CN116027033B - A method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles - Google Patents

Abstract

The present invention is applicable to the field of immunology technology, and provides a method for detecting bacteria and viral antigens and antibodies based on bacteria-like particles. The present invention uses bacterial protein (BP) or viral protein (VP) as a detection target, and uses bacteria-like particles (BLP) as a solid phase carrier. The detection content includes: fixing the recombinantly expressed antigen BP (VP)-PA through the non-covalent binding between BLP and an anchor protein (PA), generating a BLP/BP (VP)-PA antigen complex, and detecting the IgG titer of anti-BP (VP) in serum; fixing the recombinantly expressed single-chain antibody scFV-BP (VP)-PA through the non-covalent binding between BLP and an anchor protein (PA), generating a BLP/scFV-BP (VP)-PA complex, and detecting the content of BP (VP) protein antigen. The present invention realizes rapid detection, reduces the effect of detection cost, and can improve detection sensitivity.

Description

A method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles

Technical Field

The invention belongs to the technical field of immunology, and in particular relates to a method for detecting bacterial and viral antigens and antibodies based on bacterial-like particles.

Background technique

Bacteria and viruses are the main pathogens of various infectious diseases in humans and animals. The infectious diseases they cause pose a serious threat to human health. Therefore, the rapid diagnosis and identification of these infectious diseases is particularly important to prevent their spread and prevention. The diagnosis of these pathogens mainly includes antigen diagnosis and antibody diagnosis. In principle, these detection methods are mainly PCR to detect their nucleic acids and ELISA to detect their antigens and antibodies. These detection methods often have their own shortcomings, such as: ELISA method requires blocking and multiple washing, which is time-consuming and labor-intensive, and the steps are relatively cumbersome. It also requires 96-well plates and is expensive. These shortcomings need to be improved. For this reason, we propose a method for detecting bacterial and viral antigens and antibodies based on bacterial-like particles.

Summary of the invention

The purpose of the present invention is to provide a method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles, aiming to solve the problems raised in the above background technology.

To achieve the above object, the present invention provides the following technical solutions:

A method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles, comprising:

a. The specific operation of detecting the titer of pneumococcal antibody is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant antigen PspA-PA is combined to form a BLP/PspA-PA antigen complex, and this antigen complex is used to bind the anti-PspA IgG in the serum to be tested, and then the enzyme-labeled secondary antibody is used for quantitative detection;

b. The specific operation of detecting pneumococcal antigen is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant single-chain antibody scFV-PspA-PA is combined to form a BLP/scFV-PspA-PA single-chain antibody complex, and the single-chain antibody complex is used to bind to the antigen PspA or pneumococcal cells in the sample to be tested, and then the antigen PspA or pneumococcal cells are quantitatively detected using anti-PspA IgG and enzyme-labeled secondary antibodies;

c. The specific operation of detecting the antibody titer of the new coronavirus SARS-CoV-2 is: using BLP as a carrier to bind the prokaryotic expressed recombinant antigen S-PA to form a BLP/S-PA antigen complex, and then using this antigen complex to bind to the anti-S protein IgG in the serum to be tested, and then using an enzyme-labeled secondary antibody to quantitatively detect it;

d. The specific operation of detecting the novel coronavirus SARS-CoV-2 antigen is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant single-chain antibody scFV-S-PA is combined to form a BLP/scFV-S-PA single-chain antibody complex, and the single-chain antibody particles are used to bind to the antigen S protein or virus particles in the sample to be tested, and then the anti-S protein IgG and enzyme-labeled secondary antibody are used to quantitatively detect the antigen S protein or virus particles.

Furthermore, the preparation method of the BLP is: heat-treating the cultured Lactococcus lactis with trichloroacetic acid, removing impurities by washing and purification, and leaving a lifeless peptidoglycan shell, namely the BLP.

Furthermore, the preparation method of the PspA-PA recombinant antigen is as follows: a partial sequence (α-helical region and proline-rich region) of PspA family 2 subclass 4 is selected, and an anchor protein PA is fused and expressed at the C-terminus of its proline-rich region, the gene sequence is chemically synthesized, the sequence is optimized before synthesis, codons preferred by Escherichia coli are selected, and it is highly expressed in Escherichia coli, and then it is cloned and constructed on a pET20b prokaryotic expression vector, transformed into Escherichia coli BL21 for expression, the protein expression conditions are optimized, and the candidate protein is induced to express, and the target protein PspA-PA is obtained by a His Trap affinity purification column.

Furthermore, the preparation method of the BLP/PspA-PA antigen complex is as follows: take frozen BLP and PspA-PA proteins, mix them thoroughly after thawing, add 10 mg BLP to a protein solution containing 50 mg PspA-PA, shake and combine at room temperature for 1 hour, centrifuge at 8000 rpm for 20 minutes, recover the combined supernatant, absorb 6.5 mL of sterile PBS solution to resuspend the precipitated sample, and take samples for SDS-PAGE electrophoresis detection.

Furthermore, the preparation method of the S-PA recombinant antigen is as follows: the S protein antigen of SARS-CoV-2 is a partial sequence of the S protein, which is expressed and prepared in a prokaryotic system through genetic engineering technology and used as the target antigen for content determination.

Furthermore, the preparation method of the scFV-S-PA single-chain antibody is as follows: a monoclonal antibody against S protein is selected as a parent, and its heavy chain variable region is used as the N-terminus and the light chain variable region is used as the C-terminus, or its light chain variable region is used as the N-terminus and the heavy chain variable region is used as the C-terminus, and they are connected by a connecting peptide, and the anchor protein PA is fused and expressed at the C-terminus of the single-chain antibody, and the gene sequence is chemically synthesized, and the sequence is optimized before synthesis, and the codons preferred by Escherichia coli are selected to make it highly expressed in Escherichia coli, and then it is cloned and constructed on the pET20b prokaryotic expression vector, transformed into Escherichia coli BL21 for expression, the protein expression conditions are optimized, and the candidate protein is induced to express, and the target protein scFV-S-PA is obtained by a His Trap affinity purification column.

Furthermore, the preparation method of the BLP/scFV-S-PA single-chain antibody complex is as follows: 10 mg of BLP is added to a protein solution containing 50 mg of scFV-S-PA, and after shaking and binding at room temperature for 1 hour, centrifugation is performed at 8000 rpm for 20 minutes, the binding supernatant is recovered, 6.5 mL of sterile PBS solution is aspirated to resuspend the precipitated sample, and samples are taken for SDS-PAGE electrophoresis detection.

Furthermore, the optimal working concentration of the BLP/scFV-S-PA solid phase method for antigen detection is: scFV-S-PA dosage is 1 μg, and the anti-S protein serum concentration is 1:100.

Compared with the prior art, the present invention has the following beneficial effects:

This method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles does not require blocking, requires fewer washes after antibody incubation, has a shorter incubation time, simplifies the detection steps, and does not require 96-well plates, greatly reducing detection costs while ensuring indicators such as accuracy. This method has an enrichment effect on lower concentrations of antigens and antibodies, which can improve detection sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the SDS-PAGE of the PspA-PA protein induced expression in E. coli in the present invention (Lane 1: pre-stained protein marker; Lane 2: induced expression of PspA-PA protein in the E. coli system; Lane 3: protein expression in the E. coli system before induction).

Figure 2 is a Western-blot verification of the expression of PspA-PA protein in the present invention (primary antibody is anti-PspA4 polyclonal antibody; Lane 1: pre-stained protein marker; Lane 2: induced expression of PspA-PA protein in the E. coli system; Lane 3: protein expression in the E. coli system before induction).

FIG3 is an SDS-PAGE identification of BLP in the present invention (Lane 1: pre-stained protein marker; Lane 2: whole bacteria of Lactococcus lactis; Lane 3: Lactococcus lactis after acid treatment (BLP)).

FIG. 4 is a standard curve of OD600 light absorption of BLP standard in the present invention.

FIG. 5 shows the preparation and quantification of BLPs/PspA-PA in the present invention.

Figure 6 shows the effect of BLP/PspA-PA dosage on antibody titer detection in the present invention (BLP/PspA-PA dosage: (A) 38.6 μg/mL, (B) 77.2 μg/mL, (C) 154.4 μg/mL).

FIG. 7 shows the effect of blocking on antibody titer detection in the present invention ((A) using 3% bovine serum albumin for blocking, 200uL/tube, (B) not blocking).

FIG8 shows the effect of the number of PBS-T washes on antibody titer detection after incubation with multiple anti-serum in the present invention (number of washes: (A) 1, (B) 2, (C) 3).

FIG9 shows the effect of the number of PBS-T washes on antibody titer detection after incubation with enzyme-labeled secondary antibodies in the present invention (number of washes: (A) 1, (B) 2, (C) 3).

Figure 10 is the SDS-PAGE and Western Blot identification of the S protein induced to express by Escherichia coli in the present invention (primary antibody is anti-His tag monoclonal antibody; left Lane 1: before induction; right Lane 1: positive control; left Lane 2: after induction; right Lane 2: before induction; left Lane 3: supernatant after ultrasound; right Lane 3: after induction; left Lane 4: broken precipitate; right Lane 4: supernatant after ultrasound; Lane 5: broken precipitate).

Figure 11 is the SDS-PAGE and Western Blot identification of the scFV-S-PA protein induced by Escherichia coli in the present invention (primary antibody is anti-His tag monoclonal antibody; Lane 1: pre-stained protein marker; Lane 2: induced expression of PspA-PA protein in the Escherichia coli system; Lane 3: expression of protein in the Escherichia coli system before induction).

Figure 12 shows the preparation of the BLP/scFV-S-PA solid phase antibody complex of the present invention (Lane 1: BLP; Lane 2: scFV-S-PA; Lane 3: supernatant of BLP and scFV-S-PA binding; Lane 4: BLP/scFV-S-PA solid phase antibody).

FIG. 13 is a standard curve diagram of BSA standard concentration and gray value of the present invention.

Figure 14 shows the SDS-PAGE and Western Blot identification of BLP/scFV-S-PA capturing S protein in the present invention (Lane 1: positive control; Lane 2: BLP; Lane 3: scFV-S-PA; Lane 4: S; Lane 5: S-PA; Lane 6: BLP/scFV-S-PA; Lane 7: BLP/S; Lane 8: BLP/S-PA; Lane 9: BLP/scFV-S-PA+S; the primary antibody in Western Blot identification is an anti-His tag monoclonal antibody).

Figure 15 is a graph showing the standard curve of antigen detection by the BLP/scFV-S-PA solid phase method of the present invention (the plating amount of scFV-S-PA in groups A and B is 0.25 μg/well, the dilution factor of the polyclonal antibody serum is 1:100; the initial antigen concentration in group A is 66667 ng/mL, and the dilution is three-fold; the initial antigen concentration in group B is 100000 ng/mL, and the dilution is four-fold).

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

An embodiment of the present invention provides a method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles, comprising:

a. The specific operation of detecting the titer of pneumococcal antibody is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant antigen PspA-PA is combined to form a BLP/PspA-PA antigen complex, and this antigen complex is used to bind the anti-PspA IgG in the serum to be tested, and then the enzyme-labeled secondary antibody is used for quantitative detection;

b. The specific operation of detecting pneumococcal antigen is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant single-chain antibody scFV-PspA-PA is combined to form a BLP/scFV-PspA-PA single-chain antibody complex, and the single-chain antibody complex is used to bind to the antigen PspA or pneumococcal cells in the sample to be tested, and then the antigen PspA or pneumococcal cells are quantitatively detected using anti-PspA IgG and enzyme-labeled secondary antibodies;

c. The specific operation of detecting the antibody titer of the new coronavirus SARS-CoV-2 is: using BLP as a carrier to bind the prokaryotic expressed recombinant antigen S-PA to form a BLP/S-PA antigen complex, and then using this antigen complex to bind to the anti-S protein IgG in the serum to be tested, and then using an enzyme-labeled secondary antibody to quantitatively detect it;

d. The specific operation of detecting the novel coronavirus SARS-CoV-2 antigen is as follows: by using BLP as a carrier, the prokaryotic expressed recombinant single-chain antibody scFV-S-PA is combined to form a BLP/scFV-S-PA single-chain antibody complex, and the single-chain antibody particles are used to bind to the antigen S protein or virus particles in the sample to be tested, and then the anti-S protein IgG and enzyme-labeled secondary antibody are used to quantitatively detect the antigen S protein or virus particles.

In an embodiment of the present invention, the PspA protein and the S protein can be full sequences, truncated sequences or combinations thereof. Gram-positive bacteria (such as Lactococcus lactis) are acidolyzed in a high temperature environment to degrade their own nucleic acids and proteins, leaving a lifeless and loose peptidoglycan shell, which is called a bacterial-like particle (abbreviated as BLP) because it still retains the original particle form of the bacteria. BLP can connect antigens to BLP through a unique protein connector (PA). PA is the peptidoglycan binding region at the C-terminus of the bacterial cell wall hydrolase AcmA, and AcmA has a strong binding ability to the peptidoglycan structure of Lactococcus lactis. Antigens are fused and expressed with PA by a genetic recombination method, and then mixed with BLP, so that antigens are easily bound to BLP. The combination of PA and BLP is very stable and is not easy to separate after the combination.

Streptococcus pneumoniae is a Gram-positive bacterium with capsules. It usually colonizes the oral cavity and nasopharynx of healthy people and is one of the normal flora. When the human body's immunity is low, local infiltration of Streptococcus pneumoniae causes sinusitis and otitis media, and aspiration into the lungs causes pneumonia. It can also invade the body and cause diseases such as bacteremia and meningitis. There are many kinds of Streptococcus pneumoniae's own proteins, and Streptococcus pneumoniae surface protein A (Pneumococcal surface protein A, PspA) is one of them. PspA is a cholesterol-binding protein distributed on the surface of Streptococcus pneumoniae. It is a key virulence factor of Streptococcus pneumoniae and a protein necessary for survival. So far, the expression of PspA can be detected in all clinically isolated Streptococcus pneumoniae strains. PspA plays a key role in the interaction between Streptococcus pneumoniae and the host. PspA protein molecules are not completely conserved in all Streptococci pneumoniae, and there is structural variability, with molecular weights ranging from 67-100KD. The PspA molecule is divided into five regions: signal peptide region, α-helix region, proline-rich region, choline binding region and C-terminal 17 amino acid tail. Gene and protein map studies show that the key cross-protective antigenic epitope is located in the α-helix region near the proline-rich region, which is about 100 amino acids in length, known as the clade defining region (CDR). According to the difference in CDR region, PspA is divided into three families (family 1, family 2 and family 3) and six subclasses (Clade1, Clade2, Clade3, Clade4, Clade5 and Clade6), and the six subclasses belong to three families respectively; Clade1 and Clade2 belong to family 1, Clade3, Clade4 and Clade5 belong to family 2, and Clade6 belongs to family 3. The gene sequence similarity of CDR region between subclasses within each family is greater than 80%; the gene sequence similarity of CDR region between families is greater than 50%. Among the three families, the prevalence of family 1 and family 2 is greater than 95%, while family 3 rarely appears. Despite the variability in the structure and antigenicity of PspA, antibodies generated against PspA are highly cross-reactive and cross-protective.

The new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2) was named SARS-CoV-2 by the World Health Organization. It is a newly discovered member of the genus Betacoronavirus and is currently spreading around the world, posing a serious global health threat. SARS-CoV-2 has four main structural proteins: Spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N). Among them, the Spike protein is the most important protein involved in infection and immunity among the four structural proteins. The S protein can be divided into multiple functional domains. First, the globular head contains the N-terminal receptor binding domain (RBD) of the S1 region. Secondly, the stem contains the C-terminal membrane fusion domain S2 region, and the end is two heptad regions, namely the transmembrane domain and the cytoplasmic tail. The S1 region is encoded by approximately 200 amino acids, and the S1 region binds to human angiotensin converting enzyme 2 (ACE2) to mediate the virus's entry into cells. Therefore, the subunit vaccines currently under research all use the S protein as the main immunogen, and it is also the main target of antigen and antibody testing.

As a preferred embodiment of the present invention, the preparation method of BLP is: heat-treating cultured Lactococcus lactis with trichloroacetic acid, removing impurities by washing and purification, and leaving a lifeless peptidoglycan shell, namely BLP.

In the embodiment of the present invention, the quantitative operation of BLP is as follows: take an appropriate amount of BLP and freeze-dry it, and after completely removing the water, dilute the freeze-dried sample to 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, and 1 mg/mL, respectively measure the light absorption value of BLP suspensions of different concentrations at 600nm, and perform linear regression with concentration-absorbance value, and the results are shown in Figures 3-4. After mixing the prepared BLP, dilute it 10 times, measure the OD600 to be 0.6, and substitute it into the linear regression equation y=0.6488x+0.0324, and the BLP concentration is 118.3 mg/mL.

As a preferred embodiment of the present invention, the preparation method of the PspA-PA recombinant antigen is as follows (partial sequence of PspA4 protein): a partial sequence of PspA family 2 subclass 4 (α-helical region and proline-rich region) is selected, and the anchor protein PA is fused and expressed at the C-terminus of its proline-rich region, the gene sequence is chemically synthesized, the sequence is optimized before synthesis, and the codons preferred by Escherichia coli are selected to make it highly expressed in Escherichia coli, and then it is cloned and constructed on the pET20b prokaryotic expression vector (named pET20b-PspA-PA), transformed into Escherichia coli BL21 for expression, the expression conditions of the protein are optimized, and the candidate protein is induced to express, and the target protein PspA-PA is obtained by His Trap affinity purification column.

In the present embodiment, SDS-PAGE and Western Blot methods were used for identification. The results are shown in Figures 1-2. Animal immunization: Female Bal/c mice were subcutaneously immunized with the expressed and purified PspA-PA protein for a total of four immunizations, with an interval of one week between each immunization. Blood was collected from mice before immunization and one week after each round of immunization to prepare serum against PspA-PA protein.

As a preferred embodiment of the present invention, the preparation method of the BLP/PspA-PA antigen complex is as follows: take frozen BLP and PspA-PA proteins, mix them thoroughly after thawing, add 10 mg BLP to a protein solution containing 50 mg PspA-PA, shake and combine at room temperature for 1 hour, centrifuge at 8000 rpm for 20 minutes, recover the combined supernatant, absorb 6.5 mL of sterile PBS solution to resuspend the precipitated sample, and take samples for SDS-PAGE electrophoresis detection.

In the embodiment of the present invention, the quantification of BLP and PspA-PA in the solid phase BLP/PspA-PA antigen complex: dilute the BLP concentration to 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, respectively measure the light absorption value of the BLP suspension at different concentrations at 600 nm, and perform linear regression with the concentration-absorbance value, dilute the BLP/PspA-PA conjugate 10 times, measure the light absorption value at 600 nm, substitute it into the standard curve, and calculate the concentration of BLP. Grayscale analysis quantification of PspA-PA: dilute the BSA standard to 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, 1 mg/mL, make an equal volume of electrophoresis sample, and perform SDS-PAGE electrophoresis with BLP/PspA-PA, and the sample amount is 10uL/well. After electrophoresis, the gel was destained and the grayscale analysis was performed on the polyacrylamide gel with clearly visible bands. The gel was processed using Image Studio V2.1 software, and the BSA concentration and grayscale value were used as the standard curve to quantify the PspA-PA in the BLP/PspA-PA conjugate. The results are shown in Figure 5.

Investigation of BLP/PspA-PA antigen dosage: BLP/PspA-PA and BLP suspension were diluted with PBS solution to three gradients of (A) 38.6μg/mL, (B) 77.2μg/mL, and (C) 154.4μg/mL, 100uL/EP tube. An irrelevant protein was used as the negative control group, and the results of the indirect ELISA method were used as the positive control. The serum and negative serum of mice immunized four times with PspA-PA protein were serially diluted 10 times to 106 using PBS, and the serially diluted serum was added to each of the above tubes, 100uL/EP tube, vortexed to resuspend the BLP/PspA-PA particles, and incubated at room temperature for 10 minutes. Use PBS-T for the "wash-resuspend-centrifugation" process, and wash 3 times in total. Centrifuge at 8000rpm for 10 minutes and discard the supernatant. Add 1:10000 dilution of goat anti-mouse IgG/HRP labeled antibody to each tube, 100uL/tube, vortex to resuspend BLP/PspA-PA microparticles, and incubate at room temperature for 10min. Use PBS-T to perform the "wash-resuspend-centrifugation" process, and wash 3 times in total. Centrifuge at 8000rpm for 10min, discard the secondary antibody, and use PBS-T to perform the "wash-resuspend-centrifugation" process, and wash 3 times in total. Add TMB substrate to the tube, 100uL/tube, vortex to resuspend BLP/PspA-PA microparticles, shake and react in the dark for 10min, add 2M sulfuric acid to the tube to terminate the reaction, centrifuge at 8000rpm for 10min, aspirate the supernatant into a polystyrene plate, and measure OD450. The results are shown in Figure 6. It can be seen from the results that the concentration of BLP/PspA-PA is greater than 77.2μg/mL, which is excessive, consistent with the positive control, and meets the determination requirements.

The effect of blocking or not on the titer of the detected antibody: In the exploration of the dosage of BLP/PspA-PA antigen, before adding the serum and negative serum of mice immunized four times with PspA-PA protein, (A) 3% bovine serum albumin was used for blocking, 200uL/tube; (B) no blocking was performed. The other steps were the same as above. The results are shown in Figure 7. It can be seen from the results that the test results of blocking and not blocking are consistent and have no effect on the test results. Therefore, this method does not require blocking.

Effect of PBS-T washing times on the titer of the detection antibody: In the exploration of BLP/PspA-PA antigen dosage, after adding serum and secondary antibody, they were washed once, twice, and three times, respectively. The other steps were the same as above. The results are shown in Figures 8-9. It can be seen from the results that after adding serum and secondary antibody, the test results were the same after washing once to three times. Therefore, this method only needs to be washed once, and multiple repeated washings are not required.

Repeatability verification: PspA-PA protein antiserum and PBS serum from PspA-PA protein immunized mice were selected for repeatability verification. The BLP/PspA-PA complex was diluted to 77.2μg/mL with PBS solution, 100uL/tube, and the other detection steps were the same as step 5 above. The results are shown in Table 1. The results show that this method has good repeatability in detecting antiserum from PspA-PA protein immunized mice and is stable.

Table 1 Results of repeatability verification of serum titer of BLP/PspA-PA detection

As a preferred embodiment of the present invention, the preparation method of the S-PA recombinant antigen is as follows: the S protein antigen of SARS-CoV-2 is a partial sequence of the S protein, which is expressed and prepared in a prokaryotic system through genetic engineering technology as the target antigen for content determination.

As a preferred embodiment of the present invention, the preparation method of the scFV-S-PA single-chain antibody is as follows: a monoclonal antibody against S protein is selected as the parent, and its heavy chain variable region is used as the N-terminus and the light chain variable region is used as the C-terminus, or its light chain variable region is used as the N-terminus and the heavy chain variable region is used as the C-terminus, and they are connected by a connecting peptide, and the anchor protein PA is fused and expressed at the C-terminus of the single-chain antibody, and the gene sequence is chemically synthesized, and the sequence is optimized before synthesis, and the codons preferred by Escherichia coli are selected to make it highly expressed in Escherichia coli, and then it is cloned and constructed on the pET20b prokaryotic expression vector (named pET20b-scFV-S-PA), and transformed into Escherichia coli BL21 for expression, the protein expression conditions are optimized, and the candidate protein is induced to express, and the target protein scFV-S-PA is obtained by His Trap affinity purification column.

In the present embodiment, SDS-PAGE and Western Blot were used for identification. The results are shown in Figures 10-11.

As a preferred embodiment of the present invention, the preparation method of the BLP/scFV-S-PA single-chain antibody complex is as follows: 10 mg of BLP is added to a protein solution containing 50 mg of scFV-S-PA, and the mixture is shaken and combined at room temperature for 1 hour, and then centrifuged at 8000 rpm for 20 minutes, the combined supernatant is recovered, 6.5 mL of sterile PBS solution is aspirated to resuspend the precipitated sample, and samples are taken for SDS-PAGE electrophoresis detection.

In the embodiment of the present invention, the electrophoresis detection result is shown in Figure 12. Lane 2 is a scFV-S-PA protein solution with a total concentration of 0.25 mg/mL, Lane 3 is a recovered binding supernatant, and its protein concentration is measured to be 0.18 mg/mL, with a recovery rate of 72%, and Lane 4 is the prepared BLP/scFV-S-PA. It can be seen that scFV-S-PA has been captured by BLP. The BLP/scFV-S-PA solid phase antibody has been successfully prepared, and the BSA standard is serially diluted to prepare an electrophoresis sample of equal volume, which is electrophoresed together with the BLP/scFV-S-PA conjugate. The picture is processed by Image Studio V2.1 software, and the BSA concentration and the gray value of the protein band are used as a standard curve. The result is shown in Figure 13. The signal value of the sample to be tested is 18176.054. Substituting it into the standard curve, it is obtained that the concentration of the BLP/scFV-S-PA conjugate is 54μg scFV-S-PA/mg BLP.

Functional identification of BLP/scFV-S-PA binding to S protein: In order to demonstrate the function of BLP/scFV-S-PA solid phase antibody in capturing S protein, SDS-PAGE and Western Blot were used for characterization. The results are shown in Figure 14. In Lane 7, BLP could not bind directly to S protein. However, after BLP/scFV-S-PA in Lane 6 bound to S protein, the precipitate sample was taken for electrophoresis. A 27kD protein band appeared in Lane 9. Western Blot identification showed that this band was S protein, which showed that BLP could not bind directly to S protein, but BLP/scFV-S-PA solid phase antibody could capture free S protein in solution through the specific binding ability between scFV-S-PA and S protein. This provided a basis for the subsequent detection of recombinant antigen S by BLP/scFV-S-PA solid phase antibody.

As a preferred embodiment of the present invention, the optimal working concentration of the BLP/scFV-S-PA solid phase method for detecting antigens is: the amount of scFV-S-PA is 1 μg, and the anti-S protein serum concentration is 1:100.

In the embodiment of the present invention, the specific operation for determining the optimal working concentration is as follows: the BLP/scFV-S-PA conjugate is fully mixed, PBS is used as a diluent, the BLP/scFV-S-PA conjugate is diluted to a gradient of 8 μg/tube, 4 μg/tube, 2 μg/tube, 1 μg/tube, 0.5 μg/tube, 0.25/tube, centrifuged at 8000rpm for 20min, the supernatant is discarded, no blocking is required, 40 μg of S protein is added, 40 μg of irrelevant protein is added to the negative control group, vortexed to resuspend the BLP particles, and incubated at room temperature for 10min. Centrifuged at 8000rpm for 20min, the antigen is recovered, and PBS-T is used for the "wash-resuspend-centrifugation" process, and washed three times in total. Using 1% BSA as a diluent, the mouse anti-S protein serum is diluted to four gradients of 1:10, 1:100, 1:1000, and 1:10000, vortexed to resuspend the BLP particles, and incubated at room temperature for 10min. Centrifuge at 8000rpm for 15min, discard the supernatant, and repeat the "wash-resuspend-centrifuge" process with PBS-T for a total of three washes. Add 1:10000 dilution of goat anti-mouse IgG/HRP labeled antibody to the tube, 100uL/tube, vortex, and incubate at room temperature for 10min. Color development and termination: Centrifuge at 8000rpm for 15min, discard the supernatant, and use PBS-T for the "wash-resuspend-centrifuge" process for a total of 3 washes. Add TMB substrate, 100uL/tube, vortex to resuspend BLP/S-PA particles, shake and react in the dark for 10min, add 2M sulfuric acid to terminate the reaction, centrifuge at 8000rpm for 15min, aspirate the supernatant into a polypropylene plate, measure OD450 and calculate the P/N between the experimental group and the negative group. The results showed that the detection effect was best when the scFV-S-PA dosage was 1μg and the polyclonal antibody concentration was 1:100 diluted, and there was a good linear relationship between the antigen concentration and the absorbance.

Determination of the detection limit of antigen detection by BLP/scFV-S-PA solid phase method: Based on the test results, a curve of the logarithm of protein concentration and OD450 was drawn. The curve of protein concentration and OD450 is shown in Figure 15. Within a certain range, the protein concentration and OD450 are linearly related, and the detected antigen concentration range is: 30-7400ng/mL.

Repeatability verification: S protein of different concentrations was prepared and repeated determination of the above method was performed. The results are shown in Table 2. The results show that the method has good repeatability in detecting S protein and is stable.

Table 2 Results of repeatability verification of serum titer detected by BLP/scFV-S-PA

The working principle of the present invention is:

The method for detecting bacterial and viral antigens and antibodies based on bacteria-like particles can fix the recombinantly expressed antigen BP (VP)-PA or the recombinantly expressed single-chain antibody scFV-BP (VP)-PA on the surface of BLP through the non-covalent binding between BLP and anchor protein (PA), thereby generating BLP/BP (VP)-PA antigen complex or BLP/scFV-BP (VP)-PA complex, which can bind to the corresponding anti-BP (VP) IgG or BP (VP) protein in the sample to be tested, and then quantitatively detect them using enzyme-labeled secondary antibodies, so that they can be used to detect the anti-BP (VP) IgG titer or BP (VP) protein content in serum, such as the vaccine content, as well as the bacteria and virus content.

The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims (2)

1. A method for detecting bacterial and viral antigens, antibodies based on bacteria-like particles, comprising:

a. the specific operation for detecting the antibody titer of streptococcus pneumoniae is as follows: combining a prokaryotic expression recombinant antigen PspA-PA by taking BLP as a carrier to form a BLP/PspA-PA antigen complex, combining IgG of anti-PspA in serum to be detected by using the antigen complex, and quantitatively detecting the antigen complex by using an enzyme-labeled secondary antibody;

b. The specific operation of detecting the streptococcus pneumoniae antigen is as follows: combining a prokaryotic expression recombinant single-chain antibody scFV-PspA-PA by taking BLP as a carrier to form a BLP/scFV-PspA-PA single-chain antibody complex, combining an antigen PspA or a streptococcus pneumoniae cell in a sample to be detected by the single-chain antibody complex, and quantitatively detecting the antigen PspA or the streptococcus pneumoniae cell by utilizing an anti-PspA IgG and an enzyme-labeled secondary antibody;

c. The specific operation for detecting the novel coronavirus SARS-CoV-2 antibody titer is as follows: combining prokaryotic expression recombinant antigen S-PA by taking BLP as a carrier to form a BLP/S-PA antigen complex, combining IgG of an anti-S protein in serum to be detected by using the antigen complex, and quantitatively detecting the IgG by using an enzyme-labeled secondary antibody;

d. the specific operation for detecting the novel coronavirus SARS-CoV-2 antigen is as follows: combining a prokaryotic expression recombinant single-chain antibody scFV-S-PA by taking BLP as a carrier to form a BLP/scFV-S-PA single-chain antibody complex, combining the single-chain antibody particles with antigen S protein or virus particles in a sample to be detected, and quantitatively detecting the antigen S protein or virus particles by utilizing IgG of the anti-S protein and enzyme-labeled secondary antibodies;

The preparation method of the recombinant antigen PspA-PA comprises the following steps: selecting an alpha helical region and proline enrichment region sequence of PspA family 2 subclass 4, fusing and expressing an anchor protein PA at the C end of the proline enrichment region, optimizing the sequence before synthesis by chemically synthesizing a gene sequence, selecting a codon favored by escherichia coli to ensure that the gene is highly expressed in the escherichia coli, cloning and constructing the gene on a pET20b prokaryotic expression vector, transforming the gene into escherichia coli BL21 to express the gene, optimizing the expression condition of the protein, inducing and expressing a candidate protein, and obtaining a target protein PspA-PA through a His Trap affinity purification column;

The preparation method of the recombinant antigen S-PA comprises the following steps: the S protein antigen of SARS-CoV-2 is a partial sequence of S protein, expressed and prepared in a prokaryotic system by genetic engineering technology, and is used as a target antigen for content measurement;

The preparation method of the recombinant single-chain antibody scFV-S-PA comprises the following steps: a monoclonal antibody of an anti-S protein is selected as a parent, a heavy chain variable region is taken as an N end, a light chain variable region is taken as a C end, the heavy chain variable region and the light chain variable region are connected through a connecting peptide, an anchoring protein PA is fused and expressed at the C end of the monoclonal antibody, a chemical synthesis gene sequence is adopted, the sequence is optimized before synthesis, a codon favored by escherichia coli is selected to enable the codon to be highly expressed in the escherichia coli, then the clone is constructed on a pET20b prokaryotic expression vector, the expression is carried out in escherichia coli BL21, the expression condition of the protein is optimized, and an induced expression candidate protein is obtained, and a target protein scFV-S-PA is obtained through a His Trap affinity purification column.

2. The method for detecting bacterial and viral antigens and antibodies based on bacterial-like particles according to claim 1, wherein said BLP is prepared by the following method: and (3) carrying out heat treatment on the cultured lactococcus lactis and trichloroacetic acid, and washing and purifying to remove impurities, thereby leaving an inanimate peptidoglycan shell, namely BLP.