CN108728473B - Recombinant vector and recombinant strain for expressing helicobacter pylori NapA protein, and preparation method and application thereof - Google Patents

Abstract

The invention relates to a recombinant vector and a recombinant strain for expressing helicobacter pylori NapA protein, and a preparation method and application thereof, belonging to the technical field of biology. When the recombinant plasmid pNZ8110 delta sp-napA is constructed, a secretion signal sequence in pNZ8110 is removed, so that the NapA molecule expressed in the invention does not carry a secretion signal peptide and has a molecular weight of 15KD, and adverse effects on NapA bioactivity caused by the fact that the signal peptide cannot be cut in thalli when a recombinant strain fails to secrete and express protein are avoided. The lactococcus lactis recombinant strain for expressing the helicobacter pylori NapA can be used for manufacturing immune regulation medicines and producing health-care food with immune regulation efficacy, has important significance for preventing and treating diseases such as food allergy, immune suppression, tumor and the like, and can generate considerable social and economic benefits.

Description

Recombinant vector and recombinant strain for expressing helicobacter pylori NapA protein, and preparation method and application thereof

Technical Field

A recombinant vector for expressing helicobacter pylori NapA protein, a recombinant strain, a preparation method and an application thereof belong to the technical field of biology.

Background

Current studies indicate that some allergic diseases, infections and tumors are associated with immune dysfunction [ Schwartz JS, tajuden BA, Cohen na. medical management of chronic rhinonosis-a review of pathological and non-pathological therapeutics. expert Opin Investig drugs.2017 oct; 1123. 1130// Woodward J, Gkrania-Klotsas E, Kumarantane D.Chronic novirus infection and common variable immunological immunity.2017 Jun; 188(3):363-370.]. However, specific immunomodulators are often lacking in the treatment of these immune function related diseases. Research on novel immunomodulators has important implications for improving prevention and treatment technologies for these diseases [ Gernez Y, Nowak-Wegrzyn A. immunothery for Food Allergy: Are We the re YetJ Allergy Clin immunological practice.2017 Mar-Apr; (5) (2) 250-; 65(4) 388-; 51(4):299-304.]. For immune-dysregulated diseases such as food allergy, The development of Oral immunomodulators has a development prospect due to The advantages of safe, convenient and reasonable administration route, but is limited by The complexity of immune regulation mechanism, special microenvironment in gastrointestinal tract and The prior art level, and The development of Oral immunomodulators is still in The research stage [ Brotons-Canto A, Mart i N-Arbella N, Gamazo C, Irache.New pharmaceutical applications for The research of Oral immunomodulators of food allergy, Experit Optin Drug delivery, 2016Oct 27: 1-12// Z cri FJ. from Natural Product to The First animal Treatment for Multiple experiments: The Discovery of FTY720 (Gillanya^TM)？Curr Opin Chem Biol.2016Jun；32:60-6.]. Due to the degradation of the biological agents by digestive juices in the gastrointestinal tract, in order to improve the immune effect of oral biological agents, it is often necessary to deliver immunologically active proteins with the aid of bacterial vectors [ Davitt CJ, lavle ec. 91:52-69.]。

Helicobacter pylori NapA is a virulence factor that mediates adhesion of bacteria to the stomach wall by binding to hydrocarbons on the surface of gastric mucosal epithelial cells and also attracts and activates neutrophils [ Polenghi A, Bossi F. the neutrophile-activating protein of Helicobacter pylori cross to pmote neutrophile addition in v.J. Immunol,2007,178(3): 1312-1320). NapA is reported to have a dual effect on cellular DNA: in one aspect, DNA can be damaged by stimulating reactive oxygen species production; on the other hand, it can also exert a protective effect on DNA by its ferroxidase center (ferroxidase center) [ Yokoyama H, Fujii S. structures and metal-binding properties of Helicobacter pylori-activating protein with a di-nuclear ferroxidase center 2014,4(3):600-615 ]. Research finds that helicobacter pylori NapA and mucosal immune adjuvant are combined to immunize experimental animals orally, and the effect of resisting helicobacter pylori infection can be achieved; in addition, it was found through in vitro cell culture experiments that Helicobacter pylori NapA has a function of promoting the transformation of Th2 immune response into Th1 type immune response [ Satin B, Del Giudice G, Della Bianca V, et al, the neutral-activating protein (HP-NAP) of Helicobacter pylori a protective antigen and a major viral factor J Exp Med,2000,191(9): 1467-.

Lactococcus lactis (L.lactis) is a species of lactic acid bacteria, has a long history of use in food processing, belongs to food-grade probiotics, and is very safe [ Morello E, term dez-Humar. sub.n LG, Llull D, et al Lactococcus lactis, an effective cell factor for recombinant protein production and section. J. Mol. Microbiol Biotechnology, 2008,14(1-3):48-58.// Wang M, Gao Z, Zhang Y, Pan L.coli acid as microbial delivery vehicles: a recombinant therapeutic application. Microbiol. 2016biol. JL.; 100(13): 5691-; and the growth is rapid, the culture is easy, the genetic background and the regulation mechanism are relatively clear, and the protein has great potential as an oral vaccine carrier [ Stentz R, Bongaerts RJ, guining AP, Gasson M, Shearman C.controlled release of protein from viral polysaccharides cells. apple Environ Microbiol,2010,76(9): 3026-. Currently, the Lactococcus lactis NICE expression system has a significant advantage in terms of safety [ Mierau I, Kleerebezem M.10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. apple Microbiol Biotechnol,2005,68(6):705-17 ].

The authors Pengxiao swallow and others from the expression and immunological activity of helicobacter pylori neutrophil activating protein in lactic acid bacteria disclose a method for expressing NapA protein by using a lactococcus lactis system, which specifically comprises the following steps: the napA gene fragment amplified by PCR is connected between NaeI and SphI in a commercially available expression vector pNZ8110, but the NapA protein expressed by the expression vector prepared by the method fails to be secreted and only appears in a bacterial body, and carries a secretion signal peptide, so that the molecular weight is large and is 19 KD. The NapA protein expressed by the system carries a signal peptide coded by an SP sequence, can not be secreted to the outside of cells, can not be excised by bacteria, and finally exists in a fusion form of the NapA and the signal peptide, so that the biological function of the NapA protein is influenced, and the NapA protein can not be used for producing biological products related to the NapA protein, such as oral vaccines, immunoregulatory drugs, foods or biological agents.

Disclosure of Invention

The invention aims to provide a recombinant vector for expressing a helicobacter pylori NapA protein.

The invention also provides a preparation method of the recombinant vector.

The invention also provides a recombinant strain containing the recombinant vector, and the recombinant strain can express the NapA protein with biological activity and the size of 15 KD.

The invention also provides a preparation method of the recombinant strain.

The invention also provides the application of the recombinant vector and the recombinant strain.

In order to achieve the purpose, the invention adopts the technical scheme that:

a recombinant vector for expressing the NapA protein of helicobacter pylori is characterized in that a napA gene segment is inserted between a Pnis promoter and a Terminator of an expression vector pNZ8110 with a knocked-out SP functional region.

The napA gene segment is the gene segment shown as 210-644 bit in SEQ ID NO.1 or the gene segment with 95 percent of similarity with the napA gene segment.

The nucleotide sequence of the expression vector pNZ8110 without knocking out the SP functional region is shown as SEQ ID NO. 2.

Specifically, the nucleotide sequence of the recombinant vector is shown as SEQ ID NO.1, and the recombinant vector comprises a promoter Pnis (position 6-206), a replicon repC (position 1399-1608) and repA (position 1876-2574) which are regulated and controlled by nisin (nisin), a chloramphenicol resistance gene cm (position 3033-3683), a multiple cloning enzyme cutting site, a helicobacter pylori napA gene (position 210-644), and the like.

The preparation method of the recombinant vector comprises the following steps: amplifying a napA gene fragment of helicobacter pylori by PCR, continuously accessing the napA gene fragment between the NcoI enzyme cutting site and the SphI enzyme cutting site of the expression vector pNZ8110 with the SP functional region removed, and screening and identifying to obtain the product.

Specifically, the primers used for amplifying the napA gene segment are designed according to the napA gene sequence of helicobacter pylori in GenBank database (GenBank: AY366361) by using biological software Primer 5.0:

the sequence of the upstream primer is as follows: 5' -CATGCCATGG GCATGAAAACATTTGAAAT-3'；

The sequence of the downstream primer is as follows: 5' -ACGTGCATGCTTAAGCTAAATGGGCTT-3'. The upstream primer and the downstream primer contain NcoI and SphI cleavage sites, respectively.

A recombinant strain comprising the recombinant vector described above.

Specifically, the recombinant strain is constructed from L.lactis NZ 3900. The construction method comprises the following steps: and transferring the recombinant vector into a lactococcus lactis NZ3900 competent cell, and identifying and screening to obtain the recombinant vector.

More specifically, the step of constructing the recombinant strain comprises:

1) extracting genomic DNA of helicobacter pylori MEL-HP27 strain (CGMCC NO.1338) as PCR template for amplifying helicobacter pylori napA gene;

2) respectively double-enzyme cutting napA gene and expression vector pNZ8110, purifying and recovering enzyme cutting fragment,using in combination T₄Carrying out directional ligation by using DNA ligase, and converting the ligation product into a lactococcus lactis NZ3900 strain by an electroporation method;

3) screening positive transformants by selecting the medium with GM17 containing chloramphenicol (10. mu.g/ml final concentration), and identifying the recombinant strain by PCR, restriction and sequencing;

4) culturing the recombinant strain, extracting recombinant plasmid, namely a lactococcus lactis expression vector for expressing helicobacter pylori NapA protein, and naming the recombinant plasmid as follows: pNZ 8110. delta. sp-napA, lactococcus lactis containing pNZ 8110. delta. sp-napA was named: l. lactis NZ3900/pNZ 8110. delta. sp-napA.

After a plurality of positive strains are selected, a Lactococcus lactis NZ3900/pNZ8110 delta sp-napA strain with the highest NapA expression quantity is obtained and is delivered to the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 14933. Specifically, an expression vector pNZ8110 delta sp-napA is used for transforming Lactococcus lactis (Lactococcus lactis) NZ3900 strain, and the obtained positive transformation bacterium NZ3900/pNZ8110 delta sp-napA can express NapA after being induced by Nisin. The final concentration of inducer used was 40 ng/mL.

The recombinant vector is applied to the expression of NapA protein. In particular to the application of the recombinant vector in preparing an immunoregulation biological agent or food. More specifically, the recombinant vector is applied to the preparation of an immunoregulatory biological agent or food for enhancing Th17 type immune response.

The recombinant strain is applied to expressing NapA protein. In particular to application of the recombinant strain in preparing an immunoregulatory biological agent or food. More specifically, the recombinant strain is applied to the preparation of an immunoregulatory biological agent or food for enhancing Th17 type immune response.

The immune-regulatory biological agent comprises an oral vaccine, an immune-regulatory drug and the like.

Specifically, after the recombinant strain is expressed by Nisin induced NapA, the obtained bacterial liquid or a concentrated or dried product thereof is used as an immunoregulatory drug for oral administration or external use, so as to achieve the purpose of preventing or treating the immune dysregulated diseases.

The application method of the recombinant strain in food (including beverage) processing comprises the following steps: adding appropriate amount of recombinant strain into food processing material, and allowing the mixture to grow under conditions suitable for the strain (30 deg.C, 5% CO)₂Environment) to generate fermentation reaction, and adding Nisin at proper time to induce the strain to express vaccine antigen NapA. The food product may contain the strain in a viable or inactivated form.

In addition, recombinant strains are cultured, Nisin is used for inducing NapA expression, and the obtained bacterial liquid or concentrated or dried products thereof are used as immunomodulators and can be used for experimental research.

More specifically, the application of the lactococcus lactis with the preservation number of CGMCC NO.14933 in the production of the vaccine for resisting helicobacter pylori infection.

The application of lactococcus lactis with the preservation number of CGMCC NO.14933 in producing immunomodulatory drugs or food with the effect of enhancing Th17 type immune response.

At present, although there are studies showing that helicobacter pylori NapA has a phenomenon of promoting the polarization of specific immune response to Th1 type, there is no study showing or suggesting that helicobacter pylori NapA has a regulatory effect on Th17 type immune response. The invention discovers that when the recombinant lactococcus lactis strain expressing helicobacter pylori NapA is fed to a mouse, the level of interleukin IL-17 of spleen cells of the mouse can be obviously increased, and the result shows that the immune response is obviously enhanced Th17 type. Because the Th17 type immune response of the organism has the function of strengthening innate immunity and has important functions in anti-infection and tumor immune mechanisms, the immune regulation function that the helicobacter pylori NapA can strengthen the Th17 type immune response has important application value.

In practical use, the cultured lactococcus lactis recombinant bacterium can be applied to a human body to achieve the purpose of preventing or treating helicobacter pylori infection and diseases caused by the infection.

When the lactococcus lactis is used for preparing an immunoregulation biological agent, food and a biological agent for regulating Th17 type immune response, the lactococcus lactis recombinant strain is mainly used for regulating the Th17 type immune response of an organism through oral administration. In particular, the recombinant lactococcus lactis strain has the effect of regulating Th17 and/or Th1 immune response of an organism through oral administration, so that the aim of preventing or treating diseases or promoting health of a human body is fulfilled by eating the food.

The lactococcus lactis gene recombinant strain has the effect of improving the production level of the cytokine IL-17 or/and INF-gamma of spleen cells by oral administration or gastric lavage.

The food and the biological agent can be applied to human beings and other animals.

The experimental basis of the invention comprises that recombinant strain L.lactis NZ3900/pNZ8110 delta sp-napA is cultured in vitro, exogenous protein expression is induced by Nisin, and the expression of protein is analyzed and the immunocompetence of the protein is identified by SDS-PAGE and Western blot methods. As a result: l.lactis NZ3900/pNZ8110 delta sp-napA can be detected by SDS-PAGE to express the helicobacter pylori NapA protein. Detecting the expressed NapA protein in the bacterial lysis protein, wherein the molecular weight of the expressed NapA is about 15kD and accounts for 14% (w/w) of the bacterial lysis protein; western blot analysis detects that the expressed NapA can have immunoreaction with mouse serum antibody immunized with helicobacter pylori thallus lysis antigen.

BALB/c mice are immunized by gastric lavage with the recombinant strain L.lactis NZ3900/pNZ8110 delta sp-napA, spleen cells of the mice are separated, in vitro culture is carried out, helicobacter pylori thallus lysis antigen is added into a culture medium, and the result shows that the level of IL-12, IL-17 and INF-gamma produced by the spleen cells of the mice immunized by the recombinant bacteria is obviously higher than that of a control group treated by the L.lactis NZ3900/pNZ8110 and Phosphate Buffer Solution (PBS) respectively.

A BALB/c mouse is immunized by gastric lavage with a recombinant strain L.lactis NZ3900/pNZ8110 delta sp-napA, blood and excrement of the mouse are collected, and a serum IgG antibody and a secretory SIgA antibody are detected by enzyme-linked immunosorbent assay (ELISA). The results show that recombinant immunized mice had significantly higher levels of IgG and SIgA antibodies than the control group treated with l.lactis NZ3900/pNZ8110 and Phosphate Buffered Saline (PBS), respectively.

A BALB/c mouse is immunized by a recombinant strain L.lactis NZ3900/pNZ8110 delta sp-napA through gastric lavage, after the immunization program is finished, the mouse is gavaged by helicobacter pylori bacterial liquid, helicobacter pylori in mouse gastric tissue is detected after challenge infection, and the planting level of the helicobacter pylori in the mouse stomach of the recombinant bacterium immunization group is found to be remarkably lower than that of a control group treated by the L.lactis NZ3900/pNZ8110 and PBS.

The invention has the following beneficial effects:

1) the invention constructs a lactococcus lactis gene recombinant strain for expressing a Helicobacter pylori (Helicobacter pylori) neutrophil activating protein A subunit (NapA) by using a lactococcus lactis NICE expression system, provides a lactococcus lactis recombinant strain L.lactis NZ3900/pNZ8110 delta sp-napA capable of efficiently expressing Helicobacter pylori NapA protein, provides a new source for the production of the NapA protein, and can safely, simply and effectively deliver the NapA to a mucous membrane immunity site by using the strain. The lactococcus lactis belongs to probiotics, and the engineering strain established by the invention has good safety; the expressed NapA does not need to be purified, can be directly taken orally, and can also be used for fermenting dairy products to be eaten along with the dairy products; experiments prove that the lactococcus lactis recombinant strain can generate obvious immune protection effect and immune regulation effect by delivering NapA through mouth, and similar researches are not reported.

2) The engineering strain L.lactis NZ3900/pNZ8110 delta sp-napA provided by the invention has the advantages that experimental results prove that the engineering strain is applied, helicobacter pylori NapA is transmitted to a gastrointestinal tract mucous membrane immune site through oral administration or eating, the immunoregulation effect of enhancing Th17 and Th1 immune reactions is achieved, and the effect cannot be predicted before experiments based on the prior art. Experiments show that the engineering strain can improve Th17 type immune response after gastric lavage for the first time, is a Th17 type immune response enhancer, and has considerable application prospect in the production of medicines, health-care foods and biological agents. At present, under the condition that the clinical treatment of a plurality of immune disorder related diseases lacks of immune regulators, the novel immune regulator provided by the invention not only has technical progress, but also has a promoting effect on the development of biological medicines and dairy products in China, and is expected to generate good economic and social benefits.

3) The lactococcus lactis NZ3900/pNZ8110 delta sp-napA provided by the invention has the advantages that experimental results prove that the helicobacter pylori NapA antigen can be effectively delivered to gastrointestinal mucosa immune sites of human beings or other animals by oral administration or eating by applying the lactococcus lactis engineering strain, an obvious immune protection effect for resisting helicobacter pylori infection is generated, and the lactococcus lactis engineering strain can be used for preparing an oral vaccine for resisting helicobacter pylori.

In view of the fact that clinical immune dysregulated diseases are usually required to be treated by corresponding immunomodulators, the lactococcus lactis recombinant strain expressing helicobacter pylori NapA provided by the invention can be used for manufacturing immunomodulatory drugs and producing health-care foods with immunoregulation efficacy, has important significance for preventing and treating diseases such as food allergy, immunosuppression and tumor, can promote the development of biological medicines and dairy product industry, and can generate considerable social and economic benefits.

Drawings

FIG. 1 is a restriction enzyme digestion identification diagram of a recombinant vector pNZ 8110. delta. sp-napA;

FIG. 2 is a schematic structural diagram of a vector plasmid pNZ8110 and a recombinant vector pNZ8110 delta sp-napA;

FIG. 3 is an SDS-PAGE and Western blot analysis of products of lactococcus lactis induced expression;

FIG. 4 is a graph showing the results of ELISA detection of mouse anti-NapA secretory SIgA antibody and serum IgG antibody;

FIG. 5 is a graph showing the results of ELISA detection of cytokine levels in the mouse spleen cell culture supernatant;

FIG. 6 is a graph showing the results of helicobacter pylori culture and urease activity assay in mouse gastric tissue.

Detailed Description

The invention is further described below with reference to the figures and the specific examples.

(ii) strains and plasmids used in the present invention

Lactococcus lactis NZ3900 strain (lacF)^-NisNisNisK) was purchased from NIZO Food Research (Kernemseweg, Netherlands) in the Netherlands. The strain can also be obtained from Mobitec company (Germany)

Germany). Using GM containing 150ml/L glycerol₁₇The culture medium was maintained at-80 ℃.

Lactococcus lactis expression vector pNZ8110 was purchased from NIZO Food Research (Kernemseweg, Netherlands) of the Netherlands, and also from Mobitec, Germany (Mobitec: (N.E.)

Germany), the structure of which is shown in FIG. 2-A, and the nucleotide sequence of which is shown in SEQ ID NO. 2. The commercial pNZ8110 contains the promoter Pnis, the signal peptide Sequence (SP) of the lactococcus lactis usp45 gene, the chloramphenicol resistance gene (cm), the multiple cloning site, the replication genes repC and repA, a terminator and other elements.

The Lactococcus lactis strain Lactococcus NZ3900/pNZ8110 delta sp-napA provided by the invention is preserved in the China general microbiological culture Collection center (address: No.3 of West Lu 1 of the sunward area of Beijing, institute of microbiology of China academy of sciences) in 11 months and 20 days of 2017, and the preservation number is as follows: CGMCC NO. 14933. Using GM containing 150ml/L glycerol₁₇The culture medium was maintained at-80 ℃.

Helicobacter pylori (Helicobacter pylori) strain MEL-HP27 is a strain preserved in China general microbiological culture collection management center at 29.03.2005 (address: Beijing, West Lu No.1, North Cheng, the area of the republic of China, institute of microbiology, China academy of sciences), and has the following preservation number: CGMCC NO. 1338. The strain preservation solution is a mixed solution of 100g/L sucrose solution and fetal calf serum with the same volume, and the strain is preserved in a refrigerator at minus 80 ℃.

(II) preparation of the Medium of the present invention

(1) Lactic acid bacteria culture medium

GM17 medium: weigh 4.25g M₁₇Dissolving the culture medium in 80ml of distilled water, adjusting the pH value to 7.2 by using 10mol/L NaOH, fixing the volume to 100ml, carrying out autoclaving at 121 ℃ for 20min, cooling, and adding glucose to the final concentration of 5.0 g/L.

GM₁₇Culture medium plate: weighing 4.25g GM₁₇Culture medium, 1.5g agar powder, dissolved in 80ml distilled water, and treated with 10mol/L NaOHAdjusting pH to 7.2, diluting to 100ml, autoclaving at 121 deg.C for 20min, cooling to 60 deg.C, adding glucose to final concentration of 5.0g/L, mixing, and pouring the culture medium into a culture dish to obtain culture medium plate.

GSGM₁₇Culture medium: 85.50g of sucrose, 12.50g of glycine and 18.63g M g of glycine were weighed out₁₇Dissolving the culture medium in 400ml of distilled water, adjusting the pH value to 7.2 by using 10mol/L NaOH, metering to 500ml, and sterilizing under high pressure. After cooling, glucose was added to a final concentration of 5.0 g/L.

GM₁₇MC recovery medium: weighing 3.73g M₁₇Medium, 0.19g MgCl₂,0.02g CaCl₂Dissolving in 80ml distilled water, adjusting pH to 7.2 with 10mol/L NaOH, diluting to 100ml, and autoclaving. After cooling, glucose was added to a final concentration of 5.0 g/L.

(2) Helicobacter pylori culture medium

Brookfield blood plate medium: weighing 1.00g of soybean peptone, 1.00g of tryptone, 0.10g of yeast extract powder, 0.50g of sodium chloride, 0.01g of sodium sulfite and 1.50g of agar powder, adding 100ml of distilled water, adjusting the pH to 7.2, carrying out autoclaving at 121 ℃ for 20min, cooling to about 60 ℃, adding 8ml of sheep blood and 5ml of fetal calf serum, and adding the following 4 antibiotics: 0.5ml of 10mg/L vancomycin, 0.5ml of 2mg/L amphotericin, 0.1ml of 2500U/L polymyxin and 0.2ml of 5mg/L sulfanilamide synergist (TMP). Mixing, pouring into glass culture dish after high pressure sterilization.

(III) reagent used in the present invention

M₁₇Culture medium (product of Qingdao Haibo Biotech Co., Ltd.), Nisin (Sigma, USA), chloramphenicol (Amresco, USA), and T₄DNA ligase and restriction enzymes NcoI and SphI (Thermo Scientific, USA), gel recovery kit (AxyGen, Hangzhou), DNA and protein molecular weight standards and HRP-labeled goat anti-mouse IgG (Beijing kang is century Biotechnology Co., Ltd.), PCR reaction kit and RNase A (RNase A) (Beijing Tiangen Biotechnology Co., Ltd.), bacterial genomic DNA and plasmid extraction kit (Shanghai Chuang bioengineering Biotechnology Co., Ltd.), PVDF membrane (Beijing Solarbio Co., Ltd.), HRP-labeled goat anti-mouse SIgA (Abcam, UK), ELISA kit (Shanghai enzyme-Biotechnology HRP)Biotech Co., Ltd.), D-Hank's buffer solution and erythrocyte lysate, RPMI-1640 complete medium (Solambio Co., Beijing), and other common reagents are domestic analytical pure grade reagents.

The experimental materials and methods not specifically described belong to the known art. In the field of the present invention, the general reference books include molecular cloning, laboratory Manual (J. SammBruk and D.W Lassel, third edition, scientific Press, 2002), and the like.

EXAMPLE 1 construction of recombinant vectors

1. Culture of helicobacter pylori

Taking helicobacter pylori H.pylori MEL-HP27(CGMCC NO.1338) strain from a refrigerator at-80 deg.C, adding 500 μ l of the strain to Brookfield blood plate culture medium, coating the strain with glass rod, absorbing the strain by the culture medium, and making the strain pass through a microaerophilic condition (5% O) at 37 deg.C₂、10％CO₂、85％N₂) Culturing for 3-4 days.

2. Extraction of helicobacter pylori genomic DNA

(1) The cultured helicobacter pylori cells of 3-4 days were scraped into a 1.5ml centrifuge tube containing 500. mu.l of deionized water, 100. mu.l of 100g/L SDS solution was added, and the mixture was boiled for 5 min.

(2) RNase was added to a final concentration of 50. mu.g/ml for 1 hour at 37 ℃ and proteinase K (final concentration of 50. mu.g/ml) was added for 1 hour at 42 ℃.

(3) The extract was extracted once with phenol, phenol chloroform (a mixture of phenol and chloroform in equal volume), and chloroform, respectively.

(4) After addition of 2 volumes of absolute ethanol and 1/10 volumes of 2.5mol/L sodium acetate, the precipitate was precipitated at-80 ℃ for 1 h. Centrifuging at 15000r/min for 15 min.

(5) The precipitate was washed twice with 75% absolute ethanol, 100. mu.l of deionized water was added and stored at-80 ℃ for further use.

3. PCR amplification of helicobacter pylori napA gene

(1) Designing and synthesizing a PCR primer: designing PCR Primer by using biological software Primer 5.0 according to helicobacter pylori napA gene sequence (AY366361) in GenBank database, wherein the upstream Primer sequence is 5' -CATGCCATGGGCATGAAAACATTTGAAAT-3' (SEQ ID NO. 3); downstream primer sequenceThe columns are as follows: 5' -ACGTGCATGCTTAAGCTAAATGGGCTT-3' (SEQ ID NO. 4). An NcoI restriction site is introduced at the 5 'end of the upstream primer, and an SphI restriction site is introduced at the 5' end of the downstream primer. Synthesized by Shanghai Czeri bioengineering, Inc.

(2) And (3) PCR reaction system: mu.l of 8. mu. mol/L upstream primer, 1. mu.l of 8. mu. mol/L downstream primer, 25. mu.l of 2 XTAQQ PCR Master Mix, 2. mu.l of H.pylori genomic DNA template, and 50. mu.l of deionized water.

(3) The PCR cycling conditions were as follows: pre-denaturation at 95 deg.C for 4min, denaturation at 94 deg.C for 1min, annealing at 50 deg.C for 30s, extension at 72 deg.C for 2min, 30 cycles, and final extension at 72 deg.C for 10 min.

(4) Analysis of PCR products: directly taking 5 μ L of PCR reaction product, performing electrophoresis with 15g/L agarose gel at voltage of 5V/cm for 20min, and analyzing gel image with gel image analyzer.

4. Digestion and recovery of helicobacter pylori napA gene and vector pNZ8110

Taking the napA gene obtained by PCR amplification, carrying out double digestion by using restriction enzymes SphI and Nco I, and carrying out double digestion on the plasmid pNZ8110 by using SphI and Nco I. The enzyme cutting system is as follows: NcoI 1.5. mu.l, SphI 1.5. mu.l, 10 XBuffer 5. mu.l, pNZ8110 or napA gene 25. mu.l, deionized water 17. mu.l. The reaction was carried out at 37 ℃ for 12 h. The enzyme digestion fragments were purified and recovered with a gel recovery kit (Hangzhou Esquir Biotech Co., Ltd.) according to the product instructions.

5. Connection of helicobacter pylori napA gene and vector pNZ8110

The recycled napA gene and a vector pNZ8110 use T₄The DNA ligase was ligated at 16 ℃ for 16 h. Connecting a reaction system: t is₄ DNA ligase 1. mu.l, pNZ 811012. mu.l, napA gene 2. mu.l, 10 XBuffer 2. mu.l, deionized water 3. mu.l. Ligation was carried out at 16 ℃ for 16 h. Then 50 mul of absolute ethyl alcohol and 2 mul of sodium acetate solution (0.3mol/L, pH5.8) are added into a connecting reaction tube, the materials are evenly mixed and placed at minus 20 ℃ for 1h, and are centrifuged at 12000r/min for 5min, and the supernatant is discarded; precipitating with 1ml 75% ethanol once, centrifuging at 12000r/min for 5min, discarding supernatant, drying at room temperature for 20min, dissolving precipitate with deionized water, and converting lactococcus lactis.

The results are as follows:

the result of PCR amplification of the napA gene: the extracted H.pylori genomic DNA was used as a template to amplify the napA gene by PCR, and the result of agarose gel electrophoresis analysis of the amplified product showed that the length of the amplified product matched the expected length (457bp) (as shown in FIG. 1, lane M is DNAmarker; lane 2 is the PCR product of napA).

EXAMPLE 2 construction of recombinant strains of lactococcus lactis

1. Electrotransformation and screening of lactis NZ3900 Strain

(1) L.lactis NZ3900 strain was cultured with GM17 medium and l.lactis NZ3900 competent cells were prepared, as described in granted patent (ZL 201210218281.9).

(2) The stored l.lactis NZ3900 competent cells were removed from the-80 ℃ freezer and placed on ice. Mu.l of the purified ligation product from example 1 was added to 40. mu.l of L.lactis NZ3900 competent cells, mixed well and placed on ice for 5 min.

(3) Adding the bacterial liquid into a 2mm electric shock transformation cup precooled by ice; wiping water outside the electric shock cup, and putting the electric shock cup into an electric shock instrument; setting electric shock parameters to be 25 muF, 200 omega and 2500V for electric shock; after the electric shock was completed, 1ml of ice-cold GM was added rapidly₁₇Recovering the MC culture medium, uniformly mixing, transferring the bacterial liquid into a 1.5ml EP tube, and carrying out ice bath for 5-10 min; at 30 ℃ with 5% CO₂And (5) restoring the culture in the incubator for 2 h.

(4) 100. mu.l of the recovered culture broth was inoculated onto a GM17 medium plate containing chloramphenicol (final concentration: 10. mu.g/ml) at 30 ℃ with 5% CO₂Culturing in incubator for 12-14 h (see Zhang Juan, expression of helicobacter UreB and HspA protein in lactococcus lactis and immunoreactivity research thereof, university of Zhengzhou doctor academic thesis, 2008.05, Chinese academic literature web publishing headquarters).

2. Identification of Positive transformants

(1) Single colonies were picked from solid selection medium and inoculated into 5ml GM17 liquid medium at 30 ℃ with 5% CO₂Culturing in an incubator for 12 h. The recombinant plasmid was extracted using a plasmid extraction kit (Shanghai Chuangsheng bioengineering Co., Ltd.).

(2) PCR identification of positive transformants: the PCR identification is carried out by taking the extracted plasmid as a template, carrying out PCR amplification on the napA gene, and referring the reaction system and the reaction conditions. The products were analyzed by electrophoresis on a 15g/L agarose gel.

(3) Enzyme digestion identification of positive transformation bacteria: 10 mu l of plasmid identified as positive by PCR is taken to be respectively subjected to SphI single enzyme digestion and NcoI + SphI double enzyme digestion, enzyme digestion reaction is carried out for 4h at 37 ℃, and inactivation is carried out for 10min at 65 ℃. The electrophoresis analysis was carried out on a 15g/L agarose gel.

(4) Sequencing and identifying positive transformation bacteria: the napA gene in the extracted plasmid was sequenced by Shanghai Bioengineering Co.

The results are as follows:

1) screening of Positive transformants

After transformation of L.lactis NZ3900 competent cells with the product of the ligation of the napA gene with pNZ8110, the cells were grown on selection medium at 30 ℃ with 5% CO₂And (5) performing static culture in an incubator for 12-14 h to obtain a positive transformed colony.

2) Identification of Positive transformants

Colonies were picked from the selection medium and inoculated into GM17 broth containing chloramphenicol (10. mu.g/ml final concentration), cultured for 12 hours, plasmids were extracted from the broth using a plasmid extraction kit, and PCR was performed using the aforementioned napA gene amplification primers using the extracted plasmids as templates, showing that the length of PCR amplification products was consistent with that expected. The plasmid that was positive in PCR was digested with NcoI and NcoI + SphI, respectively, and the results of the restriction were correct (see FIG. 1: lane M is DNAmarker; lane 1 is recombinant plasmid pNZ 8110. DELTA. sp-napA digested with SphI and NcoI; lane 3 is pNZ8110 digested with SphI; and lane 4 is pNZ 8110. DELTA. sp-napA digested with SphI).

3) Sequencing identification of positive transformation bacteria

The plasmid which is determined to be positive by enzyme digestion is delivered to Shanghai bio-engineering company, the napA gene in the plasmid is sequenced, and the result shows that the gene sequence is consistent with the sequence (AY366361) reported by GenBank. The results show that the napA gene of H.pylori has been cloned into the L.lactis recombinant strain. The recombinant plasmid produced by ligating the napA gene with pNZ8110 was designated: pNZ8110Delta sp-napA (SEQ ID NO.1) with the structure shown in figure 2-B, wherein Pnis is a promoter regulated by nisin; SP is a signal peptide sequence of lactococcus lactis usp45 gene; napA is the napA gene coding region; napA is the napA gene coding region; t is a terminator; repA and repC are replication genes (replication genes) A and C; cm is chloramphenicol resistance gene. The l.lactis recombinant strain containing this recombinant plasmid was named: l. lactis NZ3900/pNZ 8110. delta. sp-napA. Using GM containing 150ml/L glycerol₁₇The culture medium was based on the preservation of the strain at-80 ℃.

Test example 1L. inducible expression of the lactis NZ3900/pNZ 8110. delta. sp-napA Strain

1. Inducible expression of NapA protein

(1) A single colony of strain GM17 was scraped with an inoculating loop on a plate of GM17 medium for culturing L.lactis NZ3900/pNZ 8110. delta. sp-napA and inoculated into 10ml of GM17 liquid medium at 30 ℃ with 5% CO₂And (5) an incubator for static culture for 12 hours.

(2) Adding 2ml of the bacterial liquid into 50ml of GM17 liquid medium, and culturing at 30 deg.C with 5% CO₂An incubator for static culture to the bacterial liquid OD₆₀₀When the concentration is 0.3-0.4, adding an inducer Nisin to a final concentration of 40ng/ml, and carrying out induction culture for 5 hours.

(3) Meanwhile, L.lactis NZ3900/pNZ8110 is used as a negative control for an induced expression experiment.

2. Collection of bacterial lysate protein sample

(1) After inducing for 5h, taking 10ml of bacterial liquid, centrifuging for 5min at 4 ℃ and 8000r/min, removing supernatant, and reserving thalli sediment;

(2) adding 3ml of column buffer (200mM NaCl, 20mM Tris-HCl, 1mM EDTA, 10mM beta-mercaptoethanol, pH 7.4) suspended thallus, centrifuging at 8000r/min and 4 ℃ for 5min, and removing the supernatant;

(3) adding 200. mu.l of columu buffer to resuspend the thallus, adding 22. mu.l of lysozyme (the final concentration is 10mg/ml), and carrying out water bath at 37 ℃ for 1 h;

(4) centrifuging at 8000r/min and 4 deg.C for 5min, collecting 100 μ l supernatant, adding equal volume of 2 × electrophoresis loading buffer (100mM beta-mercaptoethanol, 100mM pH 6.8Tris-HCl, 4% SDS, 20% glycerol, 0.2% bromophenol blue), boiling for 3min, and storing at-20 deg.C; another 100. mu.l of supernatant sample was used for protein content determination.

3. SDS-PAGE analysis of recombinant Strain induced products

Reference is made to the tool book [ J. SammBruk and D.W Lassel, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002], which operates as follows:

12% of separation gum and 5% of concentrated gum are used. Samples were loaded at 20. mu.g to 50. mu.g per well. The voltage is 80V when the electrophoresis is started, the voltage is changed to 120V when the bromophenol blue is electrophoresed to the separation gel, and the electrophoresis is carried out for 2 h. After the electrophoresis was completed, the concentrated gel portion was cut off, and the gel was stained with Coomassie Brilliant blue for 4 hours at room temperature. After dyeing, the gel is washed for 2 times by distilled water, decolorized by a decolorizing solution, and analyzed by a gel image analyzer after the protein band is clear.

4. Results

The result of SDS-PAGE electrophoretic analysis is shown in FIG. 3-A, M is the molecular weight standard of protein; lanes 1, 2, 3 are L.lactis NZ3900/pNZ 8110. delta. sp-napA thallus lytic protein; lane 4 is L.lactis NZ3900/pNZ8110 cytolytic protein; arrows indicate the expressed NapA protein. The results show that: after L.lactis NZ3900/pNZ8110 delta sp-napA is induced, a protein band with the molecular weight of about 15kD (kilodalton) appears in a bacterial protein sample, the molecular weight of the protein band is consistent with the molecular weight of an expected expression product, the protein band is an inducible expression product of the napA gene, and no corresponding protein band exists in an electrophoretogram of a bacterial total protein sample of a control strain L.lactis NZ3900/pNZ8110 (figure 3-A). The results show that the L.lactis NZ3900/pNZ8110 delta sp-napA strain can effectively secrete and express NapA protein, and the expressed protein accounts for about 14% of the total protein of the strain. In the process of research, other positive strains obtained by the same method can express the NapA protein, but the expression amount is low and accounts for about 7-10% of the total protein of the strain. Selecting a bacterial strain of Lactococcus lactis NZ3900/pNZ8110 delta sp-napA with the expression protein accounting for about 14% of the total protein of the thallus for preservation, wherein the preservation number is CGMCC NO. 14933.

Because the secretion signal sequence SP in pNZ8110 is removed when the recombinant plasmid pNZ8110 delta SP-napA is constructed, the molecule of the NapA expressed in the invention does not carry a secretion signal peptide (namely the peptide coded by SP), has the molecular weight of 15KD, and avoids the adverse effect on the biological activity of NapA caused by the fact that the signal peptide cannot be cut in thalli when the recombinant strain fails to secrete and express proteins.

Experimental example 2L immunological identification of lactis NZ3900/pNZ 8110. delta. sp-napA expression protein

Reference is made to the tool book [ J. SammBruk and D.W Lassel, molecular cloning, A laboratory Manual, third edition, scientific Press, 2002], which operates as follows:

1. SDS-PAGE electrophoresis: samples of total bacterial protein of L.lactis NZ3900/pNZ8110, L.lactis NZ3900/pNZ 8110. delta. sp-napA were prepared and analyzed by SDS-PAGE according to the method described in example 5 of the instructions of granted patent (ZL 201210218281.9).

2. Film transfer: after SDS-PAGE was completed, the gel was cut into a desired size, a PVDF membrane (Solambio, Beijing) and a thick filter paper were cut into the same size as the gel, and the nitrocellulose membrane was soaked in methanol for 15 seconds. The thick filter paper, the gel, the nitrocellulose membrane and the thick filter paper were stacked in this order in a Bio-Rad electrotransfer apparatus. 18V, and the film is rotated for 2h in ice bath.

3. And (3) sealing: after membrane transfer, PBS (10mM Na)₂HPO₄，2.7mM KCl，137mM NaCl，2mM KH₂PO₄pH 7.4) was rinsed once, and then the PVDF membrane was placed in a blocking solution (PBS containing 20g/L skim milk powder), slowly shaken at room temperature for 2h, and after blocking, washed 3 times with PBS, each time for 10 min.

4. Primary anti-reaction: according to the granted patent (ZL201210218281.9) specification example 4, mouse serum was prepared by immunizing helicobacter pylori thallus lysis antigen and the immune serum was used as a primary antibody. The immune serum is diluted by 1:100 of the sealing solution, and the PVDF membrane is placed in the diluted immune serum and incubated for 1h at 37 ℃. Wash 3 times with PBS for 10min each time. It was then rinsed 3 times with TBS (0.15mM NaCl, 0.1M Tris-HCl, pH 7.5) for 10min each time.

5. Secondary antibody reaction: to the PVDF membrane, horseradish peroxidase-labeled goat anti-mouse IgG diluted with 20g/L skim milk powder in TBS was added, and the mixture was rinsed 3 times with TBS solution at 37 ℃ for 10min each time.

6. Color development: putting the PVDF membrane into a plate, and developing with a DAB developing kit (Beijing kang is century science and technology Co., Ltd.), according to a product specification, the steps are as follows: and (3) putting 1ml of the reagent B into a 1.5ml centrifugal tube, adding 50 mu l of the reagent A into the centrifugal tube, uniformly mixing, dropwise adding the mixed solution onto the PVDF membrane, developing for 1-5 min, soaking the membrane into deionized water after developing, and stopping reaction.

7. The results are as follows:

western blot analysis results show that: in a total bacterial protein sample of the induced L.lactis NZ3900/pNZ8110 delta sp-napA strain, a positive reaction zone appears at the 15kD position; there was no band corresponding to the E.coli protein electrophoretogram of the control strain L.lactis NZ3900/pNZ8110 (as shown in FIG. 3-B: M is a protein molecular weight standard; lanes 1, 2, 3 are L.lactis NZ3900/pNZ 8110. delta. sp. -napA cell lysis protein; lane 4 is L.lactis NZ3900/pNZ8110 cell lysis protein; arrows indicate expressed NapA protein). The result shows that the strain L.lactis NZ3900/pNZ8110 delta sp-napA can express the protein of helicobacter pylori NapA under the action of an inducer, and the expressed NapA protein has immunoreaction activity.

Test example 3 gavage immunization of mice and detection of antibodies and cytokines

1. Lactococcus lactis bacterial liquid for immunization

Referring to test example 1, the recombinant strain and the control strain NZ3900/pNZ8110 were cultured in GM17 medium containing chloramphenicol (at a final concentration of 10. mu.g/ml), and expression was induced by Nisin. The thalli is collected by centrifugation, and the thalli is suspended by a sterilized PBS solution, and the content of the thalli in the bacterial liquid is adjusted. Bacterial solutions NZ3900/pNZ 8110. delta. sp-napA and NZ3900/pNZ8110 contain cells at 5X 10¹⁰CFU/mL。

2. Animal immunization

Animal grouping: 33 BALB/c mice were randomly grouped into groups of 11, LL-pNZ8110-napA, LL-pNZ8110 and PBS. Immunization: mice were fasted for 12h and water deprived for 4h before immunization, and 100 μ l of 3% NaHCO was applied about 30min before inoculation of the bacterial solution₃The solution is used for gastric lavage to neutralize gastric acid. Mice in the LL-pNZ8110-napA group and the LL-pNZ8110 group are respectively gavaged with NZ3900/pNZ8110 delta sp-napA bacterial liquid and NZ3900/pNZ8110 bacterial liquid for 1 time/w, 250 mu L/mouse per time and immunized for 6 times. The PBS control group was gazed with an equal volume of PBS solution.

3. Detection of serum and intestinal secretory antibodies

3.1 serum and fecal sample Collection

1w after the last immunization, blood is collected from each group of mice through eye sockets, and after the blood is coagulated, serum is collected and stored at-20 ℃ for later use. Mice were sacrificed by cervical dislocation, soaked in 75% ethanol solution for 3min, and then the spleen was separated by abdomination for use. Then, the intestinal tract was separated, 100mg of feces were collected, and 600. mu.L of sterile PBS (containing benzyl sulfonyl fluoride as a protein inhibitor to give a working concentration of 0.1mM) was added thereto and soaked overnight at 4 ℃. The next day at 12000rpm, centrifuging for 10min, collecting supernatant as sample, and storing at-20 deg.C for use (see Sun nan, construction and immunization effect of vaccine against helicobacter pylori using lactococcus lactis as carrier, Zheng Zhou university Master academic thesis, 2016.06).

3.2 antibody detection

Referring to the literature (Sun nan, construction and immune effect of a vaccine against helicobacter pylori using lactococcus lactis as a carrier, Zheng Zhou university Master's academic thesis, 2016.06), ELISA reagents were prepared and used to detect serum IgG antibody and intestinal secretory SIgA antibody, respectively, by ELISA method.

According to the literature (Kangqiaozhen, the gene clone expression and immune evaluation of the helicobacter pylori neutrophil activating protein, the university of Zhengzhou doctor academic thesis, 2005), the affinity chromatography method is used for purifying and preparing the helicobacter pylori neutrophil activating protein NapA, and the purified protein NapA is used as a detection antigen to detect a specific antibody by ELISA.

The main steps of serum IgG antibody detection are as follows:

coating 96-well ELISA plate with NapA protein (4 ug/mL), and incubating at 37 deg.C for 2 h. ② rinsing with TBST for 3 times, 5min each time. The last time, thoroughly patting dry. ③ adding 250 mu L of sealing liquid into each hole, and sealing for 1 hour at 37 ℃. Diluting the sample serum: serum samples were diluted in blocking solution at equal ratio (1:10), added to the reaction wells and incubated at 37 ℃ for 2 h. Fifthly, repeating the step II. Sixthly, 100 mu L/hole of goat anti-mouse IgG labeled by horseradish peroxidase (HRP) diluted by a sealing solution (1: 5000) is added, and the mixture is incubated for 1h at 37 ℃. And seventhly, repeating the step II. And adding a chromogenic substrate A liquid B liquid into the mixture in sequence, slightly and uniformly mixing the chromogenic substrate A liquid B liquid with 50 mu L/hole, and developing the mixture in a dark place for 15 min. And ninthly, stopping the reaction by the final 50 mu L/hole stop solution, and detecting by a microplate reader with the wavelength of 450 nm.

The detection method of the intestinal SIgA antibody in the fecal sample is similar to that of the serum sample, and the difference is that the fecal sample is added into the reaction hole as a stock solution without being diluted, and the serum sample is diluted before being added; in addition, goat anti-mouse SIgA antibodies were labeled with HRP for detection of SIgA.

4. Mouse spleen cell culture and cytokine detection

4.1 spleen cell culture

The spleen of the mouse separated above is washed 2-3 times with D-Hank's buffer solution. Spleens were ground and splenocytes rinsed with D-Hank's solution. Adding 4 times volume of erythrocyte lysate into the cell suspension, placing on ice for 15min, centrifuging at 4 ℃ for 10min at 450 Xg, and removing supernatant; washed 1 time with D-Hank's buffer. Resuspending the cells in RPMI-1640 medium and adjusting the cell concentration to 5X 10⁶cells/mL. The cell suspension was added to a 24-well plate at 400. mu.L/well. Helicobacter pylori lysis antigen (final concentration 10. mu.g/mL) is added into the hole in 5% (V/V) CO₂And cultured at 37 ℃ for 72 h. The bacterial liquid is centrifuged for 20min at 3000rpm, and the supernatant is collected for cytokine detection.

4.2 cytokine detection

The cytokines IL-4, IL-12, IL-17 and INF-gamma were detected using an ELISA kit (Shanghai enzyme-Linked Biotech) according to the product instructions. Samples were diluted 1:4 for ELISA analysis. Detecting absorbance (OD) of reaction product at 450nm by enzyme-labeling instrument₄₅₀)。

5. Statistical analysis

And (4) performing experimental data analysis by using an SAS9.13 statistical analysis software package. The mouse specific antibody concentration and cytokine level are compared by one-way anova, the measurement data are expressed by mean ± standard deviation, the Least significant difference method (LSD) is adopted for pairwise comparison between the means, and the difference is less than 0.05, which is the standard of statistical significance.

6. The results are as follows:

serum IgG antibodies (fig. 4-B) and fecal SIgA antibodies (fig. 4-a) were detected as shown in fig. 4, with ns being statistically insignificant, P <0.05 in fig. 4-a and fig. 4-B compared to the PBS group or the indicated group. The levels of mouse serum IgG antibody and fecal SIgA antibody in the LL-pNZ8110-napA group are significantly higher than those in the LL-pNZ8110 group and the PBS control group, and the difference has statistical significance (P < 0.05).

The results of the cytokine level detection in the mouse splenocyte medium supernatant are shown in fig. 5, where ns is not statistically significant, and P is <0.05, compared with the PBS group or the designated group. IL-17 (FIG. 5-C), IL-12 (FIG. 5-A), INF-gamma (FIG. 5-B) levels in mice of LL-pNZ8110-napA group were significantly higher than those of LL-pNZ8110 and PBS control group (P < 0.05); IL-4 levels in mice from the LL-pNZ8110-napA group (FIG. 5-D) were not statistically different (P >0.05) from those from the LL-pNZ8110 and PBS control groups.

At present, although there are studies showing that helicobacter pylori NapA has a phenomenon of promoting the polarization of specific immune response to Th1 type, there is no study showing or suggesting that helicobacter pylori NapA has a regulatory effect on Th17 type immune response. The inventor finds that feeding mice with the lactococcus lactis recombinant strain expressing helicobacter pylori NapA can obviously increase the level of interleukin IL-17 in spleen cells of the mice, and the mice show obviously enhanced Th17 type immune response. Because the Th17 type immune response of the organism has the function of strengthening innate immunity and has important functions in anti-infection and tumor immune mechanisms, the immune regulation function that the helicobacter pylori NapA can strengthen the Th17 type immune response has important application value.

Test example 4 measurement of immunoprotective Effect of recombinant Strain on mice

1. Animal grouping: randomly grouping 43 BALB/c mice, LL-pNZ8110-napA group, LL-pNZ8110 group and PBS group, 11 mice in each group; baseline group 10. The groups LL-pNZ8110-napA, LL-pNZ8110 and PBS were immunized as in test example 3. The Baseline group was not given immunization and challenge treatments.

2. Preparation of helicobacter pylori solution for challenge

With reference to examples 1 and 2, H.pylori was cultured for 3-5 days. Colonies were subjected to gram staining and observed under a microscope, and the cells were gram-stained negative Campylobacter pumilus. Scraping helicobacter pylori colony from culture medium plate with inoculating loop, and resuspending thallus inHelicobacter pylori is preserved in the liquid. Measuring the turbidity of the bacteria solution with spectrophotometer, calculating bacteria content of the bacteria solution, and adjusting the bacteria content to 1 × 10¹⁰CFU/mL。

3. Challenge infection in mice

1w after the last immunization, each group of mice was challenged with H.pylori solution. Before the attack, mice are placed in a clean mouse cage without padding and are fasted for 12 hours, and water is forbidden for 4 hours. Mice were gavaged with H.pylori solution 200 μ L/mouse-times, 1 challenge at 2d intervals for 4 times. Water was fasted for 4h after each challenge.

4. Evaluation of fixed planting amount of helicobacter pylori in mouse stomach

After 1w of the last challenge infection, mice were sacrificed by cervical dislocation. Fasted for 12h before sacrifice and water was withheld for 4 h. Dissecting a mouse, taking out the stomach, longitudinally cutting along the greater curvature of the stomach, flushing the stomach contents with sterile normal saline, punching two overlapped circular full-layer stomach tissues at the antrum of the stomach by using a circular puncher with the hole diameter of 5mm, placing the two tissues on a 200-mesh copper sieve, grinding, flushing the stomach tissues with 1ml of helicobacter pylori preservation solution, taking 5 mu L of filtrate, adding 100 mu L of helicobacter pylori preservation solution, uniformly mixing, transferring to a culture medium plate, culturing for 3-5 days according to the methods of the embodiment 1 and the embodiment 2, and counting the helicobacter pylori colonies. Adding 100 μ L filtrate into 500 μ L urease detection reagent, standing at room temperature for 5 hr, and determining OD₅₅₀The value is obtained. The reagent preparation is shown in the literature (Sun nan, the construction and immunization effect of a vaccine against helicobacter pylori using lactococcus lactis as a carrier, Zheng Zhou university Master academic thesis, 2016.06).

5. Statistical analysis

The statistical analysis method of the data was the same as in test example 3.

The results are as follows:

after the mice are infected by the helicobacter pylori, the evaluation and detection result of the helicobacter pylori colonization amount in the stomach is shown in figure 6, wherein A is the culture result of the helicobacter pylori in the stomach tissue of the mice, B is the urease activity detection result, compared with the PBS group or the designated group, ns is not statistically significant, and P is less than 0.05. The results of mouse gastric tissue bacteria quantitative culture and urease test show that the helicobacter pylori colonization level of the LL-pNZ8110-napA group is obviously lower than that of the LL-pNZ8110 group and the PBS control group, and the difference has statistical significance (P < 0.05).

Example 3L use of lactis NZ3900/pNZ 8110. delta. sp-napA in the preparation of oral vaccines

Referring to examples 1 and 2 and test example 1, L.lactis NZ3900/pNZ 8110. delta. sp-napA strain was cultured in GM17 medium, expression of antigen NapA was induced by Nisin, and the resulting bacterial suspension was formulated into a formulation suitable for oral administration to humans and used as an oral vaccine.

After the vaccine is orally taken by human, the vaccine can generate immunity for resisting helicobacter pylori infection and has the beneficial effects of preventing and treating helicobacter pylori related diseases.

Example 4L use of lactis NZ3900/pNZ 8110. delta. sp-napA for the production of immunomodulatory drugs

Referring to examples 1 and 2 and test example 1, L.lactis NZ3900/pNZ 8110. delta. sp-napA strain was cultured in GM17 medium, and after NapA expression was induced by Nisin, the resulting bacterial solution or its concentrated or dried product was used as an immunomodulatory drug for oral or topical administration for the purpose of preventing or treating immunomodulatory diseases.

The medicine is used for preventing and treating chronic infection or tumor with inherent hypoimmunity; or for preventing and treating food allergy based on Th2 type immune reaction. At present, clinically discovered diseases related to immune disorder are various, and safe and effective immune regulators are very lacking. The recombinant strain provided by the invention is used for preparing the immunomodulator which can be applied through the mucous membrane, the preparation process is simple and efficient, protein purification is not needed, the degradation of NapA in the digestive tract or other mucous membrane surfaces can be effectively reduced through the protection effect of bacteria, and very obvious technical progress and advantages are shown. Therefore, the recombinant strain has very considerable application prospect in the production of immunomodulatory agents.

Example 5L use of the lactis NZ3900/pNZ 8110. delta. sp-napA Strain for the production of immunomodulatory food products

The application method of the lactis NZ3900/pNZ8110 delta sp-napA strain in food (including beverage) processing comprises the following steps: adding appropriate amount of L.lactis NZ3900/pNZ8110 delta sp-napA strain into food processing material, and mixingThe strain is cultured at 30 deg.C and 5% CO under the condition suitable for growth of the strain₂And (3) performing fermentation reaction, and adding Nisin at an appropriate time to induce the strain to express the vaccine antigen NapA. The food product may contain the strain in a viable or inactivated form.

The product contains helicobacter pylori NapA protein, and has effects of enhancing innate immunity, preventing and treating immune dysregulated diseases, and preventing helicobacter pylori infection and related diseases after being eaten or drunk by human. The application of the recombinant bacterium in food processing can promote the development of food industry in China, particularly dairy industry, and is expected to produce great social and economic benefits.

Example 6L use of lactis NZ3900/pNZ 8110. delta. sp-napA for the preparation of immunomodulatory biologies

Culturing L.lactis NZ3900/pNZ8110 delta sp-napA strain, inducing NapA expression by Nisin, and using the obtained bacterial liquid or the concentrated or dried product thereof as an immunomodulator for experimental research.

The immunomodulator is used for animal intragastric administration to achieve the experimental purpose of enhancing Th17 type and/or Th1 type immune response. At present, the immunomodulator is relatively lacked, and the immunomodulator which is prepared by applying the recombinant strain and is applied through a mucous membrane has simple and efficient preparation process, does not need protein purification, and can effectively reduce the degradation of NapA in a digestive tract through the protective action of thalli, so the recombinant strain has good application prospect in the production aspect of the immunomodulator for experiments.

<110> Xinxiang medical college, Zhengzhou university

<120> recombinant vector and recombinant strain for expressing helicobacter pylori NapA protein, preparation method and application thereof

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tatagagcaa gttatgcaaa ggttcttgat gctgaaacgg gggaaataaa atgacaaaca 2580

aagaaaaaga gttatttgct gaaaatgagg aattaaaaaa agaaattaag gacttaaaag 2640

agcgtattga aagatacaga gaaatggaag ttgaattaag tacaacaata gatttattga 2700

gaggagggat tattgaataa ataaaagccc ccctgacgaa agtcgacggc aatagttacc 2760

cttattatca agataagaaa gaaaaggatt tttcgctacg ctcaaatcct ttaaaaaaac 2820

acaaaagacc acatttttta atgtggtctt tattcttcaa ctaaagcacc cattagttca 2880

acaaacgaaa attggataaa gtgggatatt tttaaaatat atatttatgt tacagtaata 2940

ttgactttta aaaaaggatt gattctaatg aagaaagcag acaagtaagc ctcctaaatt 3000

cactttagat aaaaatttag gaggcatatc aaatgaactt taataaaatt gatttagaca 3060

attggaagag aaaagagata tttaatcatt atttgaacca acaaacgact tttagtataa 3120

ccacagaaat tgatattagt gttttatacc gaaacataaa acaagaagga tataaatttt 3180

accctgcatt tattttctta gtgacaaggg tgataaactc aaatacagct tttagaactg 3240

gttacaatag cgacggagag ttaggttatt gggataagtt agagccactt tatacaattt 3300

ttgatggtgt atctaaaaca ttctctggta tttggactcc tgtaaagaat gacttcaaag 3360

agttttatga tttatacctt tctgatgtag agaaatataa tggttcgggg aaattgtttc 3420

ccaaaacacc tatacctgaa aatgcttttt ctctttctat tattccttgg acttcattta 3480

ctgggtttaa cttaaatatc aataataata gtaattacct tctacccatt attacagcag 3540

gaaaattcat taataaaggt aattcaatat atttaccgct atctttacag gtacatcatt 3600

ctgtttgtga tggttatcat gcaggattgt ttatgaactc tattcaggaa ttgtcagata 3660

ggcctaatga ctggctttta taatatgaga taatgccgac tgtacttttt acagtcggtt 3720

ttctaatgtc actaacctgc cccgttagtt gaagaaggtt tttatattac agctcca 3777

<211> 3459

<212> DNA

<213> Artificial sequence

<221> pNZ8110

<400> 2

agatctagtc ttataactat actgacaata gaaacattaa caaatctaaa acagtcttaa 60

ttctatcttg agaaagtatt ggtaataata ttattgtcga taacgcgagc ataataaacg 120

gctctgatta aattctgaag tttgttagat acaatgattt cgttcgaagg aactacaaaa 180

taaattataa ggaggcactc accatggcta aaaaaaagat tatctcagct attttaatgt 240

ctacagtgat actttctgct gcagccccgt tgtcaggtgt ttacgccggc tgcagccggc 300

gtaaacacct gacaacgggg ctgcaggcat gcggtaccac tagttctaga gagctcaagc 360

tttctttgaa ccaaaattag aaaaccaagg cttgaaacgt tcaattgaaa tggcaattaa 420

acaaattaca gcacgtgttg ctttgattga tagccaaaaa gcagcagttg ataaagcaat 480

tactgatatt gctgaaaaat tgtaatttat aaataaaaat caccttttag aggtggtttt 540

tttatttata aattattcgt ttgatttcgc tttcgataga acaatcaaag cgagaataag 600

gaagataaat cccataaggg cgggagcaga atgtccgaga ctaatgtaaa tttgtccacc 660

aattaaagga ccgataacgc gagcttctcg agtgcatatt ttcggcaatc ttctcaatga 720

gatgctcttc agcatgttca atgatgtcga ttttttatta aaacgtctca aaatcgtttc 780

tgagacgttt tagcgtttat ttcgtttagt tatcggcata atcgttaaaa caggcgttat 840

cgtagcgtaa aagcccttga gcgtagcgtg ctttgcagcg aagatgttgt ctgttagatt 900

atgaaagccg atgactgaat gaaataataa gcgcagcgtc cttctatttc ggttggagga 960

ggctcaaggg agtttgaggg aatgaaattc cctcatgggt ttgattttaa aaattgcttg 1020

caattttgcc gagcggtagc gctggaaaat ttttgaaaaa aatttggaat ttggaaaaaa 1080

atggggggaa aggaagcgaa ttttgcttcc gtactacgac cccccattaa gtgccgagtg 1140

ccaatttttg tgccaaaaac gctctatccc aactggctca agggtttgag gggtttttca 1200

atcgccaacg aatcgccaac gttttcgcca acgtttttta taaatctata tttaagtagc 1260

tttattgttg tttttatgat tacaaagtga tacactaatt ttataaaatt atttgattgg 1320

agttttttaa atggtgattt cagaatcgaa aaaaagagtt atgatttctc tgacaaaaga 1380

gcaagataaa aaattaacag atatggcgaa acaaaaaggt ttttcaaaat ctgcggttgc 1440

ggcgttagct atagaagaat atgcaagaaa ggaatcagaa caaaaaaaat aagcgaaagc 1500

tcgcgttttt agaaggatac gagttttcgc tacttgtttt tgataaggta atatatcatg 1560

gctattaaaa atactaaagc tagaaatttt ggatttttat tatatcctga ctcaattcct 1620

aatgattgga aagaaaaatt agagagtttg ggcgtatcta tggctgtcag tcctttacac 1680

gatatggacg aaaaaaaaga taaagataca tggaatagta gtgatgttat acgaaatgga 1740

aagcactata aaaaaccaca ctatcacgtt atatatattg cacgaaatcc tgtaacaata 1800

gaaagcgtta ggaacaagat taagcgaaaa ttggggaata gttcagttgc tcatgttgag 1860

atacttgatt atatcaaagg ttcatatgaa tatttgactc atgaatcaaa ggacgctatt 1920

gctaagaata aacatatata cgacaaaaaa gatattttga acattaatga ttttgatatt 1980

gaccgctata taacacttga tgaaagccaa aaaagagaat tgaagaattt acttttagat 2040

atagtggatg actataattt ggtaaataca aaagatttaa tggcttttat tcgccttagg 2100

ggagcggagt ttggaatttt aaatacgaat gatgtaaaag atattgtttc aacaaactct 2160

agcgccttta gattatggtt tgagggcaat tatcagtgtg gatatagagc aagttatgca 2220

aaggttcttg atgctgaaac gggggaaata aaatgacaaa caaagaaaaa gagttatttg 2280

ctgaaaatga ggaattaaaa aaagaaatta aggacttaaa agagcgtatt gaaagataca 2340

gagaaatgga agttgaatta agtacaacaa tagatttatt gagaggaggg attattgaat 2400

aaataaaagc ccccctgacg aaagtcgacg gcaatagtta cccttattat caagataaga 2460

aagaaaagga tttttcgcta cgctcaaatc ctttaaaaaa acacaaaaga ccacattttt 2520

taatgtggtc tttattcttc aactaaagca cccattagtt caacaaacga aaattggata 2580

aagtgggata tttttaaaat atatatttat gttacagtaa tattgacttt taaaaaagga 2640

ttgattctaa tgaagaaagc agacaagtaa gcctcctaaa ttcactttag ataaaaattt 2700

aggaggcata tcaaatgaac tttaataaaa ttgatttaga caattggaag agaaaagaga 2760

tatttaatca ttatttgaac caacaaacga cttttagtat aaccacagaa attgatatta 2820

gtgttttata ccgaaacata aaacaagaag gatataaatt ttaccctgca tttattttct 2880

tagtgacaag ggtgataaac tcaaatacag cttttagaac tggttacaat agcgacggag 2940

agttaggtta ttgggataag ttagagccac tttatacaat ttttgatggt gtatctaaaa 3000

cattctctgg tatttggact cctgtaaaga atgacttcaa agagttttat gatttatacc 3060

tttctgatgt agagaaatat aatggttcgg ggaaattgtt tcccaaaaca cctatacctg 3120

aaaatgcttt ttctctttct attattcctt ggacttcatt tactgggttt aacttaaata 3180

tcaataataa tagtaattac cttctaccca ttattacagc aggaaaattc attaataaag 3240

gtaattcaat atatttaccg ctatctttac aggtacatca ttctgtttgt gatggttatc 3300

atgcaggatt gtttatgaac tctattcagg aattgtcaga taggcctaat gactggcttt 3360

tataatatga gataatgccg actgtacttt ttacagtcgg ttttctaatg tcactaacct 3420

gccccgttag ttgaagaagg tttttatatt acagctcca 3459

<211> 29

<212> DNA

<213> Artificial sequence

<221> napA Gene upstream primer

<400> 3

catgccatgg gcatgaaaac atttgaaat 29

<211> 27

<212> DNA

<213> Artificial sequence

<221> napA Gene downstream primer

<400> 4

acgtgcatgc ttaagctaaa tgggctt 27

Claims (5)

1. The application of a recombinant strain containing a recombinant vector for expressing helicobacter pylori NapA protein in preparing an immunoregulatory biological preparation or food for enhancing Th17 type immune response; the recombinant vector is inserted with a napA gene segment between a Pnis promoter and a Terminator of an expression vector pNZ8110 of which the SP functional region is knocked out; the recombinant strain is prepared from lactococcus lactis (A)Lactococcus lactis) NZ3900 was constructed.

2. Use according to claim 1, characterized in that: the napA gene segment is the gene segment shown as 210-644 bit in SEQ ID NO. 1.

3. Use according to claim 1, characterized in that: the preparation method of the recombinant vector comprises the following steps: amplifying a napA gene fragment of helicobacter pylori by PCR, continuously accessing the napA gene fragment between the NcoI enzyme cutting site and the SphI enzyme cutting site of the expression vector pNZ8110 with the SP functional region removed, and screening and identifying to obtain the product.

4. Use according to claim 1, characterized in that: the preservation number of the recombinant strain is CGMCC NO.14933, and the recombinant strain is preserved in the China general microbiological culture Collection center.

5. Use according to claim 1, characterized in that: the preparation method of the recombinant strain comprises the following steps: transferring the recombinant vector into lactococcus lactis (Lactococcus lactis) And (4) identifying and screening NZ3900 competent cells to obtain the target.

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