Enzyme-labeled antigen for HIV (human immunodeficiency virus) diagnosis and preparation method of enzyme-labeled antigen

Abstract

The invention discloses an enzyme-labeled antigen for HIV diagnosis, the enzyme-labeled antigen is obtained by carrying out a directional coupling reaction on a gp41 recombinant antigen and an enzyme, the sequence of the gp41 recombinant antigen is shown as SEQ ID NO: 1, the enzyme is alkaline phosphatase, and the gp41 recombinant antigen and the enzyme are separated from each other. The preparation method of the enzyme-labeled antigen comprises the following steps: genetic engineering expression and purification of the antigen, modification of the antigen, activation of alkaline phosphatase, directional coupling reaction and stability optimization. The HIV recombinant antigen-alkaline phosphatase marker prepared by the invention is high in activity, strong in specificity, good in stability and good in inter-batch uniformity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an enzyme-labeled antigen for HIV diagnosis and a preparation method thereof. BACKGROUND

[0002] AIDS, namely acquired immune deficiency syndrome, is a syndrome caused by infection with human immunodeficiency virus (HIV) and leading to immune deficiency and a series of opportunistic infections and tumors, and can cause death in severe cases. HIV is divided into HIV-1 type and HIV-2 type, and the number of AIDS patients and the number of deaths have shown an increasing trend in recent years, so it is necessary to detect HIV early and carry out antiviral treatment in time.

[0003] Currently, the two most commonly used methods in AIDS screening laboratories are enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay. ELISA is the earliest serological detection technology applied to AIDS screening detection, and since the first generation of anti-HIV ELISA detection reagent was introduced in 1985, the performance of the reagent has been continuously improved. After the second generation and the third generation of simple antibody detection, the third generation and the fourth generation are currently most commonly used in China. Chemiluminescence immunoassay, referred to as chemiluminescence method, is a new type of immunological detection technology that combines luminescence reaction with immune reaction for the determination of various antigens, haptens, antibodies, etc. It is also the most advanced labeled immunoassay technology, which has high sensitivity, short detection time and high automation. Among them, HIV gp41-ALP (alkaline phosphatase labeled gp41 protein) complex plays a key role in signal amplification engine in chemiluminescence detection (CLIA), and its performance directly determines the sensitivity, speed and reliability of the detection. In AIDS prevention and control, high-performance gp41-ALP complex is the technical cornerstone to achieve the goal of "early detection and zero transmission".

[0004] In the prior art, there are many problems in the application process of HIV diagnostic reagents. On the one hand, the conservation of part of the HIV antigen sequence is insufficient, which leads to low detection coverage of different subtypes of HIV and easy missed detection; on the other hand, the coupling of antigens and alkaline phosphatase is mostly random coupling, which not only affects the immune activity of the antigen and the catalytic activity of the enzyme, but also leads to large batch-to-batch differences of the label. In addition, the existing label has poor stability and is easy to lose activity during storage and transportation, which limits its precise screening of HIV in resource-limited areas. Therefore, it is of important clinical application value to develop a gp41-ALP labeling technology based on optimized HIV antigen sequence. SUMMARY

[0005] In order to solve the problems of the prior art, the present application aims to provide an enzyme-labeled antigen for HIV diagnosis and a preparation method thereof, so as to obtain HIV recombinant antigen-alkaline phosphatase labeled antigen with high activity, strong specificity, good stability and good batch uniformity.

[0006] In order to achieve the above-mentioned target, the present application adopts the following technical solution:

[0007] An enzyme-labeled antigen for HIV diagnosis is obtained by directional coupling reaction of gp41 recombinant antigen and enzyme, the sequence of the gp41 recombinant antigen is shown in SEQ ID NO: 1, and the enzyme is alkaline phosphatase (ALP).

[0008] Preferably, the mass ratio of the aforementioned alkaline phosphatase and gp41 recombinant antigen is (1:0.5)-(1:1).

[0009] The preparation method of the enzyme-labeled antigen for HIV diagnosis comprises the following steps:

[0010] S1, genetic engineering expression and purification of antigen: obtain gp41 recombinant antigen with the sequence shown in SEQ ID NO: 1, which contains the immunodominant epitope (ELDKWA) of HIV-1 gp41 protein, and is designed by the following optimization:

[0011] (1) retain the cross-subtype conserved region (amino acids 1-30);

[0012] (2) remove the hydrophobic transmembrane region which is easy to cause non-specific reaction;

[0013] (3) add 6xHis tag at the C-terminal for easy purification;

[0014] (4) connect the functional domains through GGGGS flexible linker;

[0015] S2, modification of antigen: modify the lysine epsilon-amino group of the gp41 recombinant antigen with Traut's reagent to generate free thiol (-SH);

[0016] S3, activation of alkaline phosphatase: activate the alkaline phosphatase with SMCC, and the activated maleimide group specifically captures the thiol (-SH) of the antigen;

[0017] S4, directional coupling reaction: mix the modified gp41 recombinant antigen with the activated alkaline phosphatase to perform directional coupling, and obtain an enzyme-labeled mixture;

[0018] S5, composite stabilizer preparation: chromatographically purify the enzyme-labeled mixture, add a composite stabilizer, and obtain HIV recombinant antigen-alkaline phosphatase labeled antigen.

[0019] Preferably, in the aforementioned step S2, the molar ratio of the gp41 recombinant antigen to the Traut's reagent is (1:10)-(1:20).

[0020] Preferably, in the aforementioned step S3, the molar ratio of the alkaline phosphatase to the SMCC is (1:10)-(1:20).

[0021] Preferably, in the aforementioned step S4, the mass ratio of the alkaline phosphatase to the gp41 recombinant antigen is (1:0.5)-(1:1).

[0022] Preferably, in the aforementioned step S5, the specific method of the chromatographic purification is: desalting or dialysis treatment is performed to remove small molecular impurities; and then size exclusion chromatography is performed to separate the target marker with uniform molecular weight and remove free gp41 recombinant antigen and free alkaline phosphatase.

[0023] Preferably, in the aforementioned step S5, the concentration of the enzyme-labeled mixture after purification is 1.5-2.5 mg / mL, and the complex stabilizer includes the following components: 0.5%-1% (w / v) bovine serum albumin, 3%-5% (w / v) sucrose, 0.05%-0.2% (w / v) trehalose, 0.5-1 mM magnesium ion and 0.1% (w / v) preservative.

[0024] Use of the enzyme-labeled antigen in preparation of a reagent or medical device for diagnosing HIV infection.

[0025] An HIV detection kit, the kit comprising the enzyme-labeled antigen of claim 1.

[0026] The enzyme-labeled antigen prepared by the present application has the advantages of high activity, strong specificity, good stability and good batch uniformity, and can significantly improve the sensitivity, specificity and reliability of HIV detection, and has important application value in the field of AIDS diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a preparation process route map of the present application;

[0028] Figure 2 is an SDS-PAGE electrophoresis map of the gp41 recombinant antigen prepared in Example 1 after expression and purification;

[0029] Figure 3 is a specific and cross-reactivity competitive inhibition map of the gp41 recombinant antigen prepared in Example 1;

[0030] Figure 4 is an SDS-PAGE electrophoresis map of the ALP-gp41 marker purified in Example 2. DETAILED DESCRIPTION

[0031] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: Recombinant antigen expression and purification, including the following steps:

[0033] (1) Carrier construction:

[0034] S1. Design and synthesize a gene according to the sequence shown in SEQ ID NO: 1;

[0035] S2, clone into the pET-28a(+) vector;

[0036] S3. Select single colonies for PCR amplification;

[0037] S4. Sequencing to verify sequence correctness;

[0038] S5. Transform into chemically competent cells BL21(DE3) to obtain recombinant engineered bacteria;

[0039] (2) IPTG-induced expression:

[0040] S6. Use a pipette to add 800 μL of the corresponding antibiotic and 8 mL of bacterial culture to a 2 L Erlenmeyer flask containing 800 mL of LB culture medium.

[0041] Incubate at 37℃ and 210 rpm until the bacterial OD600 reaches 0.6-0.8 (approximately 3-4 hours).

[0042] S8. Add 160 μL of IPTG to the cultured bacterial solution to make the final concentration 0.2 mM, and incubate overnight at 13℃ and 210 rpm for 16-20 h.

[0043] S9. Centrifuge to collect bacterial cells;

[0044] (3) Antigen was purified and harvested by NI column affinity chromatography:

[0045] S10, 4℃. Take out the packing material and mix it thoroughly. Prepare the packing material according to the principle that 1 mL of packing material can purify 10-20 mg of target protein. Equilibrate the chromatography column with 5-10 times the column volume of equilibration buffer.

[0046] S11. Slowly load the processed supernatant to complete the sample loading (0.5-1 mL / min);

[0047] S12. Equilibrate the chromatography column with equilibration buffer until the A280 detection value remains essentially unchanged (usually 10-20 column volumes).

[0048] S13. Use gradient elution with 50-250mM imidazole elution buffer to collect the sample and obtain gp41 recombinant antigen.

[0049] Example 2, ALP-gp41 label preparation, including the following steps:

[0050] S14, 2 mg of the above gp41 recombinant antigen was dissolved in 2 mL of HEPES buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, pH 7.8), and modified with specific sulfhydryl reagent Traut's, the molar ratio of antigen to Traut's reagent was 1:10;

[0051] S15, dissolve ALP in 0.1 M carbonate buffer (pH 8.5), add SMCC, the molar ratio of ALP to SMCC is 1:10;

[0052] S16, the above activated ALP and gp41 recombinant antigen were directionally coupled at a mass ratio of 1:1, and were cross-linked at 4°C for 24 h;

[0053] S17, add 10 mM glycine to terminate, and obtain the ALP-gp41 label mixture.

[0054] Example 3, ALP-gp41 label purification

[0055] S18, the ALP-gp41 label mixture was subjected to PD-10 (GE Healthcare) desalting treatment or dialysis treatment to remove small molecule salts, inhibitors, etc., and was replaced with the target buffer: 50 mM HEPES + 150 mM NaCl, pH 7.2;

[0056] S19, further fine purification by size exclusion chromatography (SEC) Superdex 200 to remove free enzymes and antibodies, and obtain the ALP-gp41 label mixture.

[0057] Example 4, stability optimization of ALP-gp41 label

[0058] S20, adjust the concentration of the above purified ALP-gp41 label mixture to 2 mg / mL;

[0059] S21, add each component of the complex stabilizer to the ALP-gp41 label mixture in proportion, 0.5-1% bovine serum albumin, 3-5% sucrose, 0.05-0.2% trehalose, 0.5-1 mM magnesium ion, 0.1% preservative;

[0060] S22, 0.22 μm filtration sterilization;

[0061] S23, add an equal volume of glycerol, 37°C, 7-day accelerated evaluation, and verify the stability by repeating freezing and thawing 3 times.

[0062] Comparative Example 1: ALP-gp41 marker was prepared by traditional method, which included the following steps:

[0063] (1) 0.2 mL of ALP (containing 2 mg) was mixed with 0.1 mL of gp41 recombinant antigen (containing 1 mg), and PBS was added to 0.5 mL;

[0064] (2) 4 μL of 25% glutaraldehyde was added to the 0.5 mL ALP / antigen mixture, and it was placed at room temperature for 2 h;

[0065] (3) PBS was added to the reaction to 2 mL, and the PBS was dialyzed at 4°C overnight. The dialysate was diluted to 5 mL with Tris buffer;

[0066] (4) 0.22 μm filtration sterilization;

[0067] (5) Add an equal volume of glycerol, add 0.1% preservative, and store in aliquots.

[0068] Performance test

[0069] (1) Specificity and cross-reactivity competition inhibition experiment of gp41 recombinant antigen

[0070] The specificity of the gp41 recombinant antigen prepared in Example 1 was evaluated on a specific immune detection platform, and the cross-reactivity with Treponema pallidum antigen Tp47 and other potential interfering substances (HCV Core, BSA) was quantitatively analyzed by calculating the cross-reactivity (CR) to verify the specificity of the detection method.

[0071] Test parameters and conditions are as follows:

[0072] Label antigen working concentration: 2 μg / mL;

[0073] Competitor gradient: 0, 0.01, 0.1, 1, 10, 100 μg / mL (covering 6 orders of magnitude for standard inhibition curve construction);

[0074] Negative control (0 competitor) signal: ~ 250,000 RLU;

[0075] Duplicate well setting: 3 duplicate wells (n = 3) were set for all concentration points to ensure data reliability;

[0076] Incubation conditions: usually incubated at 37°C for 30-60 minutes, or at room temperature for 1-2 hours (the specific time and temperature were determined according to the antibody affinity optimization);

[0077] Detection instrument: chemiluminescence immunoassay analyzer (Shine i2900).

[0078] The experimental method is as follows: 50 μL of different concentrations of competitors (gradient dilutions) and 50 μL of labeled gp41 solution (2 μg / mL) are sequentially added to the microwell plate coated with anti-gp41 antibody or magnetic beads containing the capture antibody. Mix well, incubate at the specified temperature in the dark, and allow the competition reaction to reach equilibrium. Multiple washes (e.g., 5 times) are performed using a plate washer or manually to remove unbound labeled antigen and competitors. Add chemiluminescent substrate to trigger the luminescence reaction, and immediately read the relative light units (RLU) on a chemiluminescence detector. Record the RLU value of each duplicate well. The electrophoresis results are shown in Figure 2 The specific and cross-reactive competition inhibition experiment results are shown in Figure 3 .

[0079] By Figure 2 It can be seen that there is a uniform band near the 41KD molecular weight size position, and the target protein is expressed correctly. By Figure 3 It can be seen that the gp41 antigen shows excellent specificity (self-competition ICSO-0.18 μg / mL), and the cross-reactivity with syphilis Tp47 is only 0.18%, which meets the diagnostic requirements. Therefore, the gp41 recombinant antigen shows excellent specificity and low cross-reactivity.

[0080] Sequence optimization of HIV-1 gp41 protein, retaining the core immunogenic segment (such as the neutralizing epitope ELDKWAS), deleting the region known to trigger non-specific reactions (such as the homologous sequence cross-reactive with Treponema pallidum), can specifically induce neutralizing antibodies against HIV-1; at the same time, due to the removal of the hydrophobic transmembrane region and the introduction of a flexible linker, its water solubility is significantly improved, and a purification tag is added, which can be efficiently expressed in prokaryotic or eukaryotic systems and easily purified. The recombinant antigen needs to meet the following requirements at the same time: conservation (broad-spectrum), immunogenicity (detection sensitivity), low non-specificity (detection specificity), high solubility (expression feasibility), and structural stability (storage effectiveness), and there is often a conflict between these goals (such as conservation and immunogenicity, solubility and stability). Solving the above difficulties relies on the tedious and repeated experimental process of prediction-verification-reoptimization. Through sequence optimization (retaining the conserved region, removing the hydrophobic transmembrane region, and adding a purification tag), combined with process innovation (direct coupling, complex stabilizer), the prepared HIV gp41 antigen-ALP marker has been significantly improved in sensitivity, specificity, and stability.

[0081] (2) SDS-PAGE electrophoresis results of the purified ALP-gp41 marker

[0082] The SDS-PAGE electrophoresis results of the purified ALP-gp41 marker obtained in Example 3 are shown in Figure 4 .

[0083] By Figure 4 It can be seen that the reduced part has a uniform band near the positions of 41KD, 70KD and 60KD, respectively corresponding to gp41 recombinant antigen, ALP dimer monomer (molecular weight of about 70KD and 60KD), and non-reduced band position is about 170KD, there is a uniform band, which is ALP-gp41 marker mixture, that is, according to a specific connection ratio, one gp41 recombinant antigen can be connected on one ALP.

[0084] (3) Stability of ALP-gp41 marker

[0085] The stability of the ALP-gp41 marker obtained in Example 4 was evaluated under three conditions of long-term storage at 37°C after adding a stabilizer, repeated freeze-thaw cycles, and long-term storage at 37°C without adding a stabilizer to determine the best storage and processing method of the sample.

[0086] Experimental conditions and parameters:

[0087] Basic sample (as is): The original sample is directly frozen at -80°C or -20°C without any treatment.

[0088] Stress condition setting:

[0089] a. The original sample with stabilizer was placed at 37°C for 7 days; temperature: 37.0±1.0°C; time: 7 days (168 hours).

[0090] b. The original sample was repeatedly frozen and thawed 3 times: freezing condition: ≤-20°C, not less than 12 hours / time; thawing condition: room temperature (25°C) or 4°C water bath, completely thawed.

[0091] c. The original sample without stabilizer was placed at 37°C for 7 days: parameters: same as condition 1.

[0092] Experimental method: Take the original sample (as is) and immediately detect to obtain the baseline value (as is). Group 1 (with stabilizer, 37°C): Add a specified volume of stabilizer to another group of original sample samples, mix well and place in a 37°C incubator for 7 days. Group 2 (repeated freeze-thaw): Take a group of original sample samples and complete 3 complete freeze-thaw cycles. After the last thawing, take samples for detection. Group 3 (without stabilizer, 37°C): Take a group of original sample samples and directly place them in a 37°C incubator for 7 days (without adding any protective agent). All samples in each group are detected using the same detection kit and instrument within the same batch after processing to obtain RLU values. Calculate the relative deviation percentage of each sample under different processing conditions relative to its "as is". The results are shown in Table 1.

[0093] Table 1 Stability test results of ALP-gp41 marker

[0094]

[0095] Table 1 is the result of accelerated stability test and repeated freeze-thaw experiment after adding stabilizer in the labeling mixture. As can be seen from Table 1, after adding the stabilizer, the activity decreases by 4.96% at 37℃ for 7 days, and the activity of the label can still reach about 95% compared with the original sample; after repeated freeze-thaw for 3 times, the activity decreases by 1.03% compared with the original sample, and basically reaches 99%; without adding stabilizer, the activity of the label decreases by 25.01% after 7 days of 37℃ test compared with the original sample. Therefore, the results can show that the composite stabilizer provided by the present application can significantly improve the thermal stability and freeze-thaw resistance of the enzyme label.

[0096] (4) Activity of ALP-gp41 label

[0097] The relative activity of the alkaline phosphatase-gp41 (ALP-gp41) label obtained in Example 2 and Comparative Example 1 was quantitatively compared to evaluate the influence of different preparation methods on the final activity of the label.

[0098] Experimental conditions and parameters:

[0099] Label working concentration: the same dilution (such as 1:1000 dilution) was used for the labels of Example 2 and Comparative Example 1.

[0100] Detection mode: sandwich chemiluminescence immunoassay (CLIA).

[0101] Sample volume: usually 50-100 μL per step (label, sample, etc.).

[0102] Incubation conditions: 37℃ incubation for 30 minutes.

[0103] Washing times: 5-6 times.

[0104] Detection instrument: chemiluminescence immunoassay instrument (Shine i2900).

[0105] Signal reading: relative light units (RLU) were read, and the integration time was usually 0.5-1 second per well.

[0106] Experimental method: the magnetic bead plate coated with capture antibody was taken out, and the same volume of serial samples (S1-S6, P3-P12, etc.) was added. The labels of Example 2 and Comparative Example 1 were added to different wells on the same plate, and the corresponding working concentration of the labels was added, mixed, incubated at a specified temperature in the dark, and the washing steps were completed using a plate washer to completely remove unbound substances. Freshly prepared chemiluminescent substrate solution was added to each well, and the RLU value was immediately read on a chemiluminescence instrument. The comparison results are shown in Table 2.

[0107] Activity comparison of ALP-gp41 labels prepared in Example 2 and Comparative Example 1

[0108]

[0109]

[0110] From Table 2, it can be seen that Example 2 can achieve the directional coupling between ALP and gp41, because the connection point is pre-designed. Due to the specificity and directionality of the connection, the proportion of the formed conjugate ALP-gp41 is more controllable (close to 1:1), the product is more uniform, which helps to maintain the native conformation and biological activity of both. In Comparative Example 1, glutaraldehyde randomly connects any nearby amino groups, which can cause serious ALP self-crosslinking, gp41 self-crosslinking and the formation of complex ALP-gp41 multimers, and the product is a highly heterogeneous mixture. In addition, these large cross-linked polymers can produce significant steric hindrance, further hindering the substrate from approaching the active center, resulting in a significant decrease in enzyme activity, and may also mask the key antigen epitopes, reducing the binding ability of the label to the target antibody. Therefore, while preparing the ALP-gp41 label, the activity of the label obtained in Comparative Example 1 is only about 60% of the activity of the label obtained in Example 2, a decrease of 39%. The results show that the directional coupling method of the present application can significantly improve the activity of the enzyme label compared with the traditional glutaraldehyde random coupling method, and the activity is increased by more than 40%.

[0111] The above shows and describes the basic principles, main features and advantages of the present application. Those skilled in the art should understand that the above examples do not limit the present application in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of the present application.

Claims

1. An enzyme-labeled antigen for HIV diagnosis, characterized in that, A gp41 recombinant antigen with a sequence shown as SEQ ID NO: 1 is subjected to a directed coupling reaction with an enzyme, and the enzyme is alkaline phosphatase.

2. The enzyme-labeled antigen for HIV diagnosis according to claim 1, characterized in that, The mass ratio of the alkaline phosphatase and the gp41 recombinant antigen is (1:0.5)-(1:1).

3. The method for preparing an enzyme-labeled antigen for HIV diagnosis according to claim 1, characterized in that, The method comprises the following steps: S1, genetically engineering expression and purification of an antigen: obtaining a gp41 recombinant antigen with a sequence shown as SEQ ID NO: 1; S2, modification of the antigen: modifying the lysine ε-amino group of the gp41 recombinant antigen by using Traut's reagent; S3, activation of alkaline phosphatase: activating the alkaline phosphatase by using SMCC; S4, directed coupling reaction: mixing the modified gp41 recombinant antigen and the activated alkaline phosphatase to perform a directed coupling, and obtaining an enzyme-labeled mixture; S5, stability optimization: adding a composite stabilizer to the enzyme-labeled mixture after chromatographic purification, and obtaining an HIV recombinant antigen-alkaline phosphatase labeled antigen.

4. The production method according to claim 3, characterized by, In the step S2, the molar ratio of the gp41 recombinant antigen and Traut's reagent is (1:10)-(1:20).

5. The production method according to claim 3, wherein In the step S3, the molar ratio of the alkaline phosphatase and SMCC is (1:10)-(1:20).

6. The preparation method according to claim 3, characterized in that, In the step S4, the mass ratio of the alkaline phosphatase and the gp41 recombinant antigen is (1:0.5)-(1:1).

7. The preparation method according to claim 3, characterized in that, In the step S5, the specific method of chromatographic purification is as follows: first, desalting or dialysis treatment is performed to remove small molecular impurities; and then, size exclusion chromatography is performed to separate the target labeled antigen with uniform molecular weight and remove free gp41 recombinant antigen and free alkaline phosphatase.

8. The preparation method according to claim 3, characterized in that, In the step S5, the concentration of the enzyme-labeled mixture after purification is 1.5-2.5 mg / mL, and the composite stabilizer comprises the following components: 0.5%-1% bovine serum albumin, 3%-5% sucrose, 0.05%-0.2% trehalose, 0.5-1 mM magnesium ion, and 0.1% preservative.

9. Use of the enzyme-labeled antigen of claim 1 in the preparation of a reagent or medical device for diagnosing HIV infection.

10. A test kit for HIV, characterized by, The kit comprises the enzyme-labeled antigen of claim 1.

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