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Method for constructing lactobacillus expression plasmid with Nisin as natural resistance selection marker

A resistance screening marker and expression plasmid technology, which is applied in the construction of lactic acid bacteria expression plasmids, can solve the problems of inability to achieve long-term stable expression of foreign antigen genes, easy loss of plasmid vectors, and limited practical application, and achieve long-term stable expression, Not easy to lose, long-term stable expression effect

Inactive Publication Date: 2012-07-18
白杨 +2
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, since the plasmid uses the erythromycin resistance gene as a selection marker, antibiotics cannot be used as selection pressure in humans
In the absence of selection pressure, the plasmid vector is easily lost, so that the long-term stable expression of the foreign antigen gene cannot be achieved, which limits its practical application in the food and pharmaceutical industries

Method used

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  • Method for constructing lactobacillus expression plasmid with Nisin as natural resistance selection marker
  • Method for constructing lactobacillus expression plasmid with Nisin as natural resistance selection marker
  • Method for constructing lactobacillus expression plasmid with Nisin as natural resistance selection marker

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Embodiment 1

[0022] Embodiment 1 Adopt the method of the present invention, construct a kind of lactic acid bacteria expression plasmid with Nisin as natural resistance selection marker according to the following steps:

[0023] (1) Use pNZ9530 as a template to amplify the Nisin element by PCR, and insert restriction sites SphI and SacI at its 2 ends;

[0024] (2) Insert the Nisin element amplified in the above steps into the backbone vector pMG36e;

[0025] (3) The promoter element Pins1 was amplified by PCR, the restriction site EaRI was added to its 2 ends, and the Hpa I restriction site was inserted into the Pins fragment;

[0026] (4) Insert the Pins1 fragment obtained in step (3) into the backbone vector PMN-1 to obtain the vector PMN-2;

[0027] (5) Synthesize the small fragment multi-cloning site MCS and insert it into the vector PMN-2 to obtain the vector PMN-3;

[0028] (6) Synthesize the signal peptide Ins of Lactobacillus acidophilus, and insert it into the vector PMN-3 obtai...

Embodiment 2

[0030] Example 2 In combination with the method of the present invention, the development of a food-grade recombinant Helicobacter pylori bivalent Lactobacillus acidophilus live vaccine based on the Nisin system was realized according to the following steps.

[0031] 1. Construction of food-grade expression / secretion vector PMN-5:

[0032] (1) Use pNZ9530 as a template to amplify the Nisin element by PCR, and insert restriction sites SphI and SacI at its 2 ends;

[0033] (2) Insert the Nisin element amplified in the above steps into the backbone vector pMG36e;

[0034] (3) The promoter element Pins1 was amplified by PCR, the restriction site EaRI was added to its 2 ends, and the Hpa I restriction site was inserted into the Pins fragment;

[0035] (4) Insert the Pins1 fragment obtained in step (3) into the backbone vector PMN-1 to obtain the vector PMN-2;

[0036] (5) Synthesize the small fragment multi-cloning site MCS and insert it into the vector PMN-2 to obtain the vector...

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Abstract

The invention relates to a method for constructing a lactobacillus expression plasmid with Nisin as a natural resistance selection marker, belonging to the technical field of biology. The method is characterized by using a Nisin element from a lactobacillus vector pNZ9530 instead of erythromycin resistance genes of a lactobacillus plasmid pMG36e as the selection marker and realizing construction of a Nisin food-grade expression / secretion vector through PCR (polymerase chain reaction) amplification, gene cloning, synthesis of multiple cloning sites, addition of lactobacillus acidophilus signal peptide and elimination of resistance genes. The method has the following beneficial effects: the antibiotic resistance marker vector is replaced by the food-grade selection marker and the constructed food-grade expression / secretion vector is a food-grade live vector, has high safety factors to human body and environment and can be widely applied to application research of vaccines, food and medicines; and the construction method can achieve long-term stable expression of the exogenous antigen genes and obtain the target antigen proteins stably expressed for a long term, is simple and is strong in operability.

Description

technical field [0001] The invention relates to the construction of a lactic acid bacteria expression plasmid, in particular to the construction of a lactic acid bacteria expression plasmid using Nisin as a natural resistance selection marker, and belongs to the field of biotechnology. Background technique [0002] At present, the plasmid pMG36e is widely used as the expression vector of lactic acid bacteria. It is a constitutive expression vector, which consists of a strong promoter p32 and its downstream part of the open reading frame, multiple cloning sites and protease gene (prtP) from Lactobacillus subsp. The transcription terminator, the pWV01 replicon and the erythrotoxin resistance gene Emr from the plasmid pE194 are composed, and the size of the plasmid is 3.6 kb. The plasmid is convenient for the host to carry and can constitutively express the exogenous target gene. At present, a variety of exogenous genes have been successfully expressed in Lactococcus and Lact...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/64C12N15/65
Inventor 白杨龙北国范宏英
Owner 白杨
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