a whole-bacteria inactivated vaccine.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a subunit vaccine of necroptosis bovis and a preparation method thereof. Background technique [0002] Necroptosis bacillus (Fusobacterium necrophorum), also known as diphtheria calf, Corynebacterium necrosis and Actinomycetes necrosis, is a Gram-negative strict anaerobic bacterium with pleomorphic characteristics, mostly rod-shaped in shape, but does not have a capsule and spores, no motility. The bacteria are about 0.5 μm wide, about 100 μm long, and a few are 300 μm. In some cultures, rod-shaped bacteria that are twice as thick as the normal shape can also be seen, and they are more filamentous in diseased tissues and broths. Necroptosis bacillus is widely distributed in nature. It is not only the normal flora of human and animal oral cavity, respiratory tract and gastrointestinal tract, but also the primary or secondary pathogen of various suppurative ...

Claims

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Application Information

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IPC IPC(8): A61K39/05A61P31/04C12N15/70C07K14/195C12R1/19
CPCA61K39/05A61P31/04C12N15/70C07K14/195A61K2039/552A61K2039/523A61K2039/575A61K2039/572
Inventor 郭东华孙东波李春秋原东伟蒋剑成贺显晶张思瑶王志慧吕思文高晶姚爽肖佳薇汪锋锋王丽娜
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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