CN111218438A

CN111218438A - Streptococcus DNase B antigen and application thereof

Info

Publication number: CN111218438A
Application number: CN201911154845.5A
Authority: CN
Inventors: 冯晓燕; 张贺秋; 王超男; 张玲; 危利
Original assignee: Oriental Ocean Beijing Medical Research Institute Co ltd
Current assignee: Oriental Ocean Beijing Medical Research Institute Co ltd
Priority date: 2019-11-22
Filing date: 2019-11-22
Publication date: 2020-06-02
Anticipated expiration: 2039-11-22
Also published as: CN111218438B

Abstract

The invention discloses a streptococcus DNase B antigen and application thereof, belonging to the technical field of genetic engineering. The amino acid sequence of the antigen is shown as SEQ ID NO.1 in a sequence table. The DNaseB antigen can be efficiently expressed and has high activity. The double-antigen sandwich method established by the antigen is used for detecting the anti-DNaseB antibody, has high sensitivity and good specificity, and is suitable for clinical examination.

Description

Streptococcus DNase B antigen and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a streptococcus DNase B antigen and application thereof.

Background

Group A streptococcus (group A streptococcus), called group A hemolytic streptococcus or streptococcus pyogenes, is one of the most important pathogens in human bacterial infection, and the infection caused by group A streptococcus is mainly acute pharyngitis and acute tonsillitis, lung infection, scarlet fever and skin soft tissue infection, and can cause systemic infection. The bacterium is also an indirect cause of allergic diseases such as rheumatic fever and acute glomerulonephritis. The incidence of serious infections caused by group a streptococci has increased significantly in recent years, and there has been a greater concern about such bacterial infections.

Streptolysin O is an exotoxin produced by group A streptococcus, can dissolve erythrocytes, has toxic effect on various cells of an organism, is one of important metabolites of group A streptococcus, can stimulate the organism to produce anti-streptolysin O, called anti-O or ASO for short, and can generate a large amount of anti-streptolysin O antibodies in blood serum after human bodies infect hemolytic streptococcus. Detection of the antistreptolysin "O" can be used as an aid in the diagnosis of allergic diseases following streptococcal infection.

Streptococcal DNase B (DNaseB), also known as streptococcal enzyme S (DB) or MF, is a secreted protein produced mainly by group A, C, G streptococci, is highly conserved in sequence, and can degrade highly viscous DNA in pus, dilute pus and promote pathogen diffusion. DNase B has strong antigenicity to human body and stimulates the body to produce corresponding antibody, namely anti-DNase B. The anti-DNase B assay is one of two experimental diagnostic methods for streptococcal infection and its complications recommended by the Jones standards.

After the organism is infected with group A streptococcus, the organism can generate a large amount of ASO and anti-DNase B antibodies, the antibody dynamics of the ASO and the anti-DNase B antibodies are similar, the difference is mainly that the ASO appears in a short time, the peak maintaining time is short, the ASO reaches a high peak value in 3-5 weeks generally, and most ASO is reduced to be normal after 2 months; and the anti-DNA enzyme B appears later, the antibody rises slowly, the peak time is maintained longer, and the maintenance time is generally as long as 3-6 months. Because of different antibody kinetics of ASO and anti-DNase B, the ASO is in a high-titer state for acute group A streptococcus infection such as acute pharyngitis, acute tonsillitis, acute rheumatic fever and the like, so the ASO is preferred; for chronic infections and autoimmune diseases caused by infections, where ASO titers have decreased to normal, anti-DNase B should be selected because it remains in a high titer. Because the clinical manifestations of the patients with rheumatic fever are mostly slight or atypical at present, in addition, the human body has non-uniform response to the group A streptococcus antigen, and about 25 percent of the patients can have negative response to a certain antigen after being infected, the ASO and anti-DNA enzyme B combined detection can improve the positive rate. Research has proved that the positive rate of the combined detection of the acute rheumatic fever and the active rheumatic heart disease is about 90 percent, which indicates that the combined detection of ASO and DNA-resistant enzyme B has important clinical value in the diagnosis of the rheumatic fever and has wide popularization significance.

At present, natural DNaseB is mostly adopted for detecting anti-DNaseB antibodies, however, the purification process of the natural DNaseB is complex and the acquisition cost is expensive. The first fermentation of GAS to obtain large amounts of culture broth containing secreted dnase b not only requires a long growth cycle, but also presents serious safety hazards. Secondly, the purification process after obtaining the culture solution is tedious and the yield is low. Third, crude natural extracts containing both known and unknown streptococcal products interfere with the test results and amplify the test data, which are biased and thus do not meet the clinical test requirements. On the other hand, the current commercial anti-DNase B antibody detection reagent mostly adopts a micro-titration method and an immunoturbidimetry method, and not only has complex operation, but also is insensitive to the change of antibody concentration.

Disclosure of Invention

In order to overcome the problems of the prior art,

the invention firstly provides a DNaseB antigen, and the amino acid sequence of the DNaseB antigen is shown as SEQ ID NO.1 in a sequence table.

Secondly, the invention also provides a nucleic acid sequence for coding the DNaseB antigen.

As for the above nucleic acid sequence, optionally, the nucleic acid sequence is shown as SEQ ID NO.2 in the sequence table.

The invention also provides an expression vector and a microorganism for expressing the DNaseB antigen.

With regard to the above microorganism, optionally, the microorganism is a bacterium, and further, the bacterium is escherichia coli.

The invention also provides the application of the antigen, the nucleic acid sequence, the expression vector or the microorganism in the preparation of a reagent for detecting streptococcus infection.

Furthermore, the invention also provides application of the antigen, the nucleic acid sequence, the expression vector or the microorganism in preparing a reagent for detecting the anti-DNaseB antibody.

The invention also provides an anti-DNaseB antibody detection kit, which comprises the DNaseB antigen.

In the kit, optionally, the kit is an anti-DNaseB antibody double-antigen sandwich detection kit.

In the kit, optionally, the kit further comprises an enzyme-linked plate for coating the DNaseB antigen, a horseradish peroxidase (HRP) -labeled DNaseB antigen, a sample diluent, a washing solution, a TMB chromogenic substrate A solution, a TMB chromogenic substrate B solution and a reaction termination solution.

In the above kit, the above reagents, optionally, are:

sample diluent: 1% BSA, 5mM EDTA, 5% goat serum, 0.1% Proclin-300, 0.1% Triton-X100, 1.78% NaCl, 0.04% thimerosal.

Washing liquid: 5.63% Na₂HPO₄·12H₂O，0.672％NaH₂PO₄·2H₂O，17％NaCl，1％Tween-20。

TMB chromogenic substrate solution a: 0.32% citric acid (monohydrate), 0.06% hydrogen peroxide solution, 2.72% NaAc.3H₂O

TMB chromogenic substrate B solution: 0.04% EDTA-Na₂0.04% TMB, 0.19% citric acid (monohydrate), 10% glycerol (glycerin).

Reaction termination solution: 2mol/L H₂SO₄。

The invention has the beneficial effects that:

the DNaseB coding gene removes rare codons which are difficult to express, analyzes the hydrophobicity of a DNaseB amino acid sequence, removes an amino acid sequence with strong hydrophobicity which influences the expression efficiency, retains most of antigen epitopes, and finally connects a plurality of antigen dominant segments together by adopting a fusion expression technology for fusion expression to obtain the high-activity DNaseB antigen with high-efficiency expression. By utilizing the antigen, the invention establishes a double-antigen sandwich method for detecting the anti-DNaseB antibody for the first time, and the method has the advantages of simple and convenient operation, high sensitivity and good specificity, and is very suitable for clinical examination.

The DNaseB genetic engineering recombinant antigen prepared by the method has high yield, high purity, good activity, no safety risk, simple preparation method, low cost, obvious advantage over natural antigen obtained by an extraction method, and completely meets the requirement of developing an anti-DNaseB antibody immunoassay reagent.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the expressed DNase B antigens of the present application, wherein the various reference numerals are: M-Marker; 1-whole bacterium; 2-inclusion bodies; 3-purifying the antigen.

FIG. 2 shows the results of comparison of expression rates before and after antigen optimization.

FIG. 3 shows the purity analysis of the DNase B fusion dominant epitope antigen after purification.

FIG. 4 shows the identification of the activity of dominant epitope antigen of DNase B fusion of the present application, wherein each reference number is: 1-non-specific antibody; 2-anti-DNase B specific antibody.

Detailed Description

The invention is further described with reference to the following figures and specific examples. The biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art, unless otherwise specified.

Example 1 preparation of the DNaseB fusion dominant epitope antigens of the present application

First, DNase B fusion dominant epitope antigen amino acid sequence and coding sequence

The original DNaseB (streptococcal DNase B) in Genebank has 271 amino acids in total length, contains a leader peptide of 43aa and 228 amino acids of a mature protein, the sequence of the mature protein is shown as SEQ ID NO.3 in a sequence table, and the molecular weight is about 25.4 kD. The DNaseB antigen (also called DNase B fusion dominant epitope antigen, which is different from the original DNaseB) is subjected to gene optimization and epitope screening, and the amino acid sequence of the DNaseB antigen is shown as SEQ ID No.1 in a sequence table. The optimized gene sequence for coding the DNaseB antigen is shown as SEQ ID NO.2 in the sequence table.

Expression and purification of DNaseB gene engineering fusion antigen

According to the sequence, a coding gene of DNaseB antigen (shown as SEQ ID NO.2 in a sequence table) is synthesized by Zhongmeitai and biotechnology (Beijing) Limited, and the coding gene of DNaseB antigen is inserted into pBVIL1 plasmid which is subjected to double enzyme digestion by XhoI and XbaI by adopting XhoI and XbaI enzyme digestion sites through double enzyme digestion to obtain the PBVIL1/B (1-100) + (140-228) recombinant plasmid.

The recombinant expression plasmid with correct sequencing is transformed into HB101 competent cells, a single colony is selected in 3ml LB liquid culture medium containing ampicillin sodium, shaking culture is carried out at 37 ℃ overnight, the next day is inoculated in a fresh LB liquid culture medium and cultured to a logarithmic phase, and then induction is carried out at 42 ℃ overnight. Extracting the inclusion body by an ultrasonic disruption method, purifying the protein by adopting an affinity chromatography Ni column, eluting by using 25mM Tris-HCl (pH 8.5) solution containing 25mM imidazole and 250mM imidazole, respectively collecting protein peaks, carrying out electrophoretic analysis on purified products, and taking 25mM imidazole elution protein as a purified DNase B fusion dominant epitope antigen, wherein the DNase B fusion dominant epitope antigen is shown in figure 1.

The original mature DNase B antigen which is not optimized and fused is constructed according to the steps, the coding gene sequence of the antigen is shown as SEQ ID NO.4 in the sequence table, the obtained recombinant expression plasmid is PBVIL1/B (1-228), and no obvious antigen expression band is found after SDS-PAGE electrophoretic identification after induction. And scanning and measuring an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretogram by using an electrophoretic gel digital detection system, and calculating the ratio of the target protein to all mycoprotein, namely the expression rate according to the gray value of each band. The result shows that the expression rate of the non-optimized full-length DNase B antigen is only 1.73%, while the expression rate of the DNase B fusion dominant epitope antigen subjected to gene optimization and epitope screening is as high as 58.6%, as shown in FIG. 2, so that the DNase B antigen subjected to gene optimization and epitope screening can be judged to be efficiently expressed.

Then using Gel-Pro^RAnalyzer Version 3.0for Windows^TMThe software analyzed that the purity of the purified DNase B fusion dominant epitope antigen was 98.126%, and the results are shown in FIG. 3. The original mature DNase B antigen which is not optimized has very low expression level, and subsequent purification preparation cannot be carried out.

Example 2 Activity identification of DNaseB Gene engineering fusion antigens

And (3) performing 10% SDS-PAGE electrophoresis on the purified DNase B fusion dominant epitope antigen according to a conventional method, transferring a membrane, and sealing 5% skimmed milk powder at room temperature for 1 h. The specific anti-DNase B antibody and the non-specific antibody (anti-actin antibody) diluted with blocking solution, respectively, were incubated overnight at 4 ℃. After washing the membrane 3 times at room temperature with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary antibody for 1 h. TBST was washed 3 times at room temperature, positive bands were visualized by ECL, scanned and photographed. The results are shown in fig. 4, and the obtained DNase B fusion dominant epitope antigen can be well recognized by specific anti-DNase B antibody, but not by non-specific antibody, which indicates that the obtained DNase B fusion dominant epitope antigen has high antigenic activity.

Example 3 establishment of double-antigen Sandwich detection kit for anti-DNaseB antibody

Double-antigen sandwich detection kit for anti-DNaseB antibody

The double-antigen sandwich detection kit for the DNaseB antibody mainly comprises:

the kit comprises an enzyme linked plate coated with DNase B fusion dominant epitope antigen, horseradish peroxidase (HRP) labeled DNase B fusion dominant epitope antigen, sample diluent, washing liquid, TMB chromogenic substrate A liquid, TMB chromogenic substrate B liquid and reaction termination liquid.

Reaction termination solution: 2mol/L H₂SO₄。

The preparation method of the enzyme-linked plate coated with the DNase B fusion dominant epitope antigen comprises the following steps:

diluting DNase B fusion dominant epitope antigen with carbonate coating buffer solution (pH 9.6) to the concentration of 2.5 μ g/ml, coating each well with 100 μ l, and standing overnight at 4 deg.C; washing the plate with washing solution 2 times at 200. mu.l/well; add 110. mu.l/well blocking solution (0.01mol/mLPBS, 1% BSA, pH 7.2) and block for 6 hours at room temperature; washing the plate 5 times with 200. mu.l/well of washing solution; vacuum drying for 4-6 hr, packaging with packaging bag to coat enzyme linked plate of DNase B fusion dominant epitope antigen, and storing at 2-8 deg.C

The method for labeling the DNase B fusion dominant epitope antigen by using horseradish peroxidase (HRP) comprises the following steps:

5mg of HRP is weighed by an analytical balance and dissolved in 1ml of distilled water to be magnetically stirred under the condition of keeping out of the sun; adjusting the concentration of the DNase B fusion dominant epitope antigen to 5mg/ml by using a carbonate buffer solution for later use; 0.5ml of 0.06M NaIO4 aqueous solution is prepared; draw 1ml of NaIO₄Slowly dripping the aqueous solution into the HRP solution, stirring in the dark, standing in a refrigerator at 4 ℃ for 30min, and standing at room temperature for 30 min; preparing 0.16M ethylene glycol, slowly dripping 0.5ml of ethylene glycol, stirring at room temperature, and dialyzing overnight at 4 ℃ in 0.05M carbonate buffer solution; preparing NaBH4 solution with the concentration of 5mg/ml, adding 200 mul of the NaBH4 solution into the mixed solution, uniformly mixing, and standing in a refrigerator at 4 ℃ for 2 hours; finally, adding equal volume of 50% glycerol, and storing at-20 ℃ for later use.

The using method of the kit comprises the following steps:

adding 100 μ l sample diluent into each well, adding 10 μ l serum to be detected, washing the plate with washing solution for 5 times, discarding the solution at 37 deg.C for 30min, patting, adding HRFusing the P-labeled DNase B with dominant epitope antigen, and incubating for 30min at room temperature; washing the plate 5 times with 200. mu.l/well of washing solution; adding a freshly prepared TMB chromogenic substrate solution (mixing a TMB chromogenic substrate solution A and a TMB chromogenic substrate solution B in a ratio of 1: 1), incubating for 10 minutes at 100 mu l/hole in a dark place at 37 ℃; add 50. mu.l/well 2M H₂SO₄The reaction was terminated, and the absorbance of each well was measured by a microplate reader at a dual wavelength of 450nm, and the results are shown in Table 1.

The cutoff value (cutoff value) was calculated from the measured value of the healthy control serum sample, and the formula was calculated as cutoff value +3 standard deviation from the average value, and the cutoff value was taken to be 0.162. Counting results, wherein the anti-DNaseB antibody detection of the serum samples of 20 patients with positive group A streptococcus infection nucleic acid detection is positive, and the sensitivity is 100%; of the 87 healthy control serum samples, 85 were negative and 97.7% specific, with the results shown in Table 1.

TABLE 1 detection of anti-DNaseB antibodies (OD) in human serum samples by double antigen sandwich method_450nm)

The double-antigen sandwich detection method for the DNaseB antibody is established for the first time, no relevant report is found at home and abroad, and the technology has very high sensitivity and specificity and is suitable for clinical diagnosis requirements.

Sequence listing

<110> eastern ocean (Beijing) medical research institute Co., Ltd

<120> streptococcal DNase B antigen and application thereof

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Ala Gly Asp Ile Leu Tyr Ser Lys Leu Asp Glu Leu Gly Arg Thr Arg

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Gly Tyr Thr Ile Asn Tyr His Asn Gly Thr Pro Thr Gln Lys

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attactccag cactctttcc aaaagcaggt gatattctct atagcaaatt agatgagtta 180

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agtcatctca ttgcagatag tctcggtgga gatgcactca gagtcaatgc cgttacagga 420

acacgtaccc aaaatgtagg aggtcgtgac caaaaaggcg gcatgcgcta taccgaacaa 480

agagctcaag aatggttaga agcaaatcgt gatggctatc tttattatga agtcgctcca 540

atctacaacg cagacgagtt gattccaaga gctgtcgtgg tatcaatgca atcttctgat 600

aataccatca acgagaaagt attagtttac aacacagcta atggctacac cattaactac 660

cataacggta cacctactca aaaa 684

Claims

1. The amino acid sequence of the DNaseB antigen is shown as SEQ ID NO.1 in a sequence table.

2. A nucleic acid sequence encoding the dnase b antigen of claim 1.

3. The nucleic acid sequence of claim 2, wherein the nucleic acid sequence is as shown in SEQ ID No.2 of the sequence Listing.

4. An expression vector expressing the dnase b antigen of claim 1.

5. A microorganism expressing the dnase b antigen of claim 1.

6. The microorganism according to claim 5, wherein the microorganism is a bacterium, and further wherein the bacterium is Escherichia coli.

7. Use of the antigen of claim 1, the nucleic acid sequence of claims 2 to 3, the expression vector of claim 4 or the microorganism of claims 5 to 6 for the preparation of a reagent for the detection of streptococcal infections.

8. Use of the antigen of claim 1, the nucleic acid sequence of claims 2-3, the expression vector of claim 4 or the microorganism of claims 5-6 for the preparation of a reagent for the detection of anti-dnase b antibodies.

9. An anti-DNaseB antibody detection kit comprising the DNaseB antigen of claim 1.

10. The kit of claim 9, wherein the kit is an anti-dnase b antibody double antigen sandwich assay kit.