A kind of blocking protein and its preparation method and application

Abstract

The present invention provides a blocking protein and a preparation method and application, and relates to the technical field of blocking agents. The present invention constructs a blocking protein in a prokaryotic expression manner, and the blocking protein can block the binding of non-specific antibodies in the reaction system to the target on the solid phase carrier. The present invention also introduces the blocking protein into the detection system of autoantibodies to reduce the non-specific background signals in positive samples and negative samples, so that the detection results of the samples to be tested present more real signals, reduce the incidence of false positives and false negatives, and thus improve the sensitivity and specificity of the detection.

Description

Technical Field

[0001] The invention belongs to the technical field of blocking agents, and in particular relates to a blocking protein and a preparation method and application thereof. Background Art

[0002] In the commonly used methodologies in the field of in vitro diagnosis, blocking agents are often used to improve the sensitivity and specificity of diagnosis. For example, blocking agents are used to prevent nonspecific adsorption between proteins or biomolecules in the reaction system and the solid phase medium. Common protein blocking agents include skimmed milk powder, BSA, casein, fish glue and serum, and non-protein substances include formaldehyde, Polyvinylpyrrolidone (PVP), detergent (Tween-20), etc.; for example, blocking agents are used to prevent nonspecific proteins or biomolecules in the reaction system from combining with target substances. Common blocking agents (or blocking substances) are nonspecific Ig or antiserum from mice, rabbits, sheep, cattle, etc. There are also blocking agents prepared from empty cells or empty cells transfected with empty cells from the same source as the target substance to reduce nonspecific adsorption (Application No.: 2019107384768, Detection materials, preparation methods and applications of anti-AMPA1 autoantibodies in human body fluids). However, in many cases, blocking agents may not have a good blocking effect, and they are expensive and difficult to obtain. Therefore, continuous attempts are needed to find cost-effective blocking agents. In particular, in the prior art, there is no universally applicable and cost-effective blocking substance used to prevent non-specific protein antibodies in the reaction system from binding to the target substance on the solid phase carrier. In most cases, new blocking agents need to be developed to meet the needs of the reaction system, thereby improving the sensitivity and specificity of the reaction. Summary of the invention

[0003] The purpose of the present invention is to provide a blocking protein and a preparation method and application thereof, wherein the blocking protein can block the binding of non-specific antibodies in a reaction system to a target on a solid phase carrier, and the detection sensitivity and specificity can be improved by using the blocking protein to detect autoantibodies.

[0004] The present invention provides a method for constructing a blocking protein, comprising the following steps: (1) transferring the constructed prokaryotic expression vector for overexpressing a foreign gene into a prokaryotic cell, inducing expression, collecting the bacterial cells, and obtaining overexpressing cells; the foreign gene comprises GAPDH or GST;

[0005] (2) The overexpressing cells in step (1) are broken, and the upper suspension is obtained by centrifugation. The upper suspension is mixed with a protective agent to obtain a blocking protein.

[0006] Preferably, the prokaryotic expression vector in step (1) includes pET30a, pET22a, pET28a or pET40a.

[0007] Preferably, the prokaryotic cells in step (1) include BL21 (DE3), Rosetta-gami (DE3) or Arctic.

[0008] Preferably, the conditions for inducing expression in step (1) include: the concentration of the inducer is 0.1-1.5 mM; the induction temperature is 14-22°C; the OD of the bacteria during induction is 600 The OD of the bacteria during collection is 0.6-0.8. 600 It is 0.8~1.0.

[0009] Preferably, the disruption in step (2) comprises mixing the overexpressing cells with a lysis solution and then performing ultrasonic disruption;

[0010] The lysis solution is PBS buffer or a mixture of 10-100 mM Tris-HCl and 50-500 mM NaCl buffer at pH 7.0-9.0.

[0011] Preferably, the protective agent in step (2) includes trehalose, sucrose or glucose.

[0012] The present invention also provides a blocking protein prepared by the above construction method.

[0013] The present invention also provides a detection tool for detecting autoantibodies, comprising the above-mentioned blocking protein.

[0014] Preferably, the blocking protein is loaded on a solid phase carrier;

[0015] The solid support comprises a glass fiber mat.

[0016] The present invention also provides the use of the blocking protein or the detection tool in preparing an autoantibody detection kit.

[0017] Beneficial effects: The blocking process described in the technical solution of the present invention refers to preventing non-specific protein antibodies in the reaction system from binding to the target substance on the solid phase carrier. The present invention constructs a blocking protein in the form of prokaryotic expression, and the blocking protein can block the binding of non-specific antibodies in the reaction system to the target on the solid phase carrier. The present invention also introduces the blocking protein into the detection system of autoantibodies: (1) it can reduce the non-specific background signals in positive samples and negative samples, so that the detection results of the samples to be tested present a more realistic signal; (2) the use of blocking protein can turn a certain type of negative result after color development into a positive result, reducing the incidence of false negatives; (3) the use of blocking protein can turn a certain type of positive result after color development into a negative result, reducing the incidence of false positives, thereby improving the sensitivity and specificity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0019] Figure 1 The antigen distribution diagram of the membrane strip used in the embodiment of the present invention;

[0020] Figure 2 The blocking effect diagram of the blocking protein prepared for the empty bacteria;

[0021] Figure 3 The blocking effect diagram of the blocking protein prepared by the expression bacteria overexpressing the empty vector;

[0022] Figure 4 The blocking effect diagram of the blocking protein prepared for the expression bacteria overexpressing GAPDH;

[0023] Figure 5 The blocking effect diagram of the blocking protein prepared for the expression bacteria overexpressing GST;

[0024] Figure 6 This is the result diagram of the second type of negative blood after blocking with GAPDH and GST blocking proteins, where the first column is the result without adding blocking protein; the second column is the result of GAPDH blocking protein blocking; the third column is the result of GST blocking protein blocking;

[0025] Figure 7 This is a diagram showing the effect of different blocking proteins blocking false-negative serum. DETAILED DESCRIPTION

[0026] The present invention provides a method for constructing a blocking protein, comprising the following steps: (1) transferring the constructed prokaryotic expression vector for overexpressing a foreign gene into a prokaryotic cell, inducing expression, collecting the bacterial cells, and obtaining overexpressing cells; the foreign gene comprises GAPDH or GST;

[0027] (2) The overexpressing cells in step (1) are broken, and the upper suspension is obtained by centrifugation. The upper suspension is mixed with a protective agent to obtain a blocked protein.

[0028] The present invention transfers the constructed prokaryotic expression vector for overexpressing exogenous genes into prokaryotic cells, induces expression, collects bacteria, and obtains overexpressed cells; the exogenous gene includes GAPDH or GST. The present invention preferably uses a genetic engineering method to construct a prokaryotic expression vector for overexpressing exogenous genes, and the exogenous gene preferably includes GAPDH or GST, more preferably GST. In the present invention, the sequence number of the GAPDH on NCBI is: NM_002046.7, and the nucleotide sequence is shown in SEQ ID NO.1. The sequence number of the GST on NCBI is: FN315687.1, and the nucleotide sequence is shown in SEQ ID NO.2. The present invention inserts the exogenous gene into a prokaryotic expression vector, and the prokaryotic expression vector preferably includes pET30a, pET22a, pET28a or pET40a. In the embodiments of the present invention, pET30a is used as an example for illustration, but it cannot be identified as the entire protection scope of the present invention. In the embodiment of the present invention, it is preferred to insert the exogenous gene into the NheI and NotI restriction sites of pET30a to construct a prokaryotic expression vector for overexpressing the exogenous gene.

[0029] The present invention transfers a prokaryotic expression vector that overexpresses an exogenous gene into a prokaryotic cell, and the prokaryotic cell preferably includes BL21 (DE3), Rosetta-gami (DE3) or Arctic. The present invention does not specifically limit the method of transfer, and conventional methods in the art can be used. The present invention induces expression in the prokaryotic cell into which the target vector has been transferred, and the conditions for inducing expression preferably include: the concentration of the inducer is 0.1 to 1.5 mM; the induction temperature is 14 to 22°C; the OD of the bacteria during induction is 600 The OD of the bacteria during collection is 0.6-0.8. 600 is 0.8 to 1.0. The present invention collects the bacteria after the induced expression, adds a lysis solution to the collected bacteria, performs ultrasonic disruption, centrifuges, takes the upper suspension, adds a protective agent, obtains a closed protein, and stores it for later use. The lysis solution of the present invention is preferably a PBS buffer or a 10-100mM Tris-HCl+50-500mMNaCl buffer of pH7.0-9.0, and the mixing ratio of the bacteria and the lysis solution is preferably 5ml of lysis solution per 100ml of bacteria. The ultrasonic disruption of the present invention preferably includes: a power of 300w-400w, disruption for 4s, a stop of 6s, and a disruption time of 10-20min. The present invention centrifuges the upper suspension after ultrasonic disruption, and the rotation speed of the centrifuge is preferably 500-2000rmp, and the centrifugation time is preferably 5-10min.

[0030] The present invention mixes the upper suspension after centrifugation with a protective agent to obtain the closed protein, wherein the protective agent is trehalose, sucrose or glucose, and the mixing ratio of the upper suspension and the protective agent is preferably 10 to 20 g of the protective agent per 100 ml of the upper suspension. The closed protein of the present invention can be packaged in liquid form and stored at a low temperature of 4°C to -80°C; the closed protein can also be added to a solid phase carrier and dried and stored at a low temperature of 4°C to -80°C; the solid phase carrier for storing the closed protein preferably includes a glass fiber mat.

[0031] The present invention also provides a blocking protein prepared by the above construction method.

[0032] The liquid form of the blocked protein obtained by the present invention has the appearance of a suspension, which is a mixture of multiple proteins and may even contain cell debris.

[0033] The present invention also provides a detection tool for detecting autoantibodies, comprising the above-mentioned blocking protein.

[0034] When the present invention utilizes the blocking protein to detect autoantibodies, it preferably includes the following steps:

[0035] a. Add blocking protein to serum or cerebrospinal fluid samples;

[0036] b. contacting the sample to be tested prepared in step a with the target substance;

[0037] c. Wash the target substance and incubate with labeled anti-human Fc antibody;

[0038] d. Wash the target substance again and add a suitable color developer for color development.

[0039] The target substance of the present invention is preferably derived from the same expression system as the blocking protein, and can be a crude extract of the antigen, or a protein purified by a combination of one or more methods including affinity chromatography, ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography. The target substance of the present invention can be solidified on a nitrocellulose membrane, a PVDF membrane, or a polyethylene plate.

[0040] The present invention also provides the use of the blocking protein or the detection tool in preparing an autoantibody detection kit.

[0041] The application of the present invention is preferably the same as described above and will not be described in detail here.

[0042] In order to further illustrate the present invention, a blocking protein, preparation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0043] In the present invention, the membrane strips used in each embodiment are test strips fixed with 6 paraneoplastic antigens (Ri, Hu, Yo, CV2, Ma2, Amphiphysin, where Ri includes two antigens, Nova1 and Nova2, and Hu includes three antigens, HuB, HuC, and HuD). The test strips are prepared in this laboratory, and the antigens fixed on the test strips are crude extracts of prokaryotic expression proteins. The distribution of antigens on the test strips is as follows: Figure 1 As shown. Interpretation of positive results: Compared with the control point C, the color development of the antigen-coated point (Nova1, Nova2, HuB, HuC, HuD, Yo, CV2, Ma2, Amphiphysin) is stronger than that of the C area, which can be judged as positive. In the present invention, the positive blood marked in each embodiment is verified as positive by neutralization experiment and immunofluorescence method, and the negative blood marked in each embodiment is verified as negative by neutralization experiment and immunofluorescence method. In the present invention, the dot blot test strip coated with 6 paraneoplastic antigens is used as an example to illustrate the blocking effect of blocking protein. The embodiment of the present invention selects 5 representative positive sera to illustrate the effect of blocking protein, wherein the autoantibody negative antigen points and control points of 2 positive blood samples are lightly colored, and the autoantibody positive antigen points are darkly colored, and obvious positive signals can be distinguished without blocking (positive blood 1 and positive blood 3), while the autoantibody negative antigen points and control points of the other 3 positive blood samples are darker in color, and the color difference between the autoantibody positive antigen points and the control points is not very large. This result is not beautiful, and it is easy to cause misjudgment by the experimenter. After blocking, the background of the autoantibody negative antigen points is reduced, and the color becomes lighter. Compared with the control points, the color difference between the autoantibody positive antigen points and the control points increases, and the positive signal can be clearly distinguished (positive blood 2, positive blood 4 and positive blood 5). In addition, the embodiment of the present invention selects 7 representative negative sera to illustrate the effect of blocking protein, wherein the background appearing on each antigen point and the control point of 6 negative blood samples is more easily blocked by the various blocking proteins described in the present invention (negative blood 1, negative blood 2, negative blood 3, negative blood 4, negative blood 5 and negative blood 6, in Figures 2 to 5 In the figure, it is marked as the first type of negative blood), while in another negative blood, after being blocked by blocking proteins prepared from empty bacteria, empty transformed vector, and transformed GAPDH-expressing bacteria, some antigen autoantibody detection showed false positive signals, which could only be blocked by GST blocking proteins (in Figures 2 to 5 Marked as second type negative blood).

[0044] Example 1 Induced expression and application of empty bacteria

[0045] Step 1 Preparation of induced bacteria

[0046] (1) Activation of bacterial strains: Take out the frozen bacterial strains BL21 (DE3), Rosetta-gami (DE3) and Arctic (Agilent) from -80°C, streak each strain onto LB solid plates using the three-zone streak method, and culture at 37°C overnight;

[0047] (2) Expansion culture and induced expression: Pick single clones from the plate and add them to 3 mL of culture medium. Culture overnight. The next day, transfer them to 200 mL of LB liquid culture medium at a ratio of 1:100. Wait until the bacteria grow to OD 600 When the pH value was 0.6-0.8, the inducer IPTG (Sangon Biotechnology) was added, the final concentration of the inducer was 1 mM, the induction temperature was 16°C, the induction time was 16 h, and the bacteria were collected by centrifugation.

[0048] Step 2 Preparation of blocking protein

[0049] (1) Add 10 ml of PBS buffer (Solabo) to each of the three collected bacterial cells and vortex to mix;

[0050] (2) Using an ultrasonic disruptor to disrupt the bacteria in each tube, the power of the disruption is 300 W, the disruption is 4 s, the rest is 6 s, and the disruption time is 20 min;

[0051] (3) Centrifuge at 2000 rpm for 5 min at 4°C in a low-temperature centrifuge and take the upper suspension;

[0052] (4) Add sucrose (Aladdin) with a final concentration of 10% to the upper suspension as a protective agent to keep the blocked protein in suspension;

[0053] (5) The three blocking proteins prepared above were packaged and stored at 4°C for later use.

[0054] Step 3: Blocking protein is used for the detection of autoantibodies, and the reaction system is 300 μL

[0055] (1) Preparation of primary antibody:

[0056] Experimental group: Add 30 μL of liquid blocking protein to 270 μL of serum to be tested diluted with PBST (the dilution ratio of serum is 1:10) and mix thoroughly;

[0057] Control group: no blocking protein was added, and the system was supplemented to 300 μL with 30 μL PBST;

[0058] (2) Add the above mixture to the 24-well plate with the test strips placed on it, place it on a shaker, incubate at room temperature for 30 minutes, and wash with PBST for 4 minutes each time, for a total of 2 washes;

[0059] (3) AP-labeled goat anti-human IgG antibody was diluted 2000 times (the antibody was purchased from Jackson Company, and the antibody dry powder was dissolved according to the instructions), added to each well, incubated at room temperature for 30 minutes, and washed with 1% NaCl, 4 minutes / time, for a total of 2 times;

[0060] (4) Color development: After removing the washing solution in the wells, add 300 μL / well of substrate diluent BCIP / NBT (substrate: reaction buffer = 1:49), and let it stand at room temperature in the dark for 4 minutes to obtain the color development result. Discard the substrate diluent in the wells, rinse the test strip with distilled water 1 to 2 times, take it out to dry or dry it at 37°C to 45°C.

[0061] The results are as follows Figure 2 As shown, the blocking proteins prepared by the three expression bacteria have similar effects in blocking the background:

[0062] (1) From the results of positive blood 1, it can be seen that the three groups of blocking proteins in the experimental group have obvious effects and can significantly reduce the nonspecific color development of autoantibody negative antigen points. From the results of positive blood 2, it can be seen that the use of blocking proteins can significantly reduce the nonspecific color development of autoantibody negative antigen points and autoantibody positive antigen points (Yo), making the autoantibody positive antigen points (Yo) present a more realistic positive signal.

[0063] (2) From the results of negative blood, it can be seen that there are two types of negative blood. The first type of negative blood belongs to the type with easy background reduction. The use of blocking protein makes the test strip results more beautiful. The second type of negative blood has a positive signal after the use of blocking protein (autoantibodies to the six antigens HuB, HuC, HuD, Yo, and CV2 are positive). After verification, the positive signal is a false positive result.

[0064] Example 2 Inducible expression and application of empty vector

[0065] In view of the results of Example 1, the blocking proteins prepared by the three expression bacteria had similar effects. In this example, one of the expression bacteria was selected, transferred into four different expression vectors, and after inducing expression, four blocking proteins with different overexpression vectors were prepared, and the blocking effects of different blocking proteins were verified.

[0066] Step 1 Preparation of induced bacteria

[0067] (1) Preparation of competent cells: Take out the frozen strain BL21 (DE3) from -80°C and activate the strain according to the steps described in (1) of step 1 of Example 1. The activated strain is activated with CaCl 2 Method to prepare competent cells;

[0068] (2) Plasmid transformation into competent cells: Take plasmids pET30a, pET22b, pET28a, and pET40a, and transform the four plasmids into BL21 (DE3) bacteria respectively;

[0069] (3) Expansion culture and induced expression: Pick single clones from the plate and transfer them to 3 mL of culture medium, culture overnight, and transfer them to 200 mL of LB liquid culture medium at a ratio of 1:100 the next day. Wait until the bacteria grow to OD 600 When the pH value was 0.6-0.8, the inducer IPTG (Sangon Biotechnology) was added, the final concentration of the inducer was 1 mM, the induction temperature was 16°C, the induction time was 16 h, and the bacteria were collected by centrifugation.

[0070] The process of preparing the blocking protein in step 2 and using the blocking protein for the detection of autoantibodies in step 3 is the same as that in Example 1.

[0071] The results are as follows Figure 3 As shown:

[0072] (1) In positive blood 1, positive blood 3 and the first type of negative blood, the four blocking proteins had significant effects and could significantly reduce nonspecific color development. There was no significant difference between the four groups of blocking proteins, and the blocking effects were similar.

[0073] (2) The second type of negative blood still showed positive signals after using the blocking protein (the antigens that showed positive signals after the experiment using the blocking protein prepared by the expression bacteria transferred into pET22b were HuB, HuC, HuD, Yo and CV2; the antigens that showed positive signals after the experiment using the blocking protein prepared by the expression bacteria transferred into pET28a were HuB, HuC, HuD and Yo, and the color development on CV2 was weak, which was a suspected signal; the antigens that showed positive signals after the experiment using the blocking protein prepared by the expression bacteria transferred into pET30a were HuB, HuC and Yo; the antigens that showed positive signals after the experiment using the blocking protein prepared by the expression bacteria transferred into pET40a were HuB and HuC). Therefore, the blocking protein prepared by the expression bacteria with overexpression did not solve the problem of false positives in the second type of negative blood after blocking.

[0074] Example 3 Overexpression and application of GAPDH

[0075] According to the results of Example 2, the blocking effects of the four empty vectors were relatively close, so one of the vectors, pET30a, was selected to construct a recombinant vector overexpressing the GAPDH gene, and the recombinant vector overexpressing the GAPDH gene was transferred into the expression bacteria, and after inducing expression, a blocking protein was prepared, and its blocking effect was verified.

[0076] Step 1 Preparation of induced bacteria

[0077] (1) Construction of recombinant vector: The DNA sequence of GAPDH (SEQ ID NO. 1) was synthesized into pET30a by artificial synthesis and named GAPDH-pET30a, with NheI and NotI inserted into the restriction sites;

[0078] (2) Preparation of competent cells: Take out the frozen strain BL21 (DE3) from -80°C and activate the strain according to the steps described in (1) of step 1 of Example 1. The activated strain is activated with CaCl 2 Method to prepare competent cells;

[0079] (3) Plasmid transformation into competent cells: Take the recombinant plasmid GAPDH-pET30a and transform the plasmid into BL21(DE3) bacteria;

[0080] (4) Expansion culture and induced expression: Pick a single clone from the plate and add it to 3 mL of culture medium. Culture it overnight. The next day, transfer it to 200 mL of LB liquid culture medium at a ratio of 1:100. Wait until the bacteria grow to OD 600 When the pH value was 0.6-0.8, the inducer IPTG (Sangon Biotechnology) was added, the final concentration of the inducer was 1 mM, the induction temperature was 16°C, the induction time was 16 h, and the bacteria were collected by centrifugation.

[0081] The process of preparing the blocking protein in step 2 and using the blocking protein for the detection of autoantibodies in step 3 is the same as that in Example 1.

[0082] The results are as follows Figure 4 As shown:

[0083] (1) In positive blood 1, positive blood 4 and the first type of negative blood (random negative blood 3 and random negative blood 4), the blocking protein prepared by the expression bacteria overexpressing GAPDH had a significant effect and could significantly reduce nonspecific color development;

[0084] (2) After using the blocking protein, the second type of negative blood still showed positive signals (autoantibodies to the five antigens HuB, HuC, HuD, and CV2 were positive). Therefore, the blocking protein prepared by the expression bacteria overexpressing GAPDH did not solve the problem of false positives in the second type of negative blood after blocking.

[0085] Example 4 Overexpression and application of GST

[0086] From the results of Example 3, it can be seen that although the blocking protein prepared by the expression bacteria overexpressing GAPDH has a significant blocking effect, it still cannot block the false positive signal appearing on the second type negative blood. Therefore, an attempt was made to transfer the recombinant vector overexpressing the GST gene into the expression bacteria, induce the expression and prepare it into a blocking protein, and verify its blocking effect.

[0087] Step 1 Preparation of induced bacteria

[0088] (1) Construction of recombinant vector: The DNA sequence of GST (SEQ ID NO. 2) was synthesized into pET30a by artificial synthesis and named GST-pET30a, with NheI and NotI inserted into the restriction sites;

[0089] (2) Preparation of competent cells: Take out the frozen strain BL21 (DE3) from -80°C and activate the strain according to the steps described in (1) of step 1 of Example 1. The activated strain is activated with CaCl 2 Method to prepare competent cells;

[0090] (3) Plasmid transformation into competent cells: Take the recombinant plasmid GST-pET30a and transform the plasmid into BL21(DE3) bacteria;

[0091] (4) Expansion culture and induced expression: Pick a single clone from the plate and add it to 3 mL of culture medium. Culture it overnight. The next day, transfer it to 200 mL of LB liquid culture medium at a ratio of 1:100. Wait until the bacteria grow to OD 600 When the pH value was 0.6-0.8, the inducer IPTG (Sangon Biotechnology) was added, the final concentration of the inducer was 1 mM, the induction temperature was 16°C, the induction time was 16 h, and the bacteria were collected by centrifugation.

[0092] The process of preparing the blocking protein in step 2 and using the blocking protein for the detection of autoantibodies in step 3 is the same as that in Example 1.

[0093] The results are as follows Figure 5 As shown:

[0094] (1) In positive blood 1, positive blood 5 and the first type of negative blood (random negative blood 5 and random negative blood 6), the blocking protein prepared by the expression bacteria overexpressing GST had a significant effect and could significantly reduce nonspecific color development;

[0095] (2) The second type of negative blood did not show a positive signal after using the blocking protein. Therefore, the blocking protein prepared by the expression bacteria overexpressing GST solved the false positive problem of the second type of negative blood after blocking.

[0096] Example 5 Screening and verification experiment of serum using blocking protein prepared by the expression bacteria overexpressing GST prepared in Example 4

[0097] In this example, 108 normal human sera were randomly selected and serum blocking experiments were performed using blocking proteins prepared by expressing bacteria overexpressing GAPDH and GST. The results showed that 8 sera in the GAPDH blocking protein blocking group showed false positive signals (both were negative after neutralization experiment and immunofluorescence method verification), and 5 sera were positive for HuB, HuC, HuD, Yo and CV2 autoantibodies (positive signals were as follows: Figure 6 1-2), there were 3 sera with positive autoantibodies to HuB, HuC and HuD (or suspected HuD autoantibody positive) (the positive signals were as shown in Figure 6 2-2), the test results of the other samples were all negative; and these 8 samples were all judged to be negative after blocking with GST blocking protein, and no false positive signals appeared (see Figure 6 1-3 and 2-3), and the rest of the samples tested negative.

[0098] In addition, the present application also found that a very small amount of positive blood had a negative test result when no blocking protein was used in the test process, but a positive test result was obtained after the blocking protein was used ( Figure 7 ).exist Figure 7 In the experiment, the color development of Yo antigen spots was not much different from that of control spots when not blocked, but after using blocking protein, the color development of Yo antigen spots was significantly different from that of control spots, which could be judged as a positive result. The signal was verified as a positive signal by serum neutralization experiment and immunofluorescence experiment. Therefore, the use of blocking protein not only reduces the background signal, but also avoids some false negative results and improves the sensitivity of detection.

[0099] It can be seen that the blocking protein prepared by using GST-overexpressing bacteria not only reduces the background of the test sample, but also solves the problem of false positive or false negative in the detection process, improves the sensitivity and specificity of the detection, and has good application prospects.

[0100] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a blocking protein, characterized in that: The method comprises the following steps: (1) transferring the constructed GST-overexpressing pET30a into prokaryotic cells, inducing expression, collecting the bacterial cells, and obtaining overexpressing cells; The induction expression conditions include: the concentration of the inducer is 0.1-1.5 mM; the induction temperature is 14-22°C; the OD of the bacteria during induction 600 The OD of the bacteria during collection is 0.6~0.

8. 600 is 0.8-1.0; the prokaryotic cell is BL21 (DE3); (2) Disruption step (1) The overexpressed cells are centrifuged to obtain an upper suspension, and the upper suspension is mixed with a protective agent to obtain a blocked protein; the protective agent is sucrose; the centrifugal speed is 2000 rpm, and the centrifugation time is 5 to 10 min; the disruption includes mixing the overexpressed cells with a lysis solution and then performing ultrasonic disruption; the ultrasonic disruption conditions are: power 300 w to 400 w, disruption 4 s, stop 6 s, and ultrasonic disruption time is 10 to 20 min; the lysis solution is PBS buffer.

2. The blocked protein prepared by the construction method of claim 1.

3. A detection tool for detecting autoantibodies, characterized in that: The method comprises loading the blocking protein according to claim 2 onto a solid phase carrier; The solid support comprises a glass fiber mat.

4. Use of the blocking protein according to claim 2 or the detection tool according to claim 3 in the preparation of an autoantibody detection kit.

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