阅读说明：本技术 羊种布鲁氏菌疫苗株m5提取的脂多糖在制备诊断人布鲁氏菌病的产品中的应用 (Application of lipopolysaccharide extracted from Brucella melitensis vaccine strain M5 in preparation of products for diagnosing human brucellosis ) 是由 张文宝 郭刚 李军 游锡火 贾斌 齐文静 吴熙然 郭宝平 于 2020-04-27 设计创作，主要内容包括：本发明公开了羊种布鲁氏菌疫苗株M5提取的脂多糖在作为人布鲁氏菌病诊断抗原中的应用。本发明发现从羊种布鲁氏菌疫苗株M5提取的LPS抗原作为诊断抗原的反应性明显优于目前市面上布病抗体快速检测试剂盒常用的布菌牛种S19株和布菌猪种S2株提取的LPS抗原,说明羊种布鲁氏菌疫苗株M5可以作为新的布病金标诊断LPS抗原的首选来源株,其提取的LPS对人感染布病具有较好的诊断价值,为人布病感染诊断抗原的选择提供了新的途径。本发明还发现无害化处理后的羊种布鲁氏菌疫苗株M5提取的LPS抗原并不影响其抗原性。(The invention discloses application of lipopolysaccharide extracted from a Brucella melitensis vaccine strain M5 in serving as a human Brucella disease diagnosis antigen. The invention finds that the reactivity of the LPS antigen extracted from the Brucella melitensis vaccine strain M5 taken as a diagnosis antigen is obviously superior to the LPS antigen extracted from the Brucella melitensis S19 strain and the Brucella melitensis S2 strain which are commonly used in the rapid detection kit for Brucella melitensis antibodies on the market at present, which indicates that the Brucella melitensis vaccine strain M5 can be taken as a first-choice source strain of a new Brucella melitensis gold-labeled diagnosis LPS antigen, the extracted LPS has good diagnosis value on human infectious brucellosis, and a new way is provided for selecting the human Brucella melitensis infection diagnosis antigen. The invention also finds that the LPS antigen extracted from the harmless treated Brucella melitensis vaccine strain M5 does not influence the antigenicity thereof.)

1. A lipopolysaccharide is extracted from Brucella melitensis vaccine strain M5.

2. A lipopolysaccharide is extracted from Brucella melitensis vaccine strain M5 after harmless treatment.

3. Lipopolysaccharide according to claim 2, characterized in that: the harmless treatment method is to heat the bacterial liquid of the Brucella melitensis vaccine strain M5.

4. Lipopolysaccharide according to claim 3, characterized in that: the heating treatment is carried out at 95-100 deg.C for 30-35 min.

5. Use of a lipopolysaccharide according to any one of claims 1-4 in any one of the following a) -c):

a) preparing a human brucellosis diagnostic antigen;

b) as a diagnostic antigen for human brucellosis;

c) preparing a product for diagnosing brucellosis of human.

6. A colloidal gold test strip comprises a bottom plate, a sample pad, a gold-labeled pad coated with a gold-labeled antibody, a detection pad containing a detection line T and a quality control line C, and a water absorption pad, wherein the sample pad is arranged on the bottom plate;

the detection line T is coated with the lipopolysaccharide of any one of claims 1-4.

7. The colloidal gold test strip of claim 6, wherein:

the lipopolysaccharide is a lipopolysaccharide solution, and the concentration of the lipopolysaccharide in the lipopolysaccharide solution is 0.001-0.1 mg/mL;

or, the gold-labeled antibody is a colloidal gold-labeled goat anti-human IgG antibody or goat anti-human IgM antibody;

or, the quality control line C is coated with a goat anti-human IgG antibody or a goat anti-human IgM antibody;

or the sheep anti-human IgG antibody or the sheep anti-human IgM antibody is a sheep anti-human IgG antibody solution or a sheep anti-human IgM antibody solution, and the antibody concentration in the sheep anti-human IgG antibody solution or the sheep anti-human IgM antibody solution is 0.1-2 mg/mL.

8. A method for preparing the colloidal gold test strip of claim 6 or 7, comprising the steps of:

1) preparing a gold-labeled pad coated with a gold-labeled antibody and a detection pad containing a detection line T and a quality control line C;

the gold-labeled pad coated with the gold-labeled antibody is prepared as follows: uniformly mixing the colloidal gold solution and the antibody solution, and reacting to obtain a gold-labeled antibody; adding the gold-labeled antibody to a glass cellulose membrane to obtain a gold-labeled pad coated with the gold-labeled antibody;

the detection pad containing the detection line T and the quality control line C is formed by respectively coating the lipopolysaccharide and the goat anti-human IgG antibody or the goat anti-human IgM antibody of any one of claims 1 to 4 on a nitrocellulose membrane to form the detection pad containing the detection line T and the quality control line C;

2) and assembling the gold-labeled pad coated with the gold-labeled antibody, the sample pad, the detection pad containing the detection line T and the quality control line C and the water absorption pad on a bottom plate to obtain the colloidal gold test strip.

9. Use of the colloidal gold test strip of claim 6 or 7 or the colloidal gold test strip prepared by the method of claim 8 in the preparation of a product for diagnosing brucellosis in humans.

10. A harmless treatment method of pathogenic bacteria comprises the step of heating pathogenic bacteria liquid;

or, the use of the method of claim 10 in a1) or a2) as follows:

A1) eliminating the infectivity of pathogenic bacteria;

A2) preparing the antigen for diagnosing diseases caused by pathogenic bacteria.

Technical Field

The invention relates to the technical field of application of brucella live vaccines to extraction of antigens, in particular to application of lipopolysaccharide extracted from a brucella ovis vaccine strain M5 as a human brucella disease diagnosis antigen.

Background

Brucellosis (hereinafter referred to as Brucellosis) is a serious zoonosis infectious disease caused by Brucella, which causes great harm to human health and serious economic loss to animal husbandry. World statistics show that every year brucellosis causes economic losses of nearly $ 30 billion. The human wave heat and low heat caused by the Chinese medicinal composition, the heart, skeleton and nervous system pathological changes caused by chronic infection, abortion, orchitis and other diseases of ruminants cannot be cured radically until now. In addition, brucella has also been used as a biological weapon. The epidemic is particularly serious in China and listed as a national B legal infectious disease report, and has serious threats to national economic construction, healthy life of people, national safety and the like.

The Buscytitis pathogen is gram-negative coccobacillus, is parasitized in cells, and is a intracellular parasitism with a unique intracellular survival mechanism and an immunological mechanism. After invading into the body, it can escape the immune response of the body to survive and reproduce, which is also the main reason why the middle and late stage of disease distribution can not be cured. After a person is infected with bacteria, the clinical manifestation is complex, symptoms such as fever, hyperhidrosis, arthralgia and the like with different degrees can appear, and due to non-specific clinical symptoms, occult morbidity, medical conditions in farming and pastoral areas and the like, the infected person usually misses the optimal treatment time, so that the infected person is not healed for a long time, even serious complications occur, and the labor capacity is lost. Therefore, the diagnosis of the cloth disease plays a key role in the treatment and prognosis of the disease, the early diagnosis and early discovery are a key step for curing the cloth disease, and the development of a rapid diagnosis kit which can be suitable for basic use in farming and pastoral areas and has higher specificity and sensitivity is particularly important for early diagnosis and patient discovery. At present, the tiger red plate agglutination test (RBPT) is the most widely used diagnostic method, has the characteristics of low price, quick detection and the like, but also has the problems that the antigen is difficult to standardize and produce, the result judgment is influenced by human factors, and the cross reaction is easy to occur with gram-negative bacteria such as Escherichia coli and the like.

The brucella Lipopolysaccharide (LPS) is a component of endotoxin on the surface of bacteria and consists of 3 conserved domains, namely lipid a (endotoxin property), core oligosaccharide and O antigen side chain polysaccharide. The bacterium distribution rough type LPS is lack of O antigen components, and has weak toxicity relative to a smooth type. Hapten polysaccharide (NH) and polysaccharide B with surface antigen components can also be separated from the Buzhou LPS, and exposed polysaccharide chains contained in the LPS prevent the exposure of outer membrane proteins (OMPS, Bp26 and the like) of thalli, but play an important role in stimulating the body to generate antibodies, thereby becoming important components of protective immune response of the body.

Disclosure of Invention

The first object of the present invention is to provide a lipopolysaccharide.

The lipopolysaccharide provided by the invention is extracted from the Brucella melitensis vaccine strain M5 or is extracted from the Brucella melitensis vaccine strain M5 after harmless treatment.

Further, the harmless treatment method is to heat the bacterial liquid of the Brucella melitensis vaccine strain M5.

The heating treatment condition can be 95-100 deg.C for 30-60 min.

The bacterial liquid of the Brucella melitensis strain M5 is obtained by inoculating the Brucella melitensis strain M5 to a culture medium for culture.

Further, the heating treatment is carried out under conditions of 100 ℃ for 30 min.

The culture medium is LB liquid culture medium or Brucella culture medium. The LB liquid culture medium comprises the following specific formula: 10g of sodium chloride, 10g of tryptone and 5g of yeast extract, adjusting the pH to 7.0 by using 5mol/L NaOH, fixing the volume to 1L by using deionized water, and sterilizing by using high-pressure steam. The formula of the brucella culture medium is as follows: columbia agar (ISO)43g and glucose 10g, adding 1L deionized water, heating, stirring, boiling for 1min to completely dissolve, sterilizing with high pressure steam, and cooling to about 50 deg.C, adding antibiotic and inactivated horse serum.

The culture condition is shaking culture at 37 ℃ for 4 h.

The heating treatment is realized by equipment and/or instruments which can provide high-temperature environment, and the equipment and/or instruments which can provide high-temperature environment can be a water bath kettle or any other equipment and/or instruments which can heat the solution to 95-100 ℃. In practical application, the container containing the bacterial liquid of the Brucella melitensis strain M5 can be placed in a water bath for heating treatment, or the container containing the Brucella melitensis strain M5 can be heated by steam heating or direct boiling.

In the present invention, the method of the detoxification treatment is as follows: the bacterial liquid of the Brucella melitensis vaccine strain M5 is placed in a 10mL centrifugal tube, and then the centrifugal tube containing the bacterial liquid is placed in a water bath kettle and boiled for 30min at 100 ℃.

The method for extracting lipopolysaccharide can be performed by referring to a conventional method in the prior art (e.g., a method disclosed in "Liu, Y., Fratamic, P., Debroy, C., Bumbaugh, A.C., & Allen, J.W. (2008.). DNA sequencing and differentiation of diagnostic reagent O118 antigenic gene cluster and differentiation of antigenic diversity reaction on restriction in DNA sequence of the antigenic cluster of E.coli O118 and O151.foodborne Pathologen and Disease,5(4), 449."), or by using an LPS Extraction Kit (LPS Extraction Kit, manufactured by Korea: 17141).

The second object of the present invention is to provide the use of the above lipopolysaccharide in any one of the following a) to c):

a) preparing a human brucellosis diagnostic antigen;

b) as a diagnostic antigen for human brucellosis;

c) preparing a product for diagnosing brucellosis of human.

The third purpose of the invention is to provide a colloidal gold test strip.

The colloidal gold test strip provided by the invention comprises a bottom plate, a sample pad arranged on the bottom plate, a gold-labeled pad coated with a gold-labeled antibody, a detection pad containing a detection line T and a quality control line C, and a water absorption pad;

the detection line T is coated with the lipopolysaccharide.

Further, the quality control line C is coated with sheep (or any experimental animal) anti-human IgG antibody or sheep (or any experimental animal) anti-human IgM antibody.

The gold-labeled antibody is a colloidal gold-labeled sheep (or any experimental animal) anti-human IgG antibody or sheep (or any experimental animal) anti-human IgM antibody.

Further, the lipopolysaccharide is a lipopolysaccharide solution (solvent is Tris buffer or PBS or physiological saline), and the concentration of the lipopolysaccharide in the lipopolysaccharide solution can be 0.001-0.1mg/mL, specifically 0.01mg/mL or 0.02 mg/mL.

The sheep (or any experimental animal) anti-human IgG antibody or sheep (or any experimental animal) anti-human IgM antibody is a sheep (or any experimental animal) anti-human IgG antibody solution or a sheep (or any experimental animal) anti-human IgM antibody solution (the solvent is Trisbuffer or PBS or normal saline), and the antibody concentration in the sheep (or any experimental animal) anti-human IgG antibody solution or the sheep (or any experimental animal) anti-human IgM antibody solution can be 0.1-2mg/mL, specifically 1 mg/mL.

The fourth purpose of the invention is to provide a method for preparing the colloidal gold test strip.

The method for preparing the colloidal gold test strip provided by the invention comprises the following steps:

1) preparing a gold-labeled pad coated with a gold-labeled antibody and a detection pad containing a detection line T and a quality control line C;

the gold-labeled pad coated with the gold-labeled antibody is prepared as follows: uniformly mixing the colloidal gold solution and the antibody solution, and reacting to obtain a gold-labeled antibody; adding the gold-labeled antibody to a glass cellulose membrane to obtain a gold-labeled pad coated with the gold-labeled antibody;

the detection pad containing the detection line T and the quality control line C is formed by respectively coating the lipopolysaccharide and the sheep (or any experimental animal) anti-human IgG antibody or the sheep (or any experimental animal) anti-human IgM antibody on a nitrocellulose membrane to form the detection pad containing the detection line T and the quality control line C;

2) and assembling the gold-labeled pad coated with the gold-labeled antibody, the sample pad, the detection pad containing the detection line T and the quality control line C and the water absorption pad on a bottom plate to obtain the colloidal gold test strip.

In the above method, in the step 1), the preparation method (preparation ratio) of the colloidal gold solution is specifically as follows: washing a pipette with ultrapure water, repeating for 2-3 times, sucking 1mL of 1% (mass to volume) chloroauric acid by using the washed pipette, adding into a conical flask, sealing with tinfoil, heating and stirring on a magnetic stirrer to obtain a 0.01% chloroauric acid solution; after 100mL of 0.01% chloroauric acid solution is boiled, 2mL of 1% (mass to volume) trisodium citrate aqueous solution is rapidly added, and heating is continued for 10min, wherein the color change is observed; after cooling at room temperature, the volume was adjusted to 100mL with ultrapure water to obtain a colloidal gold solution.

The particle size of the colloidal gold can be 5-20 nm. Further, the particle size of the colloidal gold can be 10-20 nm. Further, the particle size of the colloidal gold is 20 nm.

The antibody solution is sheep (or any experimental animal) anti-human IgG antibody solution or sheep (or any experimental animal) anti-human IgM antibody solution. The concentration of the antibody in the sheep (or any experimental animal) anti-human IgG antibody solution or the sheep (or any experimental animal) anti-human IgM antibody solution can be 0.1-2mg/mL, and specifically 1 mg/mL.

The lipopolysaccharide is lipopolysaccharide solution (solvent is Tris buffer or PBS or physiological saline), and the concentration of lipopolysaccharide in the lipopolysaccharide solution can be 0.001-0.1mg/mL, specifically 0.01mg/mL or 0.02 mg/mL.

The sheep (or any experimental animal) anti-human IgG antibody or sheep (or any experimental animal) anti-human IgM antibody is a sheep (or any experimental animal) anti-human IgG antibody solution or a sheep (or any experimental animal) anti-human IgM antibody solution (the solvent is Trisbuffer or PBS or normal saline), and the antibody concentration in the sheep (or any experimental animal) anti-human IgG antibody solution or the sheep (or any experimental animal) anti-human IgM antibody solution can be 0.1-2mg/mL, specifically 1 mg/mL.

In the step 2), the assembling steps are as follows:

2-1) cutting the sample pad and the water absorption pad into a size of 30 multiplied by 1.7cm, and cutting the gold label pad into a size of 30 multiplied by 0.5 cm.

2-2) sticking the detection pad on the bottom plate.

2-3) sticking a gold label pad, and covering the detection pad with 1-1.5mm of the gold label pad.

2-4) sticking the sample pad, and covering 2mm of the sample pad on the gold label pad.

2-5) sticking a water absorption pad, and covering the detection pad with 1-1.5mm of the water absorption pad.

2-6) securing the engagement portion of each layer with an engagement tape.

And 2-7) cutting the assembled bottom plate into test strips with the width of 2-6mm (such as 4mm), and sealing and storing for later use to obtain the colloidal gold test strips.

The application of the colloidal gold test strip or the colloidal gold test strip prepared by the method in the preparation of products for diagnosing brucellosis of human also belongs to the protection scope of the invention.

A fifth object of the present invention is to provide a method for detoxifying pathogenic bacteria.

The method for harmless treatment of pathogenic bacteria provided by the invention comprises the step of heating the pathogenic bacteria liquid.

Further, the bacterial liquid is obtained by inoculating pathogenic bacteria to a culture medium for culture.

The pathogenic bacteria are infectious pathogenic bacteria such as Brucella, Staphylococcus aureus, Shigella flexneri, Streptococcus pneumoniae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis and the like.

The harmless treatment method is to heat the bacterial liquid.

The heating treatment condition can be 95-100 deg.C for 30-60 min.

Furthermore, the pathogenic bacteria are Brucella (such as Brucella melitensis vaccine strain M5, Brucella bovis S19 and Brucella suis S2) or Staphylococcus aureus or Shigella flexneri.

The heating treatment is carried out at 100 deg.C for 30 min.

The culture condition is shaking culture at 37 ℃ for 4 h.

The heating treatment is realized by equipment and/or instruments which can provide high-temperature environment, and the equipment and/or instruments which can provide high-temperature environment can be a water bath kettle or any other equipment and/or instruments which can heat the solution to 95-100 ℃. In practical application, the container containing the bacterial liquid of the Brucella melitensis strain M5 can be placed in a water bath for heating treatment, or the container containing the Brucella melitensis strain M5 can be heated by steam heating or direct boiling.

In the present invention, the method of the detoxification treatment is as follows: putting the bacterial liquid of the Brucella melitensis vaccine strain M5 of sheep species, the Brucella melitensis S19 of cattle species, the Brucella suis S2 of pig species, the Staphylococcus aureus bacterial liquid, the Shigella flexneri, the Escherichia coli or the Mycobacterium tuberculosis bacterial liquid into a 10mL centrifugal tube, and then putting the centrifugal tube containing the bacterial liquid into a water bath kettle to be boiled for 30min at 100 ℃.

The application of the harmless treatment method in the following A1) or A2) also belongs to the protection scope of the invention:

A1) eliminating the infectivity of pathogenic bacteria;

A2) preparing the antigen for diagnosing diseases caused by pathogenic bacteria.

Further, in the a1), the pathogenic bacteria are infectious pathogenic bacteria; the elimination of the infectivity of the pathogenic bacteria is to eliminate the infectivity of the infectious pathogenic bacteria and maintain the antigenicity of the infectious pathogenic bacteria.

The A2), wherein the pathogen-caused disease diagnostic antigen is not infectious.

Further, in the a1), the pathogenic bacteria is brucella (such as brucella ovis vaccine M5, brucella bovis S19, brucella suis S2) or staphylococcus aureus or shigella flexneri or escherichia coli or mycobacterium tuberculosis.

The A2), the disease caused by pathogenic bacteria is a disease caused by Brucella (such as brucellosis), a disease caused by staphylococcus aureus, or a disease caused by Shigella flexneri, escherichia coli or mycobacterium tuberculosis.

The invention discloses application of Lipopolysaccharide (LPS) extracted from a Brucella melitensis vaccine strain M5 in serving as a human brucellosis diagnosis antigen for the first time. The experimental result shows that the reactivity of the LPS antigen extracted from the Brucella melitensis vaccine strain M5 as a gold-labeled rapid diagnosis antigen is obviously superior to that of the LPS antigen extracted from a cattle S19 strain and a pig S2 strain commonly used in a brucellosis antibody rapid detection kit on the market at present, which indicates that the Brucella melitensis vaccine strain can be used as a first-choice source strain of a new brucellosis gold-labeled diagnosis LPS antigen, the extracted LPS has a good diagnosis value on human brucellosis, and a new way is provided for selecting the antigen for rapid diagnosis of brucellosis infection. The invention also finds that the LPS antigen extracted from the harmless treated Brucella melitensis vaccine strain M5 does not influence the antigenicity thereof. The harmless treatment can completely eliminate the infection of the Brucella, so that the subsequent treatment process has no infection harm to people and environment.

Drawings

FIG. 1 shows the comparison of the sensitivity and specificity of LPS extracted from Brucella melitensis strain M5 as diagnostic antigen with LPS from other sources. The test sera in FIG. 1A were positive sera; the test sera in FIG. 1B were negative sera. Wherein a is LPS (concentration of 0.5mg/mL) extracted after harmless treatment of the thallus of the Bulbophyllum sp S19 strain; b is LPS (concentration of 0.02mg/mL) extracted after the bacteria of the Brucella melitensis vaccine strain M5 are subjected to harmless treatment; c is LPS (low-pressure lipoprotein) extracted after the harmless treatment of the shigella flexneri thallus, wherein the concentration of the LPS is 05 mg/mL; d is peptidoglycan (concentration of 0.5mg/mL) extracted from Staphylococcus aureus thallus after innocent treatment; e is normal human serum (quality control point).

FIG. 2 shows the reactivity comparison of LPS extracted from the mycelium of Boletus suis strain S2 and Boletus niveus strain S19 after (or without) detoxification treatment and Boletus membrane protein Omp2a as diagnostic antigens. 1 is LPS (concentration of 0.5mg/mL) extracted from the strain S19 of Bulbophyllum kanehensis without harmless treatment; 2, LPS (concentration of 0.5mg/mL) extracted after harmless treatment of the strain of the Buzhou strain S2 strain; 3 is LPS finished product (concentration is 0.5 mg/mL); 4 is LPS (concentration of 0.5mg/mL) extracted after the thalli of the Bulbophyllum species S19 strain are subjected to harmless treatment; 5 is Bulbophylum Omp2a membrane protein (concentration is 0.4 mg/mL).

FIG. 3 shows the result of the colloidal gold test strip on the positive serum of Bujun.

FIG. 4 shows the result of the colloidal gold test strip on the negative serum of the bacteria distribution.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The Brucella melitensis vaccine strain M5 in the following examples is a product of Xinjiang Tiankang animal biotechnology, Inc., and the catalog number is CVCC 18.

The Bulbophyllum species S19 strain in the examples described below is a product of Australian Longjic Biometrics Co.Ltd.

The strain of Bulbophyllum commune S2 in the examples described below is a product of Auolong Biochemical Co., Ltd, Chongqing.

The finished LPS product in the examples below is a product from hungzhou hundred million minol biotechnology limited, which is extracted from the strain boswellia californica S19.

The strain Omp2a membrane protein of the following examples is described in the literature "Pathak P, Kumar A, ThavaselvamD. evaluation of recombinant protein in a protein antigen for diagnostic of Human Brucellosis [ J ]. BMC infection Diseases,2017,17(1): 485", publicly available from the Applicant, and this biomaterial is used only for repeating the relevant experiments of the present invention and is not useful for other applications.