CN100378223C - Helicobacter pylori membrane porin with adhesion function - Google Patents

Abstract

Helicobacter pylori porin with adhesion function, a DNA molecule, is characterized in that it includes: (a) the nucleotide sequence shown in SEQ ID NO: 1, (b) within the scope of genetic code degeneracy and (a) a nucleotide sequence corresponding to the sequence in (a), or, (c) a nucleotide sequence that hybridizes to the sequence in (a) or/and (b) under stringent conditions. The invention has the advantages of cloning the Helicobacter pylori membrane porin gene with adhesion function by gene recombination, and constructing a carrier to carry out sequence analysis and protein expression to improve the adhesion function, immune protection and prevention of Helicobacter pylori.

Description

Helicobacter pylori membrane porin with adhesion function

technical field

The invention relates to a Helicobacter pylori porin with adhesion function.

Background technique

Helicobacter pylori (Hp) infection is the main cause of chronic gastritis and peptic ulcer, and is also closely related to the occurrence of gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue (MALT) malignant lymphoma. In addition, serum epidemiological studies have shown that Hp infection is also related to the occurrence of circulation, respiratory, gastroduodenal and extradigestive system diseases and autoimmune diseases. With the rise of the etiological status of Hp, the research on its pathogenic mechanism is also in-depth. The pathogenic mechanism of Hp includes many links, but its adhesion mechanism is the most critical, because Hp must first be colonized in the human gastric mucosa to further play its role. Pathogenicity, and adhesion is the prerequisite for Hp colonization on the gastric mucosal surface.

Several potential H. pylori adhesins have been described so far, and a gene (hpaA) encoding the so-called N-acetylneuraminolactidine-binding hemagglutinin was cloned and sequenced (Evans et al., 1993 ). It is a protein that should recognize sialic acid-containing receptors on epithelial cells. However, the significance of this adhesin for H. pylori infection is controversial. Other potential adhesins were identified either by their molecular weight alone or by their receptor binding specificity. They include a 63 kDa protein that appears to be homologous to the extracellular enzyme S of Pseudomonas aeruginosa, an adhesin with ADP-ribosyl-transferase activity. An as yet unidentified adhesin is also suspected of mediating specific binding to the Lewis ^b blood group antigen of gastric epithelial cells (Falk et al., 1993, Boren et al., 1993).

Infection by Helicobacter pylori leads to chronic inflammation of the gastric mucosa (gastritis). In addition, specific systemic immune responses to H. pylori antigens are induced; however, the formation of secretory antibodies (slgA) in the stomach has not been uncontroversially elucidated. A consequence of the inflammation is the presence in the gastric mucosa and submucosa of a variety of immune cells such as polymorphonuclear leukocytes, monocytes, macrophages, lymphocytes and plasma cells (Blaser, 1992). Furthermore, H. pylori activates neutrophils, monocytes and macrophages in vitro (Mai et al., 1991). Experiments with specific antibodies and complement showed that neutrophils rapidly inactivate H. pylori in vitro.

Contents of the invention

The purpose of the present invention is to aim at the deficiencies of the above-mentioned prior art, to provide a Helicobacter pylori porin gene with adhesion function by genetic recombination, and to construct a vector for sequence analysis and protein expression analysis to improve adhesion. Adhesion-functional Helicobacter pylori membrane porins for attachment function and immune protection.

The object of the present invention is achieved like this: DNA molecule is characterized in that, it comprises: (a) the nucleotide sequence shown in the sequence number 1, (b) in the scope of genetic code degeneracy and (a) or, (c) a nucleotide sequence that hybridizes to the sequence of (a) or/and (b) under stringent conditions; and (a) it encodes a human cell adherent The polypeptide, (b) the vector contains at least one copy of the DNA molecule, (c) the cell is transformed by the vector; and (a) the nucleotide sequence shown in SEQ ID NO: 1, or, (b) and the sequence in (a) Nucleotide sequences with immunological cross-reactivity, (c) The polypeptide can adhere to human cells.

In addition to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence corresponding to the sequence within the degeneracy of the genetic code, the present invention also includes a DNA sequence that hybridizes to one of these sequences under stringent conditions. The present invention includes a nucleotide sequence that hybridizes to one of the nucleotide sequences shown in SEQ ID NO: 1 or to the corresponding nucleotide sequence within the range of genetic code degeneracy under certain washing conditions.

Preferably, the DNA molecule of the invention encodes a polypeptide capable of adhering to human cells, especially human gastric epithelial cells. In addition, the DNA molecule of the present invention preferably has at least 70%, particularly preferably at least 90%, homology with the nucleotide sequence shown in SEQ ID NO: 1 at the nucleotide level. Furthermore, the length of the DNA molecule is preferably at least 45 nucleotides, preferably at least 50 nucleotides.

Yet another object of the invention is a vector comprising at least one copy of the DNA molecule of the invention. The vector may be any prokaryotic or eukaryotic vector on which the DNA molecule of the invention resides, preferably under the control of expression signals (promoter, operator, enhancer, etc.). Examples of prokaryotes are chromosomal vectors such as bacteriophages (eg bacteriophage lambda) and extrachromosomal vectors such as plasmids, with circular plasmid vectors being particularly preferred.

The vectors of the present invention may also be eukaryotic vectors such as yeast vectors or vectors suitable for higher cells (eg, plasmid vectors, viral vectors, plant vectors).

Yet another object of the present invention are cells transformed with the vectors of the present invention. In a preferred embodiment, the cells are prokaryotic cells, preferably Gram-negative prokaryotic cells, particularly preferably E. coli cells. On the other hand, however, the cells of the invention may also be eukaryotic cells, eg fungal cells (eg yeast), animal or plant cells.

The invention also relates to polypeptides encoded by the DNA molecules of the invention. The polypeptide is preferably capable of adhering to human cells, and includes (a) the amino acid sequence shown in SEQ ID NO: 1, or (b) an amino acid sequence immunologically cross-reactive with the sequence in (a).

The polypeptide of the present invention preferably has at least 90% homology with the nucleotide sequence shown in SEQ ID NO:1.

The polypeptide of the present invention is preferably produced by transforming a cell with the DNA molecule or vector of the present invention, culturing the transformed cell under conditions such that the polypeptide can be expressed, and isolating from the cell and/or the culture supernatant peptide. Polypeptides of the invention can be obtained in the form of fusion polypeptides as well as non-fusion polypeptides in this method.

The polypeptides of the invention can also be used as immunogens to generate antibodies. Accordingly, the present invention also relates to antibodies raised against polypeptides of the present invention.

Another aspect of the present invention relates to a pharmaceutical composition, which comprises as an active substance the DNA molecule of the present invention, the polypeptide of the present invention or the antibody of the present invention, optionally containing common pharmaceutical auxiliary substances, diluents, additives and carrier.

In one aspect, the pharmaceutical composition of the present invention can be used for diagnosing Helicobacter pylori infection.

On the other hand, the pharmaceutical composition can also be used to prevent or treat Helicobacter pylori infection. For therapeutic applications, polypeptides or fragments thereof are used to generate active vaccines, or antibodies are used to generate passive vaccines.

The invention has the advantages of cloning Helicobacter pylori membrane porin gene by using PCR technology, and constructing a carrier for its sequence analysis and protein expression analysis, improving antiserum to prevent Helicobacter pylori from adhering to gastric mucosal epithelial cells and improving immunogenicity and activity High and high adhesion function and other advantages.

According to the results of protein electrophoresis analysis, the protein with a relative molecular weight of 56,500 was highly expressed after induction, and the protein in the conserved region of the recombinant adhesin accounted for 31.9% of the total bacterial protein, and the soluble expression accounted for 23.7% of the supernatant. Inclusion bodies accounted for 64.8% of the pellet.

In vitro adhesion experiments showed that the porin with adhesion function was located on the surface of bacteria and the antiserum of its fusion protein could completely prevent Helicobacter pylori from adhering to gastric tissue, while the results of the antiserum of the urease B fusion protein in the control experiment were consistent with This is the opposite. In addition, animals have shown that the porin with adhesion function of Helicobacter pylori can be used to prevent and treat Helicobacter pylori infection.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of PCR product of the present invention,

Fig. 2 is the dual-enzyme identification map of heavy resistance plasmid PET-22b (+)/ porin of the present invention,

Fig. 3 is the SDS-PAGE analysis figure of porin recombinant protein of the present invention,

Figure 4 is the serial number 1 of the present invention.

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings and Examples: As shown in Figures 1 to 3, the DNA molecule is characterized in that it comprises: (a) the nucleotide sequence shown in the sequence number 1, (b) a nucleotide sequence corresponding to the sequence in (a) within the scope of the degeneracy of the genetic code, or, (c) a nucleoside that hybridizes to the sequence of (a) or/and (b) under stringent conditions acid sequence; at the nucleic acid level, it has at least 90% homology with the nucleotide sequence shown in SEQ ID NO: 1; its length is at least 45 nucleotides; (a) it encodes a human The polypeptide of the cell, (b) the vector contains at least one copy of the DNA molecule, (c) the cell is transformed by the vector; the polypeptide, which is encoded by the DNA molecule, includes: (a) the nucleotide sequence shown in SEQ ID NO: 1 , or, (b) a nucleotide sequence that immunologically cross-reacts with the sequence in (a), and (c) the polypeptide can adhere to human cells. The method for producing the polypeptide comprises transforming a cell with the vector, culturing the transformed cell under the condition that the polypeptide can be expressed, and isolating the polypeptide from the cell or/and the culture supernatant. The application of the polypeptide as an immunogen to generate antibodies and the antibody to the polypeptide. Pharmaceutical composition, (a) it contains DNA molecules, polypeptides or antibodies as active substances, optionally containing common pharmaceutical auxiliary substances, diluents, additives, adhesives and carriers, (b) for pharmaceutical compositions (c) application of the pharmaceutical composition for producing a medicament for preventing or treating Helicobacter pylori infection.

The gene of porin-like adhesin was cloned and expressed by gene cloning technology, and its immune prevention and treatment effect was studied.

1 Materials and methods

1.1 Plasmids and strains

Strain BL21(DE3), plasmid pET-22b(+) and Helicobacter pylori SS1 were preserved by the Institute of Digestion, Nanfang Hospital, First Military Medical University.

1.2 Tool enzymes and reagents

Restriction enzymes Not I, Noc I and T4 DNA polymerase, Vent DNA polymerase were purchased from New England Biolabs, Taq DNA polymerase, DNA molecular weight standard λDNA/EcoR I+Hind III were purchased from Huamei Bioengineering Company, agarose, dNTPs and DNA rapid purification kits were purchased from Promega Company, the sequencing plasmid purification kit was purchased from Qiagen Company of the United States, and other reagents were domestic analytical grade.

1.3Hp Chromosomal DNA Extraction

Scrape well-growing Hp colonies from the solid medium, and prepare them according to the genomic DNA miniprep method, see reference [1] for details.

1.4 Extraction and purification of plasmid

Alkaline denaturation was used for rapid extraction and mass preparation of plasmids, see reference [2] for details.

1.5 PCR amplification of the target gene

Primers were designed, and suitable restriction enzyme cutting sites were added to their 5' ends, which were synthesized by Boya Company. The sequence is as follows: porin-like cohesin 1: 5′-TG G CC ATG GA T TGC GCT AGC ATA AGT TA-3′Nco I

Porin-like adhesin 2: 5′-AG T GC GGC CGC GAA TGA ATA CCC ATA AGA-3′Not I

PCR was performed by hot start method, denaturation at 95°C for 30s, annealing at 55°C for 50s, extension at 72°C for 90s, and extension for 10min after 35 cycles. 0.8% agarose gel electrophoresis to observe the amplification results.

1.6 Digestion, ligation, transformation and identification of positive clones of DNA fragments

The plasmid and target gene DNA were digested with Not I and noc I, and the digested fragments were recovered from glass milk, ligated for 12 hours at 16°C under the action of T4 ligase, transformed into host bacteria BL21(DE3), and positive clones were screened out through double enzyme digestion.

1.7 Sequence Determination and Analysis

A large number of recombinant cloned plasmids identified by double enzyme digestion were extracted by alkaline lysis, and sequence analysis was performed with an automatic sequencer.

1.8 Induced expression and SDS-polyacrylamide gel electrophoresis

Porin-like adhesin-positive clones were induced by IPTG and then subjected to SDS-polyacrylamide gel electrophoresis.

1.9 Animal experiments

The porin-like adhesin was given to mice infected with Helicobacter pylori through the mucosal route, and the therapeutic effect was observed. Mice not infected with Helicobacter pylori were first given porin-like adhesin through the mucosal route, and then challenged with Helicobacter pylori to observe the protective effect.

1.10 Determination of Adhesion Activity by Light Microscopy Quantitative Counting Method

MGC-803 cells were seeded in a 6-well plate with coverslips and cultured until a single cell layer was formed, the coverslips were taken out, rinsed once with PBS, and the positive clones or empty vector clones were suspended (10 ⁹ CFU/ml) Drop onto the coverslip, incubate in a humid chamber at 37°C for 40 min, rinse with PBS 6 times to remove non-adherent bacteria, dry naturally, fix, and stain with Gram. Observe the adhesion between MGC-803 and bacteria under the oil microscope, calculate the number of bacteria adhered around each cell, repeat 3 times, and take the average value. A total of 30 cells were randomly counted for each sample, and the results were expressed as mean ± standard deviation express.

1.11 Statistical processing

The independent sample t test in SPSS7.0 software was used. P<0.01 means the difference is significant.

2 results

2.1 Amplification of porin-like adhesin

Electrophoresis analysis of the PCR results revealed a band at about 1500 bp, the size was in line with the prediction, the results are shown in Figure 1.

2.2 Construction and identification of recombinant plasmids

After the PCR product was digested with Not I and Noc I, it was directional inserted into the pET-22b(+) vector that had been digested with the same double enzymes, and the recombinant plasmid was obtained and named pET-22b(+)/porin-like adhesin. The results of electrophoresis of the recombinant plasmid after Not I and Noc I double digestion are shown in Figure 2.

2.3 Sequence analysis of porin-like adhesin gene fragments

The recombinant plasmid pET-22b(+)/porin-like adhesin was directly used as a template for sequencing, and the DNA sequence of the cloned fragment was obtained. The sequence analysis results of the automatic sequencer are shown below (Figure 4).

2.4 Expression of porin-like adhesin gene in Escherichia coli

Porin-like adhesin-positive clones were cultured overnight at 37°C in LB culture medium (containing 100ug/ml ampicillin), and then transferred to LB culture medium containing ampicillin at 1%, and continued to grow until the D value was 0.6-0.8, add IPTG to a final concentration of 0.1mmol/L, induce expression for 3 hours, collect the bacteria by centrifugation, take the periplasmic osmotic shock fluid, the supernatant after ultrasound of the bacteria and the precipitate for 10% SDS-PAGE electrophoresis analysis (image 3). It was found that the relative molecular weight of the expression after induction was 56.5×10 ³

The porin-like cohesin recombinant protein accounted for 31.9% of the total bacterial protein, of which the soluble expression accounted for 23.7% of the supernatant, and the inclusion body part accounted for 64.8% of the precipitate.

2.5 Animal experiments

The effective rate of porin-like adhesin in both treatment and protection was 100%.

2.6 Light microscope adhesion test results

Compared with the empty vector clone treatment group, the number of bacteria around cells in the positive clone treatment group was significantly increased (P<0.01), and the difference was significant. The results of light microscope adhesion experiment showed that porin-like adhesin has the function of adhesion.

references

[1] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manua 1.2 ^nd ed. New York: Cold Spring Harbor Laboratory Press, 1989.35.

[2] Ausubel FM, Brent R, Kingston RE, et al. Yan Ziying, translated by Wang Hailin. The Refined Guide to Molecular Biology. Beijing: Science Press, 1998.39.

Claims (4)

1. A DNA molecule characterized in that it is selected from: (a) a nucleotide sequence as shown in Figure 4, (b) Nucleotide sequences having genetic code degeneracy with the nucleotide sequences in FIG. 4 . 2. A cell comprising a DNA molecule according to claim 1, characterized in that: (a) the vector in which said DNA molecule is contained contains at least one copy of said DNA molecule, (b) transforming cells with the vector described in (a). 3. Polypeptide characterized in that it is encoded by the DNA molecule of claim 1. 4. The method for producing the polypeptide according to claim 3, culturing the cells expressing the polypeptide according to claim 3 under the condition that the polypeptide is expressed, and isolating the polypeptide from the cells or/and the culture supernatant.

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