CN105693832B

CN105693832B - Brucella Omp10 protein antigen epitope polypeptide and application thereof

Info

Publication number: CN105693832B
Application number: CN201610226026.7A
Authority: CN
Inventors: 曹小安; 周继章; 李兆才; 娄忠子; 景志忠; 付宝权; 尚佑军
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2016-04-13
Filing date: 2016-04-13
Publication date: 2020-05-08
Anticipated expiration: 2036-04-13
Also published as: CN105693832A

Abstract

The invention discloses brucella Omp10 protein epitope polypeptide and application thereof. The polypeptide of the invention is: the amino acid sequence of the polypeptide contains SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5, or the polypeptide is formed by the sequence formed by the substitution and/or deletion of one or/and several amino acid residues of the sequence, or the sequence can be the derivative polypeptide added with the sequence with Brucella immunity source function. The polypeptide of the invention can form a recombinant vector or an expression cassette or a transgenic cell or a recombinant bacterium. The polypeptides related to the invention can be applied to the preparation of reagents or medicines for diagnosing, preventing or treating diseases caused by brucella. The screened polypeptide is chemically synthesized, can release high-concentration IL-4 after DC stimulation, and can be used as a target antigen for detecting serum infected by brucellosis or preparing an antigen of an Omp10 protein monoclonal antibody.

Description

Brucella Omp10 protein antigen epitope polypeptide and application thereof

Technical Field

The invention relates to a polypeptide, in particular to a Brucella Omp10 protein epitope polypeptide and application thereof.

Background

Brucella, a gram-negative facultative intracellular parasite, is a serious zoonosis that widely infects livestock, wild animals, and humans, and Brucella of livestock animals mainly infect sheep, cattle, and pigs, among which Brucella melitensis and Brucella abortus are the main causes of human morbidity; brucellosis infects livestock to cause epididymitis of male livestock and abortion, placental inflammation and sterility of pregnant livestock; in human beings, brucellosis causes acute inflammation and many symptoms similar to influenza infection, including wave heat, hyperhidrosis, back pain and weak constitution, in some patients, the acute clinical symptoms can last for more than one year and finally cause chronic persistent infection, the chronic clinical symptoms include irregular fever, joint pain and weakness and cause arthritis, regional peripheral nerve inflammation, rachitis, osteomyelitis and bursitis, and the prevalence of brucellosis not only harms the development of animal husbandry and causes huge economic loss, but also seriously threatens the health and public health safety of human beings.

At present, all effective vaccines for preventing brucellosis are attenuated live vaccines all over the world, the use of various vaccines not only interferes with the differential diagnosis of vaccine immunity and natural infection, but also all vaccine strains have pathogenic virulence to human in different degrees, even some vaccines have strong pathogenicity to human, the research effect of safe and reliable inactivated vaccines is poor, and the vaccine can not play an immune protection role. The research of the genetic engineering vaccine has been concerned and tried by researchers all over the world for decades, but the brucella itself has diversified immune antigens and complex structure, so that the vaccine antigens which can be used for field tests cannot be found, proteins which exist among various brucella species in a conserved way and have good immune functions are searched, and the development of a subunit vaccine with high preventive protection capability is an effective method for solving the problem.

In the prior art, most expression tools can only express one protein, and the Brucella has a plurality of proteins related to immunity, researches show that the expression of a single protein can not form an effective antigen for protecting the vaccine, and the subunit vaccine with a plurality of prevention and control functions is high in cost and complex in combined components; the components determining the function of the immune protein are tiny antigenic determinants on the protein, so that the excavation and identification of immune epitope of immune related protein can effectively extract effective immune antigen components in brucella, and meanwhile, direct guidance is provided for the research on the structure and the function of the protein, and the determination of the epitope antigen components can provide good antigen components for detection, prevention or treatment. Therefore, the screening and obtaining of the antigenic determinant polypeptides on the immunity protein provides a thought and a way for solving the defects in the prior art.

Disclosure of Invention

The invention provides brucella Omp10 protein antigen epitope polypeptide and application thereof.

The brucella Omp10 protein epitope polypeptide amino acid sequence contains SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5.

The Brucella Omp10 protein epitope polypeptide can be a polypeptide formed by a sequence formed by substituting and/or deleting one or/and several amino acid residues of a sequence shown by SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5 in amino acid sequence; or a sequence of which the amino acid sequence is shown by SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5, and a derivative polypeptide with other Brucella immunity function is added.

A recombinant vector or an expression cassette or a transgenic cell or a recombinant bacterium which is composed of SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5.

The polypeptide of the invention is applied to the preparation of reagents or medicines for diagnosing, preventing or treating diseases caused by brucella.

The polypeptide consisting of SEQ ID N0.1 or/and SEQ ID N0.2 or/and SEQ ID N0.3 or/and SEQ ID N0.4 or/and SEQ ID N0.5 can form a pharmaceutical composition for treating or preventing Brucella.

The brucella Omp10 immune protein is a first group of outer membrane protein components, is a brucella lipopolysaccharide component, can have strong reaction with brucella positive serum, and has an immune function. The invention utilizes the means of bioinformatics to analyze the composition and the function of brucella Omp10 immune protein amino acid, simulates and constructs a high-grade structure model of the protein, synthesizes the potential antigen polypeptide with immune function on the protein developed by combining the results, finally obtains the functional polypeptide through experimental verification means after chemically synthesizing the polypeptides, and the prepared brucella Omp10 immune protein polypeptide provides a new idea for the detection, the vaccine and the prevention and control of brucella, thereby having great social benefits for improving the prevention and control technical level of brucella of animals in China, ensuring the healthy development of breeding industry, providing income of farmers and herds, and ensuring public health and livestock product safety.

The invention has the beneficial effects that: 1) the screened polypeptide is chemically synthesized, but not extracted from live brucella, so that the potential safety hazards of artificial virus suppression, virus dispersion and the like are completely avoided in the production process. 2) The invention adopts the bioinformatics tool and the test verification to screen the antigen polypeptide, reduces the range of screening the antigen polypeptide and improves the screening efficiency. 3) The antigen polypeptide is screened on the dendritic cell, and the result is more close to an animal model because the dendritic cell has strong antigen processing and presenting capacity. 4) The polypeptide obtained by the invention can react with positive serum of brucellosis, so the recombinant protein can be used as a target antigen for detecting serum infected by brucellosis, such as an ELISA method and the like. 5) The target protein expressed by the invention can be directly used for preparing the antigen of the Omp10 protein monoclonal antibody.

Drawings

FIG. 1 is a diagram of a dendritic cell culture according to an embodiment of the present invention;

FIG. 2 shows the results of the flow cytometry for the detection of the phenotype of dendritic cells according to the present invention;

FIG. 3 shows the amount of IL-2 secreted by dendritic cells after the action of the polypeptide of the present invention; wherein 1-7 are tested sample polypeptides in sequence; 8 is a known epitope control; 9: and (5) negative control.

FIG. 4 shows the amount of IL-4 secreted by dendritic cells after the polypeptide of the present invention is applied thereto, wherein 1 to 7 in the figure are the polypeptides of the present invention in each test sample; 8 is a known epitope control; 9 is a negative control;

FIG. 5 shows the IFN-y secretion of a dendritic cell exposed to a polypeptide of the present invention, wherein 1 to 7 are the polypeptides of each test sample in the order named; 8 is a known epitope control; 9 is a negative control.

Detailed Description

The present invention will be further described with reference to the following examples.

Preparation of Experimental materials

1) Obtaining epitope polypeptides

The composition, the properties and the high-level structure of brucella Omp10 protein amino acid are analyzed by bioinformatics software, seven polypeptide fragments possibly having immunogenicity are obtained by mining and combining all results, and the seven polypeptide fragments are synthesized by Nanjing Kingsler Biotech Co., Ltd to obtain the polypeptide sequences as follows: TTGPGSGNAPIIAHTPAGIEGSWV (SEQ ID NO.3, polypeptide sample 1 described later);

GGIFETRTTDTNEKLAEGN (SEQ ID NO.1, polypeptide sample 2 described later);

NMRSIVRGTTSKVNCALVSPTQL (SEQ ID NO.4, polypeptide sample 3 described later);

NCTSSAGSRFSLTRRNA (SEQ ID NO.5, polypeptide sample 4 described later);

SKVNCALVSPTQLNCTSS, respectively; (SEQ ID NO.2, polypeptide sample 5 described later);

FRIVAPLALMSLALAACET (SEQ ID NO.6, polypeptide sample 6 described later);

GNYLYLSPQLVEI (SEQ ID NO.7, polypeptide sample 7 described later).

2) Culture of mouse bone marrow-derived Dendritic Cells (DCs) cervical dislocation method to sacrifice Babl/c mice, femurs and tibias were taken under sterile conditions and immersed in RPMI-1640 medium. Sucking RPMI-1640 culture solution by using a 1ml syringe, puncturing a marrow cavity from one end of a backbone, flushing marrow into a sterile culture dish, repeating for 4-6 times for each bone, collecting a marrow cell suspension in the culture dish, and centrifuging at 1500 rpm multiplied by 10 min. The supernatant was discarded, and 5 ml of sterile Tris-NH was added₄The cells were suspended in the Cl solution to lyse erythrocytes, and after they were allowed to stand at room temperature for 2 minutes to lyse erythrocytes, they were centrifuged again at 1500 rpm. times.5 min, and the supernatant was discarded. After washing with RPMI-1640 medium, the cells were suspended in complete medium and dispensed into 6-well plates, and complete medium was added to 4ml per well, followed by rmGM-CSF to a final concentration of 10ng/ml and IL-4 to a final concentration of 10 ng/ml. The cell culture plate was placed at 37 ℃ and 5% CO₂Culturing in the incubator for 48-72 hours. After the cells are lightly blown, the suspended cells are sucked away together with the culture solution, and only the adherent cells are kept. Fresh complete medium and the same concentration of rmGM-CSF was added and culture continued until day 5. Half the volume is changed and rmGM-CSF is replenished; the suspension cells were kept as much as possible. Continuing culturing for the 7 th day, gently blowing and beating by using a pipette, and collecting all suspension cells, namely, the enriched mouse Bone marrow-derived dendritic cells (BMDCs), wherein the observation period morphology of the cells under a microscope meets the characteristics of the Bone marrow-derived dendritic cells, and the reference is shown in figure 1; cell surface CD was shown by flow cytometry_11CThe antibody reaches more than 70%, and the cultured dendritic cells reach more than 70% and can meet the experimental requirements, which is shown in figure 2.

3) Interaction of Polypeptides with DCs

The DCs cultured for 7 days were collected, counted, and adjusted to 1X 10⁵Cell/ml concentration, inoculating and culturing cells at 500ul per well, adding synthetic polypeptide into cell culture system to obtain final concentration of 10ng/ml. Each polypeptide is used as 3 parallel controls, simultaneously, the identified polypeptide is used for stimulating cell culture to be used as a positive control, the cell culture without stimulation is used as a blank control, after 30 hours of action, cell culture supernatant is collected and stored at minus 80 ℃ for chip detection.

4) Chip operation process

And adding 100 muL of sample diluent into each hole, incubating for 30min on a shaking bed at room temperature, and sealing the quantitative antibody chip. Buffer solution was removed from each well, 100 μ L of standard solution and sample were added to the wells, and incubated overnight at 4 ℃.

Cleaning of

And (3) drawing out the standard substance or sample in each hole, washing with 1 Xlotion I for 3 times, shaking in a shaking table at room temperature for 10min each time, drawing out 150 muL of 1 Xlotion I in each hole, cleaning each time, and diluting with deionized water to 20 Xlotion I. And (3) extracting 1 Xwashing liquid I from each hole, adding 1 Xwashing liquid II, washing for 3 times, shaking in a shaking table at room temperature for 5min every time, extracting 150 muL of 1 Xwashing liquid II from each hole, and diluting 20 Xwashing liquid II with deionized water (all reagents are provided by the kit).

Incubation of detection antibody mixtures

The test antibody mixture vials were centrifuged and then 1.4ml of sample diluent was added, mixed well and then centrifuged quickly again. Add 80 μ L of detection antibody to each well and incubate on RT shaker for 1.5 hours. The detection antibody in each hole is pumped out, 1 Xwashing liquor I is washed for 3 times, shaking table vibration is carried out at room temperature for 10min every time, 150 muL of 1 Xwashing liquor I in each hole is washed to be pumped out and cleaned, then 1 Xwashing liquor II is added for washing for 3 times, shaking table vibration is carried out at room temperature for 5min every time, 150 muL of 1 Xwashing liquor II in each hole is washed to be pumped out and cleaned.

Incubation of Cy 3-streptavidin

The Cy 3-streptavidin vial was centrifuged, then 1.4ml of sample diluent was added, mixed well and centrifuged quickly again. Adding 80 mu L of Cy 3-streptavidin into each well, wrapping the slide with aluminum foil paper, incubating in a dark place, and incubating on an RT shaker for 1 hour. And (3) extracting Cy 3-streptavidin from each hole, washing with 1 Xwashing liquid I for 3 times, shaking in a shaking table at room temperature for 10min every time, and extracting the washing liquid from 150 mu L of 1 Xwashing liquid I in each hole every time of washing. And then adding 1 Xwashing liquid II for cleaning for 3 times, shaking the mixture in a shaking table at room temperature for 5min every time, and pumping out the washing liquid from each hole of 150 mu L of 1 Xwashing liquid II.

Fluorescence detection

The slide frame holding the chip was removed, taking care not to touch the antibody-printed side of the slide by hand. The signal is scanned with a laser scanner, e.g. InnoScan 300, using either Cy3 or green channel (excitation frequency =532 nm). Data analysis is carried out by adopting data analysis software of QAM-CYT-1, and the data obtained by analysis are shown in figures 3, 4 and 5.

II, active function identification experiment of polypeptide

Chip detection of sample cytokines

The culture supernatant after the action of the polypeptide and the DC is a sample, the sample is diluted by 10 times and then added into a well-coated cytokine detection chip, the color is developed after the reaction is carried out for 30 minutes, and the sample is placed in a detection instrument to firstly carry out color-developing scanning on different cytokines to obtain the luminous intensity value. Meanwhile, making known positive polypeptide and blank control, making quantitative control by using a standard substance, drawing a standard curve, and calculating the amount of the cell factors in the sample according to the standard curve, wherein the factors related to the B cell and T cell polypeptide comprise IL-2, IL-4 and IFN-gamma, and the specific detection values are shown in figure 3, figure 4 and figure 5.

1) IL-2 assay

IL-2 is interleukin-2 (IL-2), also known as T cell growth factor (TCRF). Cytokines with broad biological activities are produced primarily by activated CD4+ Th1 cells. Can promote Th0 and proliferation of CTLs, and thus are important factors in regulating immune responses, and are also involved in antibody responses, hematopoiesis, and tumor surveillance. Can activate T cells and promote cytokine production; stimulating NK cell proliferation, enhancing NK killing activity, generating cytokines, and inducing LAK cell generation; promoting B cell proliferation and secretion of antibodies; macrophages are activated. The main physiological role of IL-2 is to stimulate and maintain the differentiation and proliferation of T cells. Thus, the ability of a polypeptide to activate a T cell immune response in response to the level of IL-2 released by the polypeptide following stimulation with DCs is an important indicator for the evaluation of T cell polypeptides. The result shows that the concentration of the IL-2 released after the positive control polypeptide is stimulated is 0pg/ml, compared with the positive control, the concentration of the IL-2 released after the polypeptide group is stimulated by the experimental screening polypeptide group is as follows:

1.96 pg/ml（p<0.001），

5.5 pg/ml (p<0.0001)，

0.84 pg/ml(p<0.001) and the concentration of IL-2 released by the remaining test polypeptides was 0pg/ml, see FIG. 3.

2) IL-4 assay

Interleukin-4 (Interleukin-4, IL-4) is a cytokine secreted by type II helper T cells (Th2 cells). The biological actions of interleukin-4, including stimulation of activated B-and T-cell proliferation, CD4+ T cells differentiate into type II helper T cells, also play a key role in the regulation of humoral and adaptive immunity. Interleukin-4 induces class switching of B cell antibodies to IgE, a B cell growth factor-1 (B cell growth factor-1, BCGF-1). IL-4 promotes the expression of B cell MHC class II antigens, Fc epsilon RII/CD 23 and CD40, and enhances the antigen presenting ability of B cells, so that the immune system generates immune response to small amount of antigen stimulation. The functions of promoting macrophages to present antigens and kill tumor cells may be related to the regulation of MHC class II antigen and FcR expression. IL-4 has a synergistic effect with GM-CSF, IL-3 and LPS. IL-4 inducible peripheral blood mononuclearThe cells secrete G-CSF and M-CSF, enhancing neutrophil-mediated phagocytosis, killing activity and ADCC. Therefore, the level of IL-4 secretion is an important index for evaluating the immune negative and positive ability of the polypeptide B cells. The result shows that the concentration of the released IL-4 after the stimulation of the positive control polypeptide is 7.7pg/ml, and compared with the positive control, the released IL-4 after the stimulation of the polypeptide of each sample polypeptide group screened by the experiment of the invention is respectively as follows:

16.8 pg/ml（p<0.001）,

17.9 pg/ml (p<0.001)，

16.5 pg/ml(p<0.001)，

15.1 pg/ml，

13.0 pg/ml，

8.0 pg/ml，

6.9pg/ml。

4) IFN-gamma analysis

Interferon (IFN) is a broad-spectrum antiviral agent, does not directly kill or inhibit viruses, but primarily inhibits replication of hepatitis B virus by allowing cells to produce antiviral proteins through the action of cell surface receptors, and is classified into three types, α - (leukocyte), β - (fibroblast), gamma- (lymphocyte), Interferon-gamma (Interferon-gamma, IFN-gamma) which is a water-soluble dimeric cytokine and is the only member of type II Interferon, originally called macrophage activating factor, IFN-gamma which is a marker cytokine for type I helper T cells (Th1 cells), type II helper T cells (Th2 cells) release leukocytesInterleukin-4 (IL-4) and interleukin-13 (IL-13). Natural killer cells and CD8T cells also produce interferon-gamma. Interferon-gamma inhibits osteoclastogenesis by rapidly degrading TRAF6 of the RANK-RANKL signaling pathway. The release level of interferon-gamma after the polypeptide stimulates DC is positively correlated with the activation of T cells, and whether the polypeptide is T cell immune polypeptide or not is generally evaluated by the detection of interferon-gamma. The result shows that the concentration of IFN-gamma released after the positive control polypeptide is stimulated is 20pg/ml, and compared with the positive control, the concentration of IFN-gamma released after the polypeptide group polypeptide screened by the experiment is stimulated is respectively as follows:

61.8 pg/ml (p<0.001)，

43.6pg/ml(p<0.001) and the remaining samples did not release IFN- γ, see fig. 5.

The experiments show that the protein chip is used for detecting the levels shown in figures 3-5 when IL-2, IL-4 and IFN-y are secreted by cells after stimulation, and the detection result of statistical analysis shows that GGIFETRTTDTNEKLAEGN (SEQ ID NO.1, namely the polypeptide sample 2) and SKVNCALVSPTQLNCTSS (SEQ ID NO.2, namely the polypeptide sample 5) obtained from all peptide fragments can release a large amount of IL-2 and/or IFN-gamma after DC stimulation to obtain the T cell epitope polypeptide, and the peptide fragment GGIFETRTTDTNEKLAEGN, TTGPGSGNAPIIAHTPAGIEGSWV (SEQ ID NO.3, namely the polypeptide sample 1)

And NMRSIVRGTTSKVNCALVSPTQL (SEQ ID NO.4, polypeptide sample 3) showed significant release of IL-4 as a B cell epitope polypeptide upon stimulation of DCs. The excavation of the surface epitope of the protein has important significance for researching the immunological function of the protein, on the other hand, the effective epitope removes the complicated non-antigenic determinants on the protein, can intensively produce a large amount of effective epitopes to be used for disease diagnosis and vaccine development, and is a good candidate antigen for vaccines; the effective B cell epitope excavated from the protein is a good diagnostic antigen for diseases, can be used for establishing serological methods such as ELISA and the like, has higher sensitivity than the protein antigen, and screens out the preparation of monoclonal antibodies for epitope polypeptide. Experiments of the invention show that each polypeptide of SEQ ID NO.1 to SEQ ID NO.5 has an immunogenic function, can be used for forming a corresponding recombinant vector or expression cassette or transgenic cell or recombinant bacterium, and can also be applied to preparation of reagents or medicaments for diagnosing or preventing or treating diseases caused by Brucella.

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> Brucella Omp10 protein antigen epitope polypeptide and application thereof

<160>7

<210>1

<211>19

<212>PRT

<213> Artificial sequence (test specimen 3)

<400>

Gly Gly Ile Phe Glu Thr Arg Thr Thr Asp Thr Asn Glu Lys Leu Ala

1 5 10 15

Glu Gly Asn

19

<210>2

<211>18

<212>PRT

<213> Artificial sequence (test specimen 1)

<400>

Ser Lys Val Asn Cys Ala Leu Val Ser Pro Thr Gln Leu Asn Cys Thr

1 5 10 15

Ser Ser

18

<210>3

<211>24

<212>PRT

<213> Artificial sequence (test specimen 4)

<400>

Thr Thr Gly Pro Gly Ser Gly Asn Ala Pro Ile Ile Ala His Thr Pro

1 5 10 15

Ala Gly Ile Glu Gly Ser Trp Val

20 24

<210>4

<211>23

<212>PRT

<213> Artificial sequence (test specimen 9)

<400>

Asn Met Arg Ser Ile Val Arg Gly Thr Thr Ser Lys Val Asn Cys Ala

1 5 10 15

Leu Val Ser Pro Thr Gln Leu

20 23

<210>5

<211>17

<212>PRT

<213> Artificial sequence (test specimen 4)

<400>

Asn Cys Thr Ser Ser Ala Gly Ser Arg Phe Ser Leu Thr Arg Arg Asn

1 5 10 15

Ala

17

<210>6

<211>19

<212>PRT

<213> Artificial sequence (test specimen 6)

<400>

Phe Arg Ile Val Ala Pro Leu Ala Leu Met Ser Leu Ala Leu Ala Ala

1 5 10 15

Cys Glu Thr

19

<210>7

<211>13

<212>PRT

<213> Artificial sequence (test specimen book 7)

<400>

Gly Asn Tyr Leu Tyr Leu Ser Pro Gln Leu Val Glu Ile

1 5 10 13

Claims

1. The Brucella Omp10 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQID N0.1.

2. The Brucella Omp10 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQID N0.2.

3. The Brucella Omp10 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQID N0.3.

4. The Brucella Omp10 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQID N0.4.

5. The Brucella Omp10 protein epitope polypeptide is characterized in that the amino acid sequence of the polypeptide is SEQID N0.5.

6. Preparing a recombinant vector or an expression cassette or a transgenic cell or a recombinant bacterium of the Brucella Omp10 protein epitope polypeptide according to any one of claims 1 to 5.

7. Use of the polypeptide of any one of claims 1 to 5 for the preparation of an agent or medicament for the diagnosis or prevention or treatment of a disease caused by brucella.

8. A pharmaceutical composition for the treatment of brucellosis wherein there is a combination of any one or several of the polypeptides according to claims 1 to 5.

9. Use of the recombinant vector or expression cassette or transgenic cell or recombinant bacterium of claim 6 in the preparation of a reagent or medicament for the diagnosis or prevention or treatment of a disease caused by brucella.