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Lactococcus lactis expression vector and preparation method and application thereof

A technology of Lactococcus lactis and expression vector, which is applied in the field of recombinant Lactococcus lactis strains, can solve the problems that are not conducive to the effect of vaccine antigens and affect the immune effect of vaccines, and achieve the effect of great application value and outstanding safety

Active Publication Date: 2015-02-04
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as a vaccine antigen expression vector, pNZ8149 has a great defect: pNZ8149 can only express foreign proteins in the cytoplasm, but has no function of secreting and expressing foreign proteins
Lactococcus lactis has a thicker cell wall and expresses the vaccine antigen in the cytoplasm, which is not conducive to the effect of the vaccine antigen on the gastrointestinal mucosal immune system, thus affecting the immune effect of the vaccine
[0005] Apply genetic engineering technology to transform the pNZ8149 vector, construct a new expression vector with the function of secreting and expressing foreign proteins in Lactococcus lactis, and use it for the expression of vaccine antigens such as Helicobacter pylori urease (UreB), and establish a safer and more effective anti-pylorus Helicobacter vaccine strains, which are of great significance for the immune prevention and treatment of Helicobacter pylori infection and related diseases, are expected to produce considerable social and economic benefits, but have not been reported yet

Method used

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  • Lactococcus lactis expression vector and preparation method and application thereof
  • Lactococcus lactis expression vector and preparation method and application thereof
  • Lactococcus lactis expression vector and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Example 1 Construction of L. lactis expression vector pNZ8149-SPusp45

[0072] Construction of L. lactis expression vector pNZ8149-SPusp45 see figure 1 .

[0073] 1. Amplify L.lactis gene SPusp45 by PCR method

[0074] 1.1 Extraction of L.lactis NZ3900 Genomic DNA

[0075] ⑴Take out the L.lactis NZ3900 strain preservation tube from the -80°C refrigerator, and inoculate 100μl of the bacteria solution into 5ml GM 17 In liquid medium, at 30°C, 5% CO 2 After static culture for 12 hours, take 1.5ml~3ml of bacterial liquid, centrifuge at 5000r / min for 5min, and collect the bacterial cells.

[0076] (2) Wash the bacteria once with 500μl TE buffer (10mM Tris-Cl, 1mM EDTA, pH8.0).

[0077] (3) Resuspend the cells in 50 μl TE buffer, add lysozyme to a final concentration of 10 mg / ml, and bathe in water at 37°C for 60 minutes.

[0078] (4) Add proteinase K to a final concentration of 50 μg / ml, mix well, add 100 g / L SDS to a final concentration of 10 g / L, and bathe in water ...

Embodiment 2

[0143] Example 2 Construction of L.lactis expression vector pNZ8149-SPusp45-ureB

[0144] Construction of L. lactis expression vector pNZ8149-SPusp45-ureB see image 3 .

[0145] 1. Culture of Helicobacter pylori

[0146] Take out the Helicobacter pylori H.pylori MEL-HP27 strain from the refrigerator at -80°C, take 500 μl of the bacterial liquid and add it to the Brucella blood plate medium, spread the bacterial liquid evenly with a glass rod, and wait for the bacterial liquid to be absorbed by the medium 37°C, under microaerobic conditions (5%O 2 , 10%CO 2 , 85%N 2 ) for 3 to 4 days.

[0147] 2. Extraction of Helicobacter pylori Genomic DNA

[0148] (1) Scrape the Helicobacter pylori cells cultured for 3-4 days into a 1.5ml centrifuge tube filled with 500μl of deionized water, add 100μl of 100g / L SDS solution, and boil for 5min.

[0149] (2) Add RNase to a final concentration of 50 μg / ml, 37°C for 1 hour, add proteinase K to a final concentration of 50 μg / ml, and act a...

Embodiment 3

[0185] Example 3 Preparation of recombinant strain L.lactis NZ3900 / pNZ8149

[0186] The preparation of the recombinant strain L.lactis NZ3900 / pNZ8149 can either adopt the technical method provided by the strain L.lactis NZ3900 seller NIZO Food Research in the Netherlands or Mobitec in Germany, or the following method:

[0187] 1. Preparation of L.lactis NZ3900 Competent Cells

[0188] Method is with embodiment 1.

[0189] 2. Electrotransformation of competent cells of L.lactis NZ3900 strain

[0190] 1 μl of the purchased expression vector pNZ8149 was mixed with 40 μl of L. lactis NZ3900 competent cells, and the electrotransformation of L. lactis and the screening method of positive transformants were the same as in Example 1.

[0191] 3. Identification of L. lactis positive transformants

[0192] 3.1 From the GM of screening positive transformants 17 Pick a single yellow colony on the culture plate and inoculate it into 5ml GM 17 culture medium, put CO 2 Incubator, 30°C,...

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Abstract

The invention relates to a food-grade expression vector capable of realizing secretory expression of heterologous proteins in lactococcus lactis and a construction method and application thereof. The vector contains a nucleotide sequence as shown in SEQ ID (sequence identity) NO. 1. The construction method comprises the following steps of: amplifying signal peptide genes SPusp45 of lactococcus lactis Usp45 proteins through a PCR (polymerase chain reaction) method, connecting with a food-grade expression vector pNZ8149, using a connection product to transform Escherichia coli, screening and identifying positive transformation bacteria, and extracting recombinant plasmids to obtain a lactococcus lactis secretory expression vector pNZ8149-SPusp45. The invention further discloses the application of the pNZ8149-SPusp45 as a heterologous protein expression vector, including recombinant lactococcus lactis which is constructed by taking the pNZ8149-SPusp45 as the vector and has the secretory expression of helicobacter pylori UreB proteins and the application thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an expression vector capable of secreting and expressing foreign proteins in Lactococcus lactis, and at the same time, also relates to a construction method and application of the expression vector, especially a Helicobacter pylori expression vector constructed by using the vector The recombinant Lactococcus lactis strain of mycoprotein and the application of the strain. Background technique [0002] Current research shows that: for some human digestive tract pathogen infections, such as Helicobacter pylori (H. More effective prevention and treatment of such diseases can only be hoped for the success of vaccine research. In vaccine research, oral vaccines have attracted attention due to their safety, convenience, and rationality [Levine MM. "IDEAL" vaccines for resource poor settings. Vaccine, 2011, 29 Suppl 4:D116-25.]. However, the lack of safe and effective vaccine carriers has hi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74C12N15/66C12N1/21A61K39/02A61P31/04G01N33/569A23C9/12A23L1/00C12R1/01
Inventor 段广才张荣光乔丹范清堂张卫东郗园林陈帅印
Owner ZHENGZHOU UNIV
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