Human cytomegalovirus UL135 protein antibody and its preparation method and application

Abstract

The present application proposes a human cytomegalovirus UL135 protein antibody and its preparation method and application, which relate to the field of immune prevention and treatment technology. The preparation method is: according to the sequence of the human cytomegalovirus UL135 gene, an antigenic epitope is selected, and the gene sequence of the antigenic epitope is shown in SEQ ID NO.1; the antigenic epitope polypeptide is synthesized in solid phase using the above antigenic epitope and purified; the above antigenic epitope polypeptide is immunized in animals to produce human cytomegalovirus UL135 antiserum; the above human cytomegalovirus UL135 antiserum is purified to obtain a specific UL135 protein antibody. The present application develops an HCMV tissue detection antibody based on the HMCV latent infection-related protein UL135, and the protein antibody can be used for the detection of latent HCMV infection in tumor tissue. It has the advantages of high operational sensitivity and high specificity, and is suitable for clinical immunohistochemistry detection.

Description

Technical Field

[0001] The present application relates to the technical field of immune prevention and treatment, and specifically to an antibody against human cytomegalovirus UL135 protein and a preparation method and application thereof. Background Art

[0002] Human cytomegalovirus (HCMV) belongs to the herpesviridae family and is an opportunistic pathogen. HCMV infection is quite common in the human population. HCMV exists in a latent form in various cells of the human body. In humans with a normal immune system, latent infection is not pathogenic, but HCMV infection can lead to serious consequences, including death, in immunocompromised populations such as pregnant women and infants. In recent years, an increasing number of studies have found that chronic HCMV infection is associated with various diseases, including tumors. HCMV infection can occur in multiple organs and induce HCMV-related malignancies. HCMV infects a variety of adult stem cells, expressing HCMV proteins associated with latency, promoting stem cell proliferation, causing genomic instability, and leading to precancerous lesions (such as colon polyps) and cancer. HCMV can infect neurons and glial stem cells in the brain, enhancing the survival potential of stem cells and increasing the risk of cancer. IE1 protein maintains the survival of glioblastoma stem cells by inducing the expression of Sox-2, Nanog and Oct3 / 4; pUS28 chemokine receptor induces the expression of COX-2, IL-6 and the activation of NF-κB and STAT-3, driving the oncogenic pathway; vIL-10 chemokine is expressed in latent tumor cells and can increase the invasiveness of tumor cells.

[0003] Detection methods for HCMV primarily include serological testing, viral isolation and culture, and nucleic acid testing. Viral isolation and culture is the gold standard for laboratory diagnosis of HCMV. Specimens such as urine, saliva, and cerebrospinal fluid can be collected for testing. The presence of cytomegalovirus inclusion bodies in the specimen after inoculation is considered a positive result, indicating the presence of cytomegalovirus in the body. However, because HCMV only proliferates slowly in human fibroblasts, testing takes 7-21 days and is prone to contamination, making it currently used only in research. Serological and nucleic acid testing are important tools for the clinical diagnosis of cytomegalovirus infection. However, due to the high prevalence of HCMV infection in the human population, serological and nucleic acid testing cannot accurately reflect HCMV infection in tumor tissue. Therefore, a rapid and sensitive tissue HCMV detection method is urgently needed. Currently, clinically used HCMV immunohistochemical detection antibodies use HCMV PP65 as the antigen. PP65 is a late protein expressed by HCMV and an early marker of active HCMV infection. However, HCMV in tumor tissues is mostly latently infected, and the positive rate of PP65 detection is extremely low, which cannot accurately reflect the situation of latent HCMV infection in tumor tissues. Summary of the Invention

[0004] To solve the problem in the prior art that PP65 is used as an antigen to detect HCMV with a low positive rate and cannot accurately reflect the status of latent HCMV infection in tumor tissues, the present application provides a human cytomegalovirus UL135 protein antibody and its preparation method and application.

[0005] In order to solve the above technical problems, the technical solutions adopted in this application are:

[0006] In one aspect, the present application provides a method for preparing an antibody against human cytomegalovirus UL135 protein, comprising the following steps:

[0007] S1. Select an antigenic epitope based on the sequence of the human cytomegalovirus UL135 gene, wherein the amino acid sequence of the antigenic epitope is shown in SEQ ID NO.1;

[0008] S2. solid-phase synthesis of antigen epitope polypeptides using the above antigen epitopes and purification;

[0009] S3, immunizing animals with the antigen epitope polypeptide to produce human cytomegalovirus UL135 antiserum;

[0010] S4. Purify the human cytomegalovirus UL135 antiserum to obtain specific UL135 protein antibodies.

[0011] On the other hand, the present application provides an antibody against human cytomegalovirus UL135 protein prepared by the above preparation method.

[0012] In another aspect, the present application also provides a use of an antibody against human cytomegalovirus UL135 protein in a kit for detecting latent HCMV infection in tumor tissue.

[0013] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0014] This application develops an HCMV tissue detection antibody based on the HMCV latent infection-associated protein UL135. This protein antibody can be used to detect latent HCMV infection in tumor tissues. Compared to traditional detection methods using HCMV PP65 as an antigen, this application has the advantages of high sensitivity and specificity, making it suitable for clinical immunohistochemical testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The three-dimensional protein structure of the alphafold of human cytomegalovirus UL135 in Example 1 of this application is predicted;

[0017] Figure 2 This is the antigen score curve of human cytomegalovirus UL135 in Example 1 of the present application;

[0018] Figure 3 This is a graph showing the antibody titer of the serum from rabbits immunized with the polypeptide from Example 1 of this application using an ELISA.

[0019] Figure 4 This is a WB detection image of the UL135 antibody prepared in Example 1 of the present application;

[0020] Figure 5 This is a diagram of the clinical sample analysis and detection of the antibody in Example 2 of this application. DETAILED DESCRIPTION

[0021] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0022] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0023] A method for preparing an antibody against human cytomegalovirus UL135 protein comprises the following steps:

[0024] S1. Select an antigenic epitope based on the sequence of the human cytomegalovirus UL135 gene, wherein the amino acid sequence of the antigenic epitope is shown in SEQ ID NO.1;

[0025] S2. solid-phase synthesis of antigen epitope polypeptides using the above antigen epitopes and purification;

[0026] S3, immunizing animals with the antigen epitope polypeptide to produce human cytomegalovirus UL135 antiserum;

[0027] S4. Purify the human cytomegalovirus UL135 antiserum to obtain specific UL135 protein antibodies.

[0028] In some embodiments of the present application, the above-mentioned step S1 specifically involves using alphafold to predict the protein structure of human cytomegalovirus UL135 and select antigenic epitopes.

[0029] In some embodiments of the present application, the purity of the antigen polypeptide in the above step S2 is above 95%.

[0030] In some embodiments of the present application, a step of verifying whether the animal immunization is successful is also included between the above steps S3 and S4.

[0031] In some embodiments of the present application, the above-mentioned step S4 further includes a verification experiment step of whether the UL135 protein antibody is specific.

[0032] A human cytomegalovirus UL135 protein antibody is prepared by the above method.

[0033] Application of an antibody against human cytomegalovirus UL135 protein in a kit for detecting latent HCMV infection in tumor tissue.

[0034] The features and performance of the present application are further described in detail below with reference to the embodiments.

[0035] Example 1

[0036] A human cytomegalovirus UL135 protein antibody, the preparation method of which is:

[0037] S1. The amino acid sequence of the human cytomegalovirus UL135 gene (MVWLWLGVGLLGGTGLASLVLAISLFTQRRGRKRSDETSSRGRLPGA ASDKRGACACCYRIPKEDVVEPLDLELGLMRVATHPPTPQVPRCTSLYIGEDGLPIDKPEFPPARFEIPDVSTPGTPTSIGRSPSHCSSSSSLSSSASVDTVLHQPPPSWKPPPPPGRKKRPPTPPVRAPTTRLSSHRPPTPIPAPRKNLSTPPTKKTPPPTKPKPVGWTPPVTPRPFPKTPTPQKPPRNPRLPRTVGLENLSKVGLSCPCPRPRTPTEPTTLPIVSVSELAPPPRWSDIEELLEKAVQSVMKDAESMQMT, where LWLGVGLLGGTGLASLVLAISLF is the transmembrane region) was analyzed, and the three-dimensional protein structure was predicted using alphafold, as shown in FIG. Figure 1 As shown, and possible antigenic epitopes are predicted. Figure 2 The curve graph shows the antigen score, among which SEQ ID NO.1 (amino acid sequence is PTKPKPVGWTPPVTPR) has good antigenicity and is used as the preferred antigen epitope of the present application to continue the experiment.

[0038] S2. Solid-phase synthesis of the epitope peptide using the above epitope and purification to a purity of over 95%. The specific synthesis and purification steps are as follows: 1) Weigh dichlororesin, add it to a reactor, swell it with dichloromethane for 10 minutes, and remove the dichloromethane solvent. 2) Dissolve a mixture of F-moc amino acid and DIEA (2M) in dichloromethane and add it to the reactor for 1 hour. 3) Wash with dichloromethane 3-5 times and block with blocking solution (DCM:MEOH:DIEA = 17:2:1) for 10-15 minutes. After blocking, wash with dichloromethane 3-5 times and DMF 3-5 times, each for 1 minute. 4) Cleave F-moc using a 20% piperidine solution (piperidine:DMF = 1:4) for 30 minutes. Wash with DMF 3-5 times, each for 1 minute. 5) Weigh another amino acid and dissolve a mixture of HBTU and DIEA in a ratio of 1:1:2 in DMF. Add the mixture to the reactor and allow to react for 2 hours. Wash with DMF 3-5 times. Cleave the Fmoc residue with 20% piperidine for 30 minutes, then wash with DMF 3-5 more times (repeat this step for subsequent amino acids). 6) Add a cleavage agent (95% TFA + 2.5% H2O + 2.5% TIS) to the reactor and allow to cleave for 30-45 minutes. 7) Collect the cleavage agent and wash it with a 1% TFA solution in dichloromethane 3-5 times, 1 minute each time. Collect the entire solution and evaporate to dryness on a rotary evaporator. Add anhydrous ether to the dried residue. If a solid precipitates, blow off the ether and collect the solid, which is the synthesized peptide. 9) LC-MS: Confirm the quality and purity of the product.

[0039] S3. Eight (2.5 ± 0.2) kg New Zealand white rabbits were randomly divided into an immunization group and a PBS control group. 1 ml of Freund's complete adjuvant was thoroughly mixed with 1 ml of the prepared antigen epitope peptide to produce a milky white color. The rabbits were carefully removed from their cages and injected into four sites on the back of each rabbit, 250 μl per site. The needle was inserted 1-2 cm subcutaneously at a 45-degree angle, and the injection was held for several seconds to prevent antigen leakage. The above immunization steps were repeated four times, with weekly immunizations and blood samples collected seven days after immunization.

[0040] S4. Add 100 μl of 1 μg / ml UL135 protein to each well of a 96-well plate and incubate at 37°C for 2 hours. Discard the antigen solution and add 200 μl of blocking solution to each well. Incubate at 37°C for 2 hours. Discard the blocking solution and tap the plate on absorbent paper to remove as much residual liquid as possible. Wash the plate three times with washing solution, patting as much residual liquid as possible each time. If storing for a period of time, dry the plate at 37°C and seal it in a sealed bag at -20°C. Add 100 μl of negative reference solution to the first well, 100 μl of positive reference solution to the second well, and the test antiserum at a 1:500 dilution to the third well. Make serial dilutions of the test antiserum in subsequent wells. Incubate at 37°C for 1 hour. Discard the solution, wash the plate three times with washing solution, and pat dry. Add 100 μl of HRP-conjugated mouse anti-rabbit IgG (1:2000 dilution) to each well and incubate at 37°C for 45 minutes. Discard the solution, wash the plate three times with washing solution, and pat dry. Add 100ul of TMB substrate to each well and incubate at 37℃ for 20min. Add 50ul of stop solution (2NH2SO4) to each well and read the absorbance at 450nm on a microplate reader. Analyze the antibody titer. The results are as follows: Figure 3 As shown, from Figure 3 Elisa results showed that the antigen-immunized serum had a good recognition titer for UL135 protein, indicating that the polypeptide immunization in the rabbit in step S3 successfully produced an immune response and antibodies were successfully produced in the rabbit serum, and the next step of blood collection and purification could be continued.

[0041] At week 5, rabbits were anesthetized and blood was collected from the heart. The collected venous blood was allowed to stand at 37°C for 1 hour, then centrifuged at 1500 × g for 15 minutes, and the supernatant was collected. 1 mg of agarose gel was added to 2 mmol / L hydrochloric acid to obtain 3 ml of swollen gel. The gel was transferred to a purification column and the medium was washed three times with approximately 20 ml of 2 mmol / L hydrochloric acid. The medium was washed once with coupling buffer. 10 mg of polypeptide antigen dissolved in 5 ml of coupling buffer was added to the gel. Gently mix and shake at room temperature for 2-4 hours. The medium was washed once with 20 ml of coupling buffer. 15 ml of 1% BSA solution was added and incubated at room temperature for 2 hours. The medium was washed three times with phosphate buffered saline (PBS), 15 ml each time. The antigen affinity column was washed with 10 volumes of PBS to equilibrate the column. 10 ml of diluted antiserum was added to the equilibrated column. Gently mix and shake at room temperature for 2-4 hours. The antigen column was washed with 10 volumes of PBS to remove contaminants bound to the column. Wash the column with 2 volumes of antibody elution buffer to obtain specific antibodies.

[0042] S6. Construct HCMV UL135 overexpression plasmid and transfect tumor cells. UL135 protein is expressed 48 hours later. After total protein extraction, WB analysis is performed. The UL135 antibody purified in step S5 is used as the primary antibody for detection to verify its antibody specificity. Figure 4As shown, Figure 4 WB detection showed that the UL135 antibody could specifically recognize the two subtypes of UL135 protein, and no nonspecific bands appeared, indicating that the S5 step successfully prepared a specific human cytomegalovirus UL135 protein antibody.

[0043] Example 2

[0044] This example verifies the use of the human cytomegalovirus UL135 protein antibody prepared in Example 1 in a kit for detecting latent HCMV infection in tumor tissue.

[0045] Pathological sections of clinical patients were collected and placed in a 65°C oven for 2 hours. They were then soaked in xylene three times for 5 minutes each time. They were then soaked in graded alcohol (95%, 90%, 80%, and 70%), each gradient allowing to stand for 5 minutes. They were then soaked in distilled water for 5 minutes. The sections were placed in a pressure cooker containing citrate buffer and heated under high pressure until the valve core began to spray air for 3 minutes. The sections were then cooled to room temperature until the valve core stopped spraying air. The pressure cooker was opened and rinsed with running water to cool the pressure cooker. When the water temperature dropped to 50°C, the sections were removed and washed with distilled water. 3% H2O2 was added and incubated at room temperature for 10 minutes. The sections were then washed three times with PBST buffer for 5 minutes each time. P-type cholinesterase inhibitors were used for the analysis of the pathological sections. BST buffer, add 5% goat serum for blocking, incubate at room temperature for 30 minutes; remove goat serum, add purified UL135 antibody, incubate at room temperature for 3 hours, rinse 3 times with PBST; add secondary antibody, incubate at room temperature for 30 minutes, rinse 3 times with PBST; incubate with DAB colorimetric solution for 10 minutes; hematoxylin staining for 5 minutes, rinse 3 times with PBST buffer, differentiate with hydrochloric acid alcohol for 3 seconds, rinse with tap water for 5 minutes; soak in gradient alcohol (70%, 80%, 90%, 95%, 100%), let each gradient stand for 5 minutes, soak in xylene for 5 minutes, wait for the slices to dry, and seal with neutral resin for storage. Analyze the detection efficiency of clinical samples of antibodies, such as Figure 5 The results showed that the UL135 antibody showed specific staining in tumor tissues and was localized in the cytoplasm, indicating that the HCMV tissue detection antibody developed in this application based on the HMCV latent infection-related protein UL135 can detect HCMV in tumor tissues.

[0046] In summary, the human cytomegalovirus UL135 protein antibody, preparation method, and application described in the embodiments of this application have the following advantages: This application develops an HCMV tissue detection antibody based on the HMCV latent infection-associated protein UL135, which can be used to detect latent HCMV infection in tumor tissue. Compared to traditional detection methods using HCMV PP65 as an antigen, this application has the advantages of high operational sensitivity and specificity, making it suitable for clinical immunohistochemical testing.

[0047] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Claims

1. A method for preparing an antibody against human cytomegalovirus UL135 protein, characterized in that: The following steps are involved: S1. Select an antigenic epitope based on the sequence of the human cytomegalovirus UL135 gene, wherein the amino acid sequence of the antigenic epitope is shown in SEQ ID NO.1; S2. solid-phase synthesis of antigen epitope polypeptides using the above antigen epitopes and purification; S3, immunizing animals with the antigen epitope polypeptide to produce human cytomegalovirus UL135 antiserum; S4. Purify the human cytomegalovirus UL135 antiserum to obtain specific UL135 protein antibodies.

2. The method for preparing an antibody against human cytomegalovirus UL135 protein according to claim 1, characterized in that: The S1 step specifically involves using alphafold to predict the protein structure of human cytomegalovirus UL135 and select antigenic epitopes.

3. The method for preparing an antibody against human cytomegalovirus UL135 protein according to claim 1, characterized in that: The purity of the antigen polypeptide in step S2 is above 95%.

4. The method for preparing an antibody against human cytomegalovirus UL135 protein according to claim 1, characterized in that: Between steps S3 and S4, there is also a step of verifying whether the animal immunization is successful.

5. The method for preparing an antibody against human cytomegalovirus UL135 protein according to claim 1, characterized in that: The S4 step also includes a verification experiment step of whether the UL135 protein antibody is specific.

6. An antibody against human cytomegalovirus UL135 protein, characterized in that: The method according to any one of claims 1 to 5 is used for preparation.

7. Use of the human cytomegalovirus UL135 protein antibody according to claim 6 in a kit for detecting latent HCMV infection in tumor tissue.

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