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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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 Protecti

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

Example Embodiment

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

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

[0058] 1.1 PCR primer design and synthesis

[0059] The La2 gene sequence of Avian paragallinarum in Example 1, which is shown in SEQ ID NO. 2, was subjected to full gene synthesis. And use the biology 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'. Introduce the NcoI restriction site at the 5'end of the upstream primer, and introduce the SacI restriction site at the 5'end of the downstream primer. The length of the target product is 1702bp. The primers and Avian Bacillus paragallina La2 gene sequence were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.

[0060] 1.2 PCR amplificat...

Example Embodiment

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

[0108] The construction schematic diagram of Lactococcus lactis expression vector pNZ8149-La2 is as follows image 3 As shown, the specific 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 the GM17 solid medium, inoculated into 5mL GM17 liquid medium, and cultured overnight at 30°C. The plasmid was extracted according to the instruction of QIAprep Spin MiniprepKit kit from QIAGEN, and the steps were the same as 1.4.

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

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

[0113] PUC-57-La2 recombinant plasmid ...

Example Embodiment

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

[0150] 3.l La2 protein inducer concentration screening

[0151] (1) Use an inoculating loop to scrape a single colony on the GM17 medium plate of the culture strain L.lactis NZ3900 / pNZ8149-La2 to inoculate 10ml GM17 liquid medium at 30℃, 5% CO 2 Incubate for 12h.

[0152] (2) Take 2ml of bacterial liquid and add it to 50ml of GM17 liquid medium, at 30℃, 5% CO 2 In the incubator, when the OD of the bacterial solution reaches 0.3-0.5, add the inducer Nisin (Sigma) to the final concentration of 0.5ng / ml, 1ng / ml, 5ng / ml, 10ng / ml, 20ng / ml, 40ng / ml, 60ng / ml, induction culture for 4h, at the same time, take the negative control before induction.

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

[0154] 3.2 Induction time screening of La2 protein

[0155] (1) Use an inoculating loop to scrape a single colony on the GM17 medium plate of the cul...

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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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