Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier

A technology of Helicobacter pylori and Lactococcus lactis is applied in the biological field to achieve considerable social and economic benefits

Inactive Publication Date: 2012-11-28
ZHENGZHOU UNIV
View PDF6 Cites 8 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression vectors pNZ8110 and pNZ8149 of the NICE system have been reported to express Helicobacter pylori antigens, but these two expression vectors can only express foreign proteins in the cytoplasm or secrete them outside the cells [Zhang XJ, Duan G, Zhang R, Fan Q.Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis nisin-controlled

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier
  • Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier
  • Carrier capable of showing and expressing helicobacter pylori protein on surface of lactococcus lactis, and preparation method and application of carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Construction of Lactococcus lactis expression vector pNZ8110-hpaA-lysM

[0054] For the construction of Lactococcus lactis expression vector pNZ8110-hpaA-lysM see figure 1 .

[0055] 1 Preparation of Helicobacter pylori genomic DNA

[0056] 1.1 Use Brooke's blood plate medium, at 37 ° C, 5% CO 2 , under microaerophilic conditions, culture the Helicobacter pylori MEL-HP27 strain for 3-4 days, scrape 1-2 rings of bacteria with an inoculation loop, add 0.5ml sterilized deionized water and 100μl 100g / L SDS solution, boil at 100℃ for 5min .

[0057] 1.2 Add RNase A (to a final concentration of 50 μg / ml) in a 37°C water bath for 1 hour, then add proteinase K to a final concentration of 50 μg / ml, and place in a 42°C water bath for 1 hour.

[0058] 1.3 Use equal volumes of phenol, phenol-chloroform (a mixture of equal volumes of phenol and chloroform), and chloroform to extract once respectively.

[0059] 1.4 Add 2 times the volume of ice-cooled absolute ethanol...

Embodiment 2

[0109] Example 2 Construction of L.lactis NZ3900 / pNZ8110-hpaA-lysM

[0110] 1. Preparation of Competent Cells of Lactococcus lactis L.lactis NZ3900 Strain

[0111] (1) Inoculate 5ml GSGM with 200μl bacterial solution from the L.lactis NZ3900 strain tube stored at -80℃ 17 culture medium, put CO 2 Incubator, 30°C, 5% CO 2 Stand overnight culture;

[0112] (2) Add 5ml of the overnight cultured bacterial solution to 50ml of GSGM 17 medium, 30°C, 5% CO 2 Static culture for 12h~14h;

[0113] (3) Add 50ml cultured bacteria solution to 400ml GSGM 17 Medium, 30°C, 5% CO 2 static culture to OD 600 about 0.3;

[0114] (4) Centrifuge the bacterial liquid at 4°C and 4000r / min for 20min to collect the bacterial cells;

[0115] (5) Wash once with ice-cold 400ml lotion I (0.5mol / L sucrose, 100ml / L glycerol), centrifuge at 4000r / min, 4°C for 20min, and collect the bacterial solution;

[0116] (6) Resuspend the bacteria in 200ml ice-cold washing solution II (0.5mol / L sucrose, 100ml / L...

Embodiment 3

[0133] Example 3 Preparation of recombinant strain L.lactis NZ3900 / pNZ8110

[0134] The preparation of the recombinant strain L.lactis NZ3900 / pNZ8110 can refer to the technical method provided by the seller of the L.lactis NZ3900 strain, NIZO Food Research in the Netherlands or Mobitec in Germany, or the following method:

[0135] 1. Preparation of L.lactis NZ3900 Competent Cells

[0136] Method is with embodiment 2.

[0137] 2. Electrotransformation of L.lactis NZ3900 strain competent cells

[0138] Take 1 μl of the expression vector pNZ8110 (10ng~20ng) and mix it with 40 μl of L.lactis NZ3900 competent cells, and refer to Example 2 for the electrotransformation of L.lactis and the screening method for positively transformed bacteria.

[0139] 3. Identification of L. lactis positive transformants

[0140] 3.1 GM containing 10 μg / ml chloramphenicol from screening positive transformants 17 Pick a single colony on the culture plate and inoculate it in 5ml liquid GM 17 Mediu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a carrier capable of showing and expressing helicobacter pylori protein on the surface of lactococcus lactis, and a construction method and application of the carrier. The construction method of the carrier comprises the following steps of: amplifying helicobacter pylori adhesin A (hpaA) gene by taking helicobacter pylori genomic deoxyribose nucleic acid (DNA) as a template by a polymerase chain reaction (PCR) method; performing enzyme digestion on the obtained hpaA gene and an expression carrier pNZ8110-lysM and connecting, wherein a connection product is used for transforming escherichia coli; screening and identifying positive transformation bacteria, and extracting recombinant plasmid pNZ8110-hpaA-lysM from the positive transformation bacteria; transferring the recombinant plasmid into a lactococcus lactis NZ3900 strain by an electroporation method, and screening and identifying to obtain a recombinant strain L.1actis NZ3900/pNZ8110-hpaA-lysM; and culturing the recombinant strain, and introducing the expression of the hpaA gene by using an inductive agent Nisin. Western blots analysis shows that the protein of the cell wall of the recombinant strain contains the expressed HpaA protein which has immunocompetence. The expression carrier and the recombinant strain containing the carrier can be applied to preparation of helicobacter pylori resistance vaccine and a diagnostic reagent or food processing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an expression vector capable of expressing Helicobacter pylori protein in Lactococcus lactis and displaying the expressed protein on the surface of bacteria, and also relates to a preparation method and application of the vector. Background technique [0002] Helicobacter pylori (H. pylori) is a Gram-negative pathogenic bacteria that infects more than 50% of the world's population. Helicobacter pylori infection is closely related to the occurrence and development of human chronic gastritis, peptic ulcer, gastric cancer and other diseases [Kitajima Y, Ohtaka K, Mitsuno M, et al. Helicobacter pylori infection is an independent risk factor for Runx3 methylation in gastric cancer. Oncol Rep,2008,19(1):197-202], prevention and treatment of Helicobacter pylori infection can reduce the incidence of these diseases. Since the use of antibiotics to treat Helicobacter pylori infection is prone t...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/74C12N1/21C12N15/66A61K39/02A61P31/04G01N33/68A23C9/123A23L1/00C12R1/01
Inventor 段广才张荣光程文滨范清堂张卫东郗园林陈帅印
Owner ZHENGZHOU UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap