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Recombinant lactobacillus plantarum for expressing newcastle disease virus antigen gene and fermentation process and application of recombinant lactobacillus plantarum

A technology of Lactobacillus plantarum and Newcastle disease virus, which is applied in the field of microorganisms, can solve the problems of the influence of foreign protein expression of recombinant bacteria, and achieve the effects of strong acid resistance and strong bile salt resistance

Active Publication Date: 2021-02-02
JILIN AGRICULTURAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Recombinant lactobacillus plantarum for expressing newcastle disease virus antigen gene and fermentation process and application of recombinant lactobacillus plantarum
  • Recombinant lactobacillus plantarum for expressing newcastle disease virus antigen gene and fermentation process and application of recombinant lactobacillus plantarum
  • Recombinant lactobacillus plantarum for expressing newcastle disease virus antigen gene and fermentation process and application of recombinant lactobacillus plantarum

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 lactobacillus plantarum for expressing a newcastle disease virus antigen gene and a fermentation process and application of the recombinant lactobacillus plantarum, and belongs to the technical field of microorganisms. The recombinant lactobacillus plantarum NC8-pSIP409-pgsA' (ata)-HN-DCpep is successfully constructed. Tests prove that the recombinant lactobacillus plantarum can efficiently express the HN antigen protein, and the protein has immunogenicity. Compared with an original starting strain NC8, the recombinant lactobacillus plantarum has better growth, fermentation, storage performance and the like, meanwhile, SPF chicks are immunized with recombinant lactobacillus plantarum freeze-dried bacterial powder, the safety performance is good, and thechicks has no abnormal reaction after large-dose immunization. The immune performance of the mucous membrane of an organism can be remarkably improved by adopting low-dose immunization, the effect ofresisting the NDV-VII toxic strain is achieved, the survival rate of chickens counteracting toxic substances is 71.6%, and therefore good practical application value is achieved.

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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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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