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A recombinant Lactobacillus plantarum expressing the antigenic gene of Newcastle disease virus and its fermentation process and application

A technology of Lactobacillus plantarum and Newcastle disease virus, applied in the field of microorganisms, can solve problems such as the influence of exogenous protein expression of recombinant bacteria, and achieve the effect of improving mucosal immunity, good safety performance and optimal growth.

Active Publication Date: 2021-09-21
JILIN AGRICULTURAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poly-γ-glutamic acid synthetase A (Poly-γ-Glutamic Acid SynthetaseA, pgsA) comes from Bacillus subtilis (Bacillus subtilis), which can anchor foreign proteins on the surface of host bacteria, but the inventors found that the The longer gene sequence (1143bp) of the protein affects the construction of recombinant bacteria and the expression of foreign proteins

Method used

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  • A recombinant Lactobacillus plantarum expressing the antigenic gene of Newcastle disease virus and its fermentation process and application
  • A recombinant Lactobacillus plantarum expressing the antigenic gene of Newcastle disease virus and its fermentation process and application
  • A recombinant Lactobacillus plantarum expressing the antigenic gene of Newcastle disease virus and its fermentation process and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1. Construction of recombinant Lactobacillus plantarum BLCC2-0394

[0082] 1 Materials and methods

[0083] 1.1 Materials

[0084] 1.1.1 Strains and plasmid vectors

[0085] Newcastle disease virus VII strain (NDV-VIINA-1) allantoic fluid, Lactobacillus plantarum NC8 (Lactobacillus plantarum NC8), PUC Vector (pMD18-T Simple Vector), shuttle expression vector (Escherichia coli / lactic acid bacteria) pSIP409, E. coliχ6212 and the plasmid pSIP409-pgsA'-EGFP(ata) containing alanine racemase gene alr were constructed or preserved by the laboratory of Professor Wang Chunfeng of Jilin Agricultural University, so the above materials can be obtained from the laboratory of Professor Wang Chunfeng of Jilin Agricultural University. room obtained.

[0086] 1.1.2 Enzymes and main test reagents

[0087] Restriction enzymes (XbaI and HindⅢ), SppIP were purchased from Takara Bioengineering Dalian Co., Ltd.; T4 DNAligase, DNA Marker (DL-2000, λ-HindⅢ) were purchased from Takar...

Embodiment 2

[0151] Example 2 Morphological analysis and immunogenicity analysis of recombinant Lactobacillus plantarum BLCC2-0394

[0152] 1 Materials and methods

[0153] 1.1 Materials

[0154] Bacterial strain: the recombinant Lactobacillus plantarum BLCC2-0394 obtained in Example 1 of the present invention.

[0155] Medium: MRS medium: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.2g / L, ammonium citrate 2g / L, Sodium acetate 5g / L, Tween-80 1ml / L, pH value 6.0±0.05, sterilized at 121°C for 20min. Solid medium needs to add 1.2% agar.

[0156] 1.2 Method

[0157] 1.2.1 Morphological analysis

[0158] Take 10 μL of the glycerol tube preservation solution of recombinant Lactobacillus plantarum BLCC2-0394 and spread it on the MRS agar plate, and culture it at 30°C for 48h. Pick a typical single colony of Lactobacillus growing on the plate (short rod, medium size, raised, slightly white with neat edges) for Gram staini...

Embodiment 3

[0183] Example 3. Analysis of biological characteristics of recombinant Lactobacillus plantarum BLCC2-0394

[0184] 1 Materials and methods

[0185] 1.1 Materials

[0186] Bacterial strain: the recombinant Lactobacillus plantarum BLCC2-0394 obtained in Example 1 of the present invention.

[0187] Medium: MRS medium: glucose 20g / L, peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.2g / L, ammonium citrate 2g / L, Sodium acetate 5g / L, Tween-80 1ml / L, pH value 6.0±0.05, sterilized at 121°C for 20min. Solid medium needs to add 1.2% agar.

[0188] 1.2 Method

[0189] 1.2.1 Acid resistance test of recombinant Lactobacillus plantarum BLCC2-0394

[0190] Inoculate 0.5mL of recombinant Lactobacillus plantarum BLCC2-0394 glycerol tube preservation solution into 100mL MRS medium, cultivate overnight at 30°C, transfer 2% of the inoculum to freshly sterilized MRS medium, and culture overnight at 30°C Take 5 mL of bacterial solution, cent...

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Abstract

The invention provides a recombinant plant lactobacillus expressing Newcastle disease virus antigen gene and its fermentation process and application, belonging to the technical field of microorganisms. The present invention successfully constructs a recombinant plantarum Lactobacillus NC8-pSIP409-pgsA'(ata)-HN-DCpep. Tests have proved that the recombinant plantarum lactobacillus obtained in the present invention can not only highly express the HN antigen protein, but also has immunogenicity. At the same time, compared with the original starting strain NC8, it has better growth, fermentation and storage performance, etc. At the same time, the use of the recombinant Lactobacillus plantarum freeze-dried bacterial powder to immunize SPF chicks has good safety performance, and large-dose immunization has no abnormalities in chickens reaction. Low-dose immunization can significantly improve the immune function of the body's mucosa, and has the effect of resisting NDV‑VII strains. The survival rate of chickens after challenge is 71.6%, so it has good practical application value.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a recombinant plant lactobacillus expressing a Newcastle disease virus antigen gene and its fermentation process and application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Newcastle disease (ND) is a kind of highly contagious infectious disease caused by Newcastle disease virus (NDV), which can cause a variety of poultry to have febrile, acute, and septic symptoms, and the mortality rate is extremely high. , also known as pseudo chicken plague or Asian chicken plague, is one of the important diseases that seriously endanger the poultry farming industry in the worl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C07K19/00C12N9/24A23K10/16A61K39/17A61P31/14C12R1/25
CPCC12N9/2402C12Y302/01018C12N15/746A23K10/16A61K39/12A61P31/14C12N2800/22C07K2319/33C07K2319/035A61K2039/542A61K2039/552A61K2039/523C12N2760/18134A23V2400/169
Inventor 王春凤单宝龙刘乃芝亓秀晔郭杨丽姜延龙陈雷谷巍徐海燕张建梅程福亮李丹王红宋翔宁扬侯玉凤李鹏陈晓雯
Owner JILIN AGRICULTURAL UNIV
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