CN111220799B

CN111220799B - Application of HSP90AB1 protein in identifying meningitis of streptococcus suis

Info

Publication number: CN111220799B
Application number: CN202010233086.8A
Authority: CN
Inventors: 雷连成; 刘洪涛; 吴桐; 姜合祥; 贾丽; 孙莹莹; 孙长江
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-03-29
Filing date: 2020-03-29
Publication date: 2021-11-05
Anticipated expiration: 2040-03-29
Also published as: CN111220799A

Abstract

The invention discloses an application of HSP90AB1 protein in identifying meningitis of streptococcus suis, and provides a new application of the HSP90AB1 protein, namely, the HSP90AB1 protein is used as a target for detection, and the SS2 meningitis is newly diagnosed in an auxiliary way according to a result; compared with the traditional method, the method is more accurate and sensitive, and the protein does not show high expression in the serum of non-meningitis type infection or healthy pigs, so that SS2 infection can be determined, meanwhile, meningitis can be assisted to diagnose when clinical meningitis symptoms are not shown, and targeted treatment can be conveniently and timely carried out; the invention is simple and convenient for detecting the protein expression level and can obtain accurate results in a short time. And the corresponding commercial reagent and kit are easy to manufacture aiming at protein detection, and can be more widely applied to the detection of sick pigs.

Description

Application of HSP90AB1 protein in identifying meningitis of streptococcus suis

Technical Field

The invention discloses application of HSP90AB1 protein in identifying streptococcus suis meningitis, wherein the pig streptococcus meningitis is identified through changes of protein in blood; belongs to the technical field of pig disease identification methods.

Background

Streptococcus suis type 2 (SS 2) is an important zoonotic pathogen and can cause serious clinical symptoms such as meningitis, arthritis, endocarditis and septicemia of human or pigs. Not only brings huge economic loss to the pig industry all over the world, but also seriously threatens public health safety. Compared with non-meningitis type SS2 infection, meningitis caused by meningitis type SS2 infection has the most serious damage to organisms, can cause encephaledema, infiltration of inflammatory cells in brains, and neurological symptoms, even death of animals, and is often accompanied with serious sequelae such as hearing loss and the like even if being cured. However, there is currently a lack of rapid and accurate diagnostic methods for distinguishing between meningitis and non-meningitis SS2 infections in sick pigs, which results in lack of specificity in clinical treatment and increased mortality from meningitis-type SS2 infections.

HSP90AB1 protein (heat shock protein 90 kDA alpha, class B, chamber 1), also known as HSP90beta, is one of the members of the HSPs protein family; by binding to receptor proteins (including kinases, ubiquitin ligases, transcription factors, etc.), proper protein folding is guided and protein stability is maintained. On the other hand, HSP90AB1 has close connection with the occurrence and development of various cancers, and promotes the growth of tumors and the spread of cancers. By virtue of the variety of receptor proteins and actions, HSP90AB1 becomes a regulator which plays an important role in a variety of biological processes.

In the research so far, no report exists that the diagnostic reagent is used as an auxiliary diagnostic index for meningitis.

Disclosure of Invention

The invention provides application of HSP90AB1 protein in identification of streptococcus suis meningitis, discloses significant differential expression of the protein in meningitis type and non-meningitis type SS2 infected pig serum, and provides HSP90AB1 as an auxiliary diagnosis index of streptococcus suis meningitis.

According to the invention, the HSP90AB1 protein is expressed in serum of a sick pig after infection of meningitis type SS2, and the expression condition of the protein in a sample is detected, so that the auxiliary diagnosis can be carried out on whether the sick pig suffers from meningitis caused by streptococcus infection.

The protein antibody can be prepared to prepare an ELISA kit, the protein level of HSP90AB1 in a serum sample of a sick pig is detected, and the protein level is compared with positive and negative control values for diagnosis.

In the serum of non-meningitis type infection or healthy pigs, the HSP90AB1 protein does not show high expression, so that SS2 infection can be determined, meanwhile, meningitis can be diagnosed in an auxiliary mode when clinical meningitis symptoms are not shown, and targeted treatment can be conveniently and timely carried out.

The invention has the positive effects that:

provides a new medical application of HSP90AB1 protein, namely HSP90AB1 protein is used as a target to be detected, and SS2 meningitis is newly diagnosed in an auxiliary way according to the result; compared with the traditional method, the method is more accurate and sensitive, and the protein does not show high expression in the serum of non-meningitis type infection or healthy pigs, so that SS2 infection can be determined, meanwhile, meningitis can be assisted to diagnose when clinical meningitis symptoms are not shown, and targeted treatment can be conveniently and timely carried out; the invention is simple and convenient for detecting the protein expression level and can obtain accurate results in a short time. And the corresponding commercial reagent and kit are easy to manufacture aiming at protein detection, and can be more widely applied to the detection of sick pigs.

Drawings

FIG. 1 shows the level of HSP90AB1 of the invention in sera of each group.

Detailed Description

The present invention is further illustrated by the following examples, which do not limit the present invention in any way, and any modifications or changes that can be easily made by a person skilled in the art to the present invention will fall within the scope of the claims of the present invention without departing from the technical solution of the present invention.

Example 1

Method and device

1. Establishment of strain source and streptococcus suis type 2 infection model

The strain source is as follows: the SS2 strain was isolated from SS2 infected pigs. Strain JZLQ022 was isolated from brain tissue of a swine with meningitis; strain JZLQ001 was isolated from the mandibular lymph node of arthritic pigs.

Grouping experiments: 18 healthy Bama minipigs aged 26 days were randomly divided into three groups of 6 each, namely a meningitis group, a non-meningitis group and a healthy control group.

Preparing a model: pig ear intravenous injection of 200 μ L containing 5 × 10⁶PBS of CFUs JZLQ 022; non-meningitis group pig ear intravenous injection of 200. mu.L containing 5X 10⁶PBS of CFUs JZLQ 001; healthy control pigs were injected intravenously with 200 μ L PBS.

After infection, each group of pigs was monitored for clinical symptoms including appetite, mental state, body temperature.

2. Serum sample collection and processing

Blood was collected from the successfully established infection model and serum was isolated. The serum was added to appropriate doses of lysis buffer (containing 7M urea, 2M thiourea, 0.1% propanesulfonic acid), mixed well by vortexing, and incubated at 25 ℃ for 30 min. Then, the mixture was centrifuged at 15000g at 4 ℃ for 20min, and the supernatant was collected and dispensed into 1.5ml centrifuge tubes. And (3) carrying out proteomics analysis on the sample by utilizing an LC-MS/MS technology.

3. Proteomics data analysis

Raw data were processed by Maxquant software and Sus scrofa protein database on Uniprot Website (http:// www.uniprot.org /). Fold protein differences were determined between groups of sera based on label-free qualitative (LFQ) intensity. Proteins with fold differences of >1.5 fold, <0.66 fold, P value <0.05 were identified as proteins with meaningful differences in abundance.

4. Enzyme-linked immunosorbent assay (ELISA) verification of target protein differential expression

Serum samples were tested using an ELISA kit, the expression level of HSP90AB1 protein was tested in the exact protocol of the ELISA kit, and the expression levels of the protein in the sera of three groups of pigs were compared.

Second, result in

1. Proteomics results

Three groups of pig sera were collected for proteomics analysis and each serum sample was tested three times. 705 proteins are detected by using an LC-MS/MS technology, iBAQ and LFQ algorithm calculation is carried out on the proteins by Maxquant software, and finally 345 meaningful differential expression proteins are selected from pig serum of meningitis groups and non-meningitis groups. Among the 345 differentially expressed proteins, 283 were included, with up-regulated expression and 62 with down-regulated expression.

2. Verification of up-regulation of HSP90AB1 protein expression

HSP90AB1 is one of the 283 proteins whose expression is up-regulated in the meningitis group. The expression level of HSP90AB1 in the serum of each group was measured by enzyme-linked immunosorbent assay (ELISA), and found to have the same trend as that of proteomic results (LFQ values), and the expression level of HSP90AB1 in the serum was increased after SS2 infection in the meningitis group compared with that in the non-meningitis group (table 1).

Compared with the HSP90AB1 protein content among groups, the protein content is consistent with the up-regulation trend of the omics result, and the HSP90AB1 protein content in the serum of the meningitis group is obviously higher than that in the serum of the non-meningitis group (figure 1);

table 1: protein level trend:

M/N: the results of the analysis of the meningitis pig serum were compared to the non-meningitis pig serum.

Third, conclusion

The HSP90AB1 protein has obvious expression difference in serum of a pig suffering from meningitis and a pig suffering from non-meningitis caused by SS2 infection, has obvious up-regulated expression in the serum of the pig suffering from meningitis, and can be used as a diagnosis marker for diagnosing meningitis type streptococcal infection, so that the detection of the expression level of HSP90AB1 in the serum can be used as an auxiliary diagnosis method for meningitis.

Example 2

Method and device

1. Production of antibody-coated ELISA plate

[ solution ] Rabbit anti-porcine HSP90AB1 antibody was collected, the antibody concentration was detected by BCA, and a coating buffer (1.59 g/L Na) was used₂CO₃，2.93g/L NaHCO₃，1L ddH₂O, PH = 9.4) adjusting the antibody concentration to 7 μ g/mL, to obtain an antibody coating solution;

② adding the antibody coating solution into a blank ELISA plate at 100 mu L/hole, and incubating overnight at 4 ℃.

2. ELISA detection of porcine serum sample HSP90AB1 protein level

Taking overnight coated enzyme label plate, discarding coating liquid, adding washing buffer solution (8.0 g/L NaCl, 0.2g/L KCl, 3.58g/L Na) into 200 mu L/hole₂HPO₄，0.27g/L KH₂PO₄500 mu L/L Tween-20, 1L ddH 2O), shaking and washing for 3 times, 5min each time, and beating to dry;

② 150 mu L/hole is added with sealing liquid (prepared by 0.05g/mL skim milk), and sealed for 1h at 37 ℃;

thirdly, removing the sealing liquid and washing (the method is the same as the above);

taking 10 parts of positive serum sample of meningitis type SS2 infected pig and 10 parts of negative serum sample of non-meningitis type SS2 infected pig, adding the serum samples into an enzyme label plate at 100 mu L/hole, and incubating for 1h at 37 ℃;

fifthly, discarding serum and washing (the same method is used above);

sixthly, taking a mouse anti-porcine HSP90AB1 antibody, 1: diluting with 5000, adding 100 μ L/well of enzyme label plate, and incubating at 37 deg.C for 1 h;

seventhly, removing the antibody, and washing (the method is the same as the above);

eighthly, taking a goat anti-mouse IgG enzyme-labeled secondary antibody, 1: diluting with 5000, adding 100 μ L/well of enzyme label plate, and incubating at 37 deg.C for 1 h;

ninthly, removing the enzyme-labeled secondary antibody, and washing (the method is the same as the above);

adding TMB substrate color development solution into the red (100 μ L) per well, and incubating for 10-30min in dark;

⑪ 50 mu L/well, stop solution (2M H)₂SO₄Solution);

⑫ and reading OD value at 450nm by using an enzyme-labeling instrument within 15min after the stop solution is added.

Second, result in

The serum ELISA detection results (Table 2) show that the positive serum OD value is significantly higher than that of the negative serum, and the ratio of the positive serum OD value to the negative serum OD value is more than 2.1, so that the method has practical significance.

Table 2: ELISA method for detecting positive and negative serum HSP90AB1 protein level

Positive serum	1	2	3	4	5	6	7	8	9	10
											OD₄₅₀Value of	0.634	0.625	0.603	0.537	0.547	0.693	0.626	0.545	0.515	0.512
Negative serum	1	2	3	4	5	6	7	8	9	10
											OD₄₅₀Value of	0.185	0.202	0.104	0.124	0.113	0.111	0.132	0.142	0.19	0.166

Third, conclusion

The experiments and results show that the ELISA kit prepared by aiming at HSP90AB1 protein has obvious difference in positive and negative serum detection results, can accurately diagnose meningitis type SS2 infection, and can be used as an auxiliary diagnosis method.

Claims

1. Use of porcine HSP90AB1 protein in the preparation of a reagent for identifying meningitis associated with streptococcus suis type 2 infection.