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Protective antigen of avibacterium paragallinarum and expression and application of protective antigen

A technology of protective antigens and bacilli, which is applied in the field of poultry infectious disease prevention, can solve the problems of difficult operation, time-consuming, laborious and other problems, and achieve the effect of strong immunogenicity and excellent protective effect

Active Publication Date: 2018-01-02
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when designing recombinant antigens for the preparation of vaccines, due to the difficulty in screening antigenic epitopes that are common to the three serotypes of Apg and have neutralizing activity, they can simultaneously resist the three serotypes of A. paragallinarum A, B, and C Protective antigens are rarely reported
In addition, the recombinant protein expressed by E. coli prokaryotic contains a large amount of endotoxin. When the recombinant protein is used as an antigen, it must be accompanied by a special removal process, which is time-consuming, laborious and difficult to operate.

Method used

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  • Protective antigen of avibacterium paragallinarum and expression and application of protective antigen
  • Protective antigen of avibacterium paragallinarum and expression and application of protective antigen
  • Protective antigen of avibacterium paragallinarum and expression and application of protective antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of cloning vector PUC-57-La2

[0057] The schematic diagram of the construction of the PUC-57-La2 cloning vector is as follows: figure 1 As shown, the specific operation steps are as follows:

[0058] 1.1 Design and synthesis of PCR primers

[0059] The whole gene synthesis was carried out to the La2 gene sequence of Avionas paragallinarum in Example 1, that is, as shown in SEQ ID NO.2. And use biological software Primer 5.0 to design PCR primers, the upstream primer sequence is (SEQ ID No.3): 5'-CATGGCTG GCA CAG AGC GTA AAA ACC AAC TTT-3'; the downstream primer is (SEQ ID No.4): 5 '-TCGAG ATTTGC GGT GGT TGC ACC GCC TTG GGT-3'. A NcoI restriction site was introduced at the 5' end of the upstream primer, and a SacI restriction site was introduced at the 5' end of the downstream primer. The target product is 1702bp in length. The primers and the La2 gene sequence of Avibacterium paragallinarum were synthesized by Shanghai Sangon Bioengineering ...

Embodiment 2

[0107] Example 2 Construction of recombinant Lactococcus lactis expressing La2 gene

[0108] The schematic diagram of the construction of Lactococcus lactis expression vector pNZ8149-La2 is as follows: image 3 As shown, the specific operation steps are as follows:

[0109] 2.1 Extraction of Lactococcus lactis pNZ8149 plasmid

[0110] A single colony (Lactococcus lactis expression vector pNZ8149) was picked from GM17 solid medium, inoculated in 5 mL GM17 liquid medium, and cultured overnight at 30°C. Plasmids were extracted according to the instructions of the QIAprep Spin MiniprepKit kit from QIAGEN, and the steps were the same as 1.4.

[0111] 2.2 Double digestion and recovery of cloning vector and expression vector pNZ8149

[0112] The recombinant cloning vector prepared in Example 1, that is, the PUC-57-La2 recombinant plasmid and the expression vector pNZ8149, were digested with restriction enzymes NcoI and SacI. The restriction enzyme digestion system was as follows: ...

Embodiment 3

[0149] Example 3 Induced expression of strain L.lactis NZ3900 / pNZ8149-La2

[0150] 3.1 Inducer concentration screening of La2 protein

[0151] (1) On the GM17 medium plate of cultured bacterial strain L.lactis NZ3900 / pNZ8149-La2, use an inoculation loop to scrape a single colony and inoculate it in 10ml GM17 liquid medium, at 30°C, 5% CO 2 Incubator, static culture 12h.

[0152] (2) Take 2ml of bacterial liquid and add it to 50ml GM17 liquid medium, at 30°C, 5% CO 2 Incubator, static culture until the OD of the bacterial solution reaches 0.3-0.5, add the inducer Nisin (Sigma company) to the final concentration of 0.5ng / ml, 1ng / ml, 5ng / ml, 10ng / ml, 20ng / ml, 40ng / ml, 60ng / ml, induced culture for 4 hours, and take before induction as a negative control.

[0153] (3) At the same time, L.lactisNZ3900 / pNZ8149 was used as the negative control of the induced expression experiment.

[0154] 3.2 Induction time screening of La2 protein

[0155] (1) On the GM17 medium plate of cultur...

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Abstract

The invention relates to the field of poultry infectious disease prevention, and in particular to a protective antigen of avibacterium paragallinarum and expression and application of the protective antigen. The amino acid sequence of the protective antigen of avibacterium paragallinarum is of SEQ ID NO.1 as shown in the specification. The invention further discloses an expression vector for expressing the protective antigen of avibacterium paragallinarum, and an establishment method of the expression vector. The expression vector can be expressed in food-grade lactococcus lactis. The invention further provides application of the protective antigen of avibacterium paragallinarum, that is, application in preparing a chicken infectious rhinitis vaccine. By adopting the vaccine, the resistance of chickens to three serum type avibacterium paragallinarum bacteria A, B and C is remarkably improved, the protection effect of the vaccine is prior to those of whole-bacterial inactivated vaccines, and the vaccine can be adopted to prevent chicken infectious rhinitis caused by different serum type avibacterium paragallinarum bacteria.

Description

technical field [0001] The invention relates to the technical field of epidemic prevention of poultry infectious diseases, in particular to a protective antigen of Avian bacilli paragallinarum, its expression and application. Background technique [0002] Avian infectious coryza is one of the most important respiratory diseases of poultry caused by Avibacterium paragallinarum (Apg). Affected chickens mainly showed clinical symptoms such as runny nose, eyelid swelling and epiphora. Infectious rhinitis in chickens can lead to delay in laying period and decrease in egg production, thus causing significant economic losses to farmers. [0003] Apg belongs to the short Gram-negative bacilli of Pasteurellaceae. The basic characteristics are non-motility, pleomorphic bacteria, and virulent strains with capsules. Page et al. divided Apg into three serotypes: A, B, and C. Studies have shown that the three serotypes of Apg have different degrees of pathogenicity, but there is no inte...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/74C12N1/21A61K39/02A61P31/04C12R1/46
Inventor 王宏俊梅晨李淑芳张培君龚玉梅黄明明李桂萍
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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