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Helicobacter pylori NapA protein expressed recombinant vector, recombinant strain and preparation methods and application thereof

A Helicobacter pylori and recombinant vector technology, applied in the biological field, can solve the problems of NapA protein biological function influence, failure to be excised by bacteria, unusable and other problems, achieve obvious immune protection effect, obvious immune regulation effect, and improve production level. Effect

Active Publication Date: 2018-11-02
XINXIANG MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] "Expression and Immunological Activity of Helicobacter Pylori Neutrophil Activating Protein in Lactic Acid Bacteria", Peng Xiaoyan, etc., disclosed a method for expressing NapA protein using Lactococcus lactis system, specifically: the napA gene fragment amplified by PCR It is obtained by connecting between NaeI and SphI in the commercially available expression vector pNZ8110, but the NapA protein expressed by the expression vector prepared by this method fails to secrete, only appears in the bacteria, and carries a secretion signal peptide, so the molecular weight is relatively large , for 19KD
The NapA protein expressed by this system has a signal peptide encoded by the SP sequence, which can neither be secreted outside the cell nor be excised by the bacteria, and finally exists in the fusion form of NapA and signal peptide, making the biological function of NapA protein The function is affected and cannot be used for the production of biological products related to NapA protein, such as oral vaccines, immunomodulatory drugs, food or biological agents

Method used

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  • Helicobacter pylori NapA protein expressed recombinant vector, recombinant strain and preparation methods and application thereof
  • Helicobacter pylori NapA protein expressed recombinant vector, recombinant strain and preparation methods and application thereof
  • Helicobacter pylori NapA protein expressed recombinant vector, recombinant strain and preparation methods and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1 Construction of recombinant vector

[0074] 1. Culture of Helicobacter pylori

[0075] Take out the Helicobacter pylori H.pylori MEL-HP27 (CGMCC NO.1338) 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 until the bacterial liquid is completely After the medium was absorbed, at 37°C, under microaerophilic conditions (5% O 2 , 10% CO 2 , 85%N 2 ) for 3 to 4 days.

[0076] 2. Extraction of Helicobacter pylori genomic DNA

[0077] (1) Scrape Helicobacter pylori cells cultured for 3-4 days into a 1.5ml centrifuge tube filled with 500μl deionized water, add 100μl 100g / L SDS solution, and boil for 5min.

[0078] (2) Add RNase to a final concentration of 50 μg / ml, 37°C for 1 hour, add proteinase K (final concentration 50 μg / ml), and act at 42°C for 1 hour.

[0079] (3) Use equal volumes of phenol, phenol chloroform (equal volume ...

Embodiment 2

[0093] Example 2 Construction of Lactococcus lactis recombinant strain

[0094] 1. Electrotransformation and screening of L. lactis NZ3900 strain

[0095] (1) Referring to the instructions of the authorized patent (ZL201210218281.9), culture L. lactis NZ3900 strain with GM17 medium, and prepare L. lactis NZ3900 competent cells.

[0096] (2) Take out the stored L.lactis NZ3900 competent cells from the -80°C refrigerator and put them on ice. Take 1 μl of the purified ligation product in Example 1 and add it to 40 μl L. lactis NZ3900 competent cells, mix well, and place on ice for 5 minutes.

[0097] (3) Add the bacterial solution to the 2mm electric shock conversion cup pre-cooled with ice; wipe off the water outside the electric shock cup, and put it into the electric shock instrument; set the electric shock parameters to 25μF, 200Ω, 2500V, and perform electric shock; Add 1ml ice-cold GM 17 MC recovery medium, mix well, transfer the bacterial solution to a 1.5ml EP tube, and...

Embodiment 3

[0174] Example 3 Application of L.lactis NZ3900 / pNZ8110Δsp-napA in the preparation of oral vaccines

[0175] Referring to Examples 1 and 2 and Test Example 1, the L.lactis NZ3900 / pNZ8110Δsp-napA strain was cultured in GM17 medium, the expression of the antigen NapA was induced with Nisin, and the bacterial solution was made into a dosage form suitable for oral administration by humans for use as an oral vaccine.

[0176] After oral administration of the vaccine, humans can develop immunity against Helicobacter pylori infection and have beneficial effects in preventing and treating Helicobacter pylori-related diseases.

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Abstract

The invention relates to a helicobacter pylori NapA protein expressed recombinant vector, a recombinant strain and preparation methods and application thereof and belongs to the technical field of biology. According to the helicobacter pylori NapA protein expressed recombinant vector disclosed by the invention, when recombinant plasmid pNZ8110 delta sp-napA is constructed, a secretion signal sequence in the pNZ8110 is removed; thus, NapA molecules expressed by the recombinant vector disclosed by the invention does not carry secretion signal peptide, molecular weight is 15KD, adverse effects toNapA biological activity are avoided, and the adverse effects are caused by the fact that signal peptide cannot be excised in thalli when the recombinant strain fails in secretion expression protein.The helicobacter pylori NapA expressed lactococcus lactis recombinant strain disclosed by the invention not only can be applied to preparing immune adjusting drugs, but also can be applied to producing health care food with an immune adjusting effect; the helicobacter pylori NapA expressed lactococcus lactis recombinant strain also has important significance in preventing and treating diseases offood allergy, immunosuppression, tumor and the like and can generate considerable social and economic benefits.

Description

technical field [0001] The invention relates to a recombinant vector for expressing Helicobacter pylori NapA protein, a recombinant bacterial strain and a preparation method and application thereof, belonging to the field of biotechnology. Background technique [0002] Current research shows that some allergic diseases, infections and tumors are related to immune dysfunction [Schwartz JS, Tajudeen BA, Cohen NA. Medical management of chronic rhinosinusitis-a review of traditional and novel medical therapies. Expert Opin Investig Drugs. 10):1123-1130. / / Woodward J, Gkrania-Klotsas E, Kumararatne D. Chronic norovirus infection and common variable immunodeficiency. Clin Exp Immunol. 2017 Jun; 188(3):363-370.]. However, there is often a lack of specific immunomodulators for the treatment of these immune function-related diseases. The study of new immunomodulators is of great significance to improve the prevention and treatment of these diseases [Gernez Y, Nowak-Wegrzyn A. Immunot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C07K14/195A61K38/16A61P37/02C12R1/46
CPCA23L33/195C12N15/746C07K14/195A23V2002/00A61K38/00A23V2200/324
Inventor 张荣光段广才彭晓燕王辉王琛余飞燕牛志国梁文娟陈帅印范清堂张凌寒张卫东杨海燕郗园林
Owner XINXIANG MEDICAL UNIV
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