A hepatitis B virus-specific peptide and tetramer and a kit for detecting hepatitis B virus-specific CD8+T cells

Abstract

The present invention relates to a hepatitis B virus-specific peptide segment, a tetramer and a kit for detecting hepatitis B virus-specific CD8+T cells, and belongs to the field of medical technology. The present invention provides a specific hepatitis B virus peptide segment, and the sequence of the peptide segment is shown in SEQ ID NO.1. A tetramer is constructed according to the peptide segment, and the obtained tetramer is stable, has a high detection rate for hepatitis B virus-specific CD8+T cells, and provides a basis for the detection of hepatitis B virus-specific immune cells in the later stage.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a hepatitis B virus-specific peptide segment and tetramer and a kit for detecting hepatitis B virus-specific CD8+T cells. Background Art

[0002] Viral hepatitis B is an infectious disease caused by the hepatitis B virus and mainly causes liver lesions. Clinically, it manifests as loss of appetite, nausea, upper abdominal discomfort, liver pain, and fatigue. Some patients have jaundice, fever, and hepatomegaly. Some patients may develop chronic diseases and even cirrhosis, and a few may develop liver cancer. It can be seen that viral hepatitis B should not be underestimated. Early detection and early treatment can reduce the losses of hepatitis B patients.

[0003] Hepatitis B virus-specific T cells are closely related to chronic hepatitis B virus infection and immune-mediated liver damage and disease progression. Hepatitis B virus-specific CD8+T cells are the main effector cells of host immune control. The methods used to detect hepatitis B virus-specific CD8+T cells at this stage are histocompatibility antigen (HLA)-peptide tetramers combined with flow cytometry, mass spectrometry, enzyme-linked immunospot technology, etc., but mass spectrometry and enzyme-linked immunospot (Elispot) technology require mass spectrometry flow cytometers or enzyme-linked immunospot analyzers, which have high equipment requirements, and the detection rate and sensitivity of hepatitis B virus-specific CD8+T cell detection need to be further improved. The detection of hepatitis B virus-specific CD8+T cells using histocompatibility antigen (HLA)-peptide tetramers combined with flow cytometry is restricted by HLA typing, and there is currently a lack of HLA-peptide tetramers that can identify the common genotypes of hepatitis B virus A, B, C, and D. Based on this, the present invention is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a hepatitis B virus-specific peptide segment and tetramer and a kit for detecting hepatitis B virus-specific CD8+T cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a hepatitis B virus-specific peptide segment, the sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a tetramer of a hepatitis B virus-specific peptide segment, comprising the peptide segment.

[0008] Preferably, the preparation method of the tetramer is:

[0009] (1) mixing the peptide fragment with dimethyl sulfoxide to obtain a peptide solution with a concentration of 8 to 12 mg / mL;

[0010] (2) Mix the peptide solution with Quick Switch Tm , peptide exchange factors, and incubate in dark for 3.5-4.5 hours to obtain a protein complex;

[0011] (3) The protein complex is mixed with streptavidin-phycoerythrin to obtain a tetramer.

[0012] Preferably, the peptide solution and Quick Switch Tm The volume ratio of the mixture is 1:45-55;

[0013] The volume ratio of the peptide solution to the peptide exchange factor mixture is 1:1-2.

[0014] Preferably, the peptide exchange factor is sodium azide buffer;

[0015] The concentration of the sodium azide buffer is 0.06-0.08 wt %.

[0016] Preferably, the molar concentration ratio of the protein complex to streptavidin-phycoerythrin is 4-5:1.

[0017] The present invention also provides a kit for detecting hepatitis B virus-specific CD8+T cells, the kit comprising the following components:

[0018] Tetramer, 96-well culture plate, negative control, positive control, culture medium, washing solution, CD3 labeled with Percp-Cy5.5, and CD8 labeled with APC.

[0019] Preferably, the negative control is prepared by extracting peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulating the peripheral blood mononuclear cells with stimulator 2, adding interleukin-2 on the third day of stimulation culture, and continuing stimulation culture for 3 to 5 days to obtain a negative control;

[0020] The stimulant 2 includes the following components:

[0021] 1640 medium, interleukin-12, and bovine serum albumin;

[0022] The mass volume ratio of interleukin-12 to 1640 culture medium is 1 μg: 9-11 mL;

[0023] The mass volume ratio of the bovine serum albumin to the 1640 culture medium is 1 g: 9-11 mL;

[0024] The volume ratio of the stimulator 2 to the peripheral blood mononuclear cells is 1:0.8-1.2;

[0025] The concentration of peripheral blood mononuclear cells is 2 to 4×10 6 Pieces / mL;

[0026] The final concentration of interleukin-2 is 50 IU / mL.

[0027] Preferably, the preparation method of the positive control is: extracting peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulating the peripheral blood mononuclear cells with a stimulator 3, adding interleukin-2 on the third day of stimulation culture, and continuing the stimulation culture for 3 to 5 days to obtain a positive control;

[0028] The stimulant 3 comprises the following components:

[0029] 1640 medium, interleukin-12, bovine serum albumin, and phytohemagglutinin;

[0030] The mass volume ratio of interleukin-12 to 1640 culture medium is 1 μg: 9-11 mL;

[0031] The mass volume ratio of the bovine serum albumin to the 1640 culture medium is 1 g: 9-11 mL;

[0032] The mass volume ratio of the plant lectin to 1640 culture medium is 25 μg: 9-11 mL;

[0033] The volume ratio of the stimulator 3 to the peripheral blood mononuclear cells is 1:0.8-1.2;

[0034] The concentration of peripheral blood mononuclear cells is 2 to 4×10 6 Pieces / mL;

[0035] The final concentration of interleukin-2 is 50 IU / mL.

[0036] Preferably, the culture medium is 1640 culture medium containing fetal bovine serum;

[0037] The final concentration of the fetal bovine serum is 9-11wt%;

[0038] The washing solution is phosphate buffered saline.

[0039] The present invention provides a hepatitis B virus-specific peptide segment and tetramer and a kit for detecting hepatitis B virus-specific CD8+T cells. The peptide segment of the present invention is specific to the hepatitis B virus polymerase. After being prepared into a tetramer, it can be used to detect the hepatitis B virus CD8+T cell immune response reaction. The tetramer is prepared into a kit for flow cytometry detection of hepatitis B virus, and the detection rate of hepatitis B virus detection is higher than that of the Elispot method. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the histogram of hepatitis B virus-specific tetramer detected by flow cytometry.

[0041] Figure 2 This is a scatter plot of flow cytometry detection of hepatitis B virus-specific CD8+T cells in HLA-A2 positive patients.

[0042] Figure 3 This is the frequency chart of hepatitis B virus-specific CD8+T cells in clinical positive samples. DETAILED DESCRIPTION

[0043] The present invention provides a hepatitis B virus-specific peptide segment, the sequence of which is shown in SEQ ID NO. 1. In the present invention, the peptide segment is a peptide segment of segments 422 to 430 of hepatitis B virus polymerase; the sequence of the peptide segment is NLSWLSLDV, and the peptide segment is highly conserved in hepatitis B virus genotypes A, B, C, and D. In the present invention, the peptide segment is a synthetic peptide segment.

[0044] The present invention also provides a hepatitis B virus-specific peptide tetramer, comprising the peptide segment.

[0045] In the present invention, the preparation method of the tetramer is:

[0046] (1) mixing the peptide fragment with dimethyl sulfoxide to obtain a peptide solution with a concentration of 8 to 12 mg / mL;

[0047] (2) Mix the peptide solution with Quick Switch Tm , peptide exchange factors, and incubate in dark for 3.5-4.5 hours to obtain a protein complex;

[0048] (3) The protein complex is mixed with streptavidin-phycoerythrin to obtain a tetramer.

[0049] In the present invention, the concentration of the peptide solution is preferably 10 m / mL; the peptide solution and the Quick Switch Tm The volume ratio of the mixture is 1:45-55, preferably 1:50; the volume ratio of the peptide solution to the peptide exchange factor mixture is 1:1-2, preferably 1:1.5. In the present invention, the peptide exchange factor is a sodium azide buffer; the concentration of the sodium azide buffer is 0.06-0.08wt%, preferably 0.07wt%.

[0050] In the present invention, the molar concentration ratio of the protein complex and streptavidin-phycoerythrin is 4-5:1, preferably 4.5:1.

[0051] The present invention also provides a kit for detecting hepatitis B virus-specific CD8+T cells, the kit comprising the following components:

[0052] Tetramer, 96-well culture plate, negative control, positive control, culture medium, washing solution, CD3 labeled with Percp-Cy5.5, and CD8 labeled with APC.

[0053] In the present invention, the preparation method of the negative control is as follows: extract peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulate the peripheral blood mononuclear cells with a stimulator 2, add interleukin-2 on the third day of stimulation culture, and continue stimulation culture for 3 to 5 days to obtain a negative control; the stimulator 2 includes the following components: 1640 culture medium, interleukin-12 and bovine serum albumin; the mass volume ratio of interleukin-12 to 1640 culture medium is 1 μg: 9 to 11 mL, preferably 1 μg: 10 mL; the mass volume ratio of bovine serum albumin to 1640 culture medium is 1 g: 9 to 11 mL, preferably 1 g: 10 mL; the volume ratio of the stimulator 2 to the peripheral blood mononuclear cells is 1: 0.8 to 1.2, preferably 1: 1.0; the concentration of the peripheral blood mononuclear cells is 2 to 4×10 6 / mL, preferably 3×10 6 / mL; the final concentration of interleukin-2 is 50IU / mL. .

[0054] In the present invention, the preparation method of the positive control is as follows: extract peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulate the peripheral blood mononuclear cells with a stimulant 3, add interleukin-2 on the third day of stimulation culture, continue stimulation culture for 3 to 5 days, and obtain a positive control; the stimulant 3 comprises the following components: 1640 culture medium, interleukin-12, bovine serum albumin and phytohemagglutinin; the mass volume ratio of the interleukin-12 to the 1640 culture medium is 1 μ g:9-11mL, preferably 1μg:10mL; the mass volume ratio of bovine serum albumin to 1640 culture medium is 1g:9-11mL, preferably 1g:10mL; the mass volume ratio of phytohemagglutinin to 1640 culture medium is 25μg:9-11mL, preferably 25μg:10mL; the volume ratio of stimulator 3 to peripheral blood mononuclear cells is 1:0.8-1.2, preferably 1:1.0; the concentration of peripheral blood mononuclear cells is 2-4×10 6 / mL, preferably 3×10 6 / mL; the final concentration of interleukin-2 is 50IU / mL.

[0055] In the present invention, the culture medium is 1640 culture medium containing fetal bovine serum; the final concentration of the fetal bovine serum is 9-11wt%, preferably 10wt%; and the washing solution is phosphate buffer.

[0056] In the present invention, the detection method of the kit comprises the following steps:

[0057] Peripheral blood mononuclear cells are extracted from a blood sample, and the peripheral blood mononuclear cells and the tetramer are incubated in a culture medium for 25 to 40 minutes to obtain an intermediate; the intermediate is washed 1 to 2 times in a washing solution, mixed with CD3 labeled with Percp-Cy5.5 and CD8 labeled with APC, and detected by flow cytometry.

[0058] In the present invention, the method for extracting peripheral blood mononuclear cells from a blood sample is Ficoll density gradient centrifugation; the mass ratio of the peripheral blood mononuclear cells to the tetramer added is 1:1; and the incubation temperature is 35-39°C, preferably 37°C.

[0059] In the present invention, the detection method of the kit is flow cytometry. The detection kit and the detection method of the present invention are for non-disease diagnosis purposes.

[0060] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0061] Example 1

[0062] The peptide NLSWLSLDV of the 422-430 segment of the hepatitis B virus polymerase was artificially synthesized for the experiment. The peptide was mixed with dimethyl sulfoxide to prepare a peptide solution with a concentration of 8-12 mg / mL; 1 μL of the peptide solution was mixed with 50 μL of Quick Switch Tm Mix with 1 μL of peptide exchange factor (sodium azide solution with a concentration of 0.08 wt%) and incubate at 37°C in the dark for 4 h to obtain a protein complex. Take 10 μL of the protein complex and mix with 2 μL of streptavidin-phycoerythrin to obtain a tetramer. Flow cytometry was used to detect the quality and quantity of the tetramer. The test results are as follows: Figure 1 shown.

[0063] from Figure 1 It can be seen that stable tetramers can be synthesized using the peptide segments and methods of the present invention.

[0064] Example 2

[0065] Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples of patients with hepatitis B using Ficoll density gradient centrifugation. PBMCs (3×10 6 On the third day of culture, interleukin-2 was added to make the concentration of interleukin-2 reach 50 IU / mL. The co-stimulation culture was continued for 7 days.

[0066] Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples of patients with hepatitis B using Ficoll density gradient centrifugation. PBMCs (3×10 6 On the third day of culture, interleukin-2 was added to make the concentration of interleukin-2 reach 50 IU / mL. The co-stimulation culture was continued for 7 days to obtain a negative control.

[0067] Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood samples of patients with hepatitis B using Ficoll density gradient centrifugation. PBMCs (3×10 6 On the third day of culture, 1 mL of interleukin-2 was added to make the concentration of interleukin-2 reach 50 IU / mL. The co-stimulation culture was continued for 7 days to obtain a positive control.

[0068] The stimulated peripheral blood mononuclear cells were incubated with the tetramer of Example 1 in a culture medium (1640 culture medium containing 10 wt% fetal bovine serum) for 30 min to obtain an intermediate; the intermediate was washed twice in a phosphate buffer, mixed with CD3 labeled with Percp-Cy5.5 and CD8 labeled with APC, and detected by flow cytometry using positive control and negative control as controls. The flow cytometric graph of peripheral blood mononuclear cell expression is shown in FIG. Figure 2 As shown in A; forward scatter (FSC) VS side scatter (SSC) dot plot, set the gate to select lymphocytes and the results are as follows Figure 2 As shown in B; forward scatter (FSC) VS Percp-Cy5.5-labeled CD3 dot plot, set the gate to select CD3+T lymphocytes, the results are as follows Figure 2 As shown in C; APC-labeled CD8 VS tetramer dot plot, set a gate to select tetramer-positive CD8+ T lymphocytes, the results are as follows Figure 2 As shown in D.

[0069] Figure 2It shows that the method of the present invention can detect hepatitis B virus-specific CD8+T cells.

[0070] Example 3

[0071] 15 patients with acute hepatitis B and 45 patients with chronic hepatitis B were selected. The 60 patients were tested using the method of Example 2 and the Elispot method was used to compare the number of hepatitis B virus-specific CD8+T cells detected by the two methods. The final results are shown in Table 1 and Figure 3 The clinical sample patient data are shown in Table 2.

[0072] Table 1 Comparison of the detection results of hepatitis B patients by two different methods

[0073]

[0074] Table 1 shows that the number of positive patients detected by the method of Example 2 is more than that of the positive patients detected by the Elispot method. According to the positive detection rate = (positive detection number / total detection number) × 100%, the positive detection rate of the method of Example 2 is 45%, and the detection rate of the Elispot method is 33.3%.

[0075] Table 2 Patient data

[0076]

[0077]

[0078]

[0079] The "neg" indicates a negative test result, and the "pos" indicates a positive test result; M indicates male, and F indicates female; AHB indicates acute hepatitis B, and CHB indicates chronic hepatitis B; and NA indicates that the value cannot be detected.

[0080] As can be seen from the above embodiments, the present invention provides a kit for detecting hepatitis B virus-specific CD8+T cells, and the kit. The peptide segment of the present invention is specific to the hepatitis B virus polymerase. After being prepared into a tetramer, it can be used to detect the hepatitis B virus-specific CD8+T cell immune response. The tetramer is prepared into a kit for flow cytometry detection of hepatitis B virus-specific CD8+T cells, which has a higher detection rate than the Elispot method. It provides a scientific basis for the detection of virus-specific immune responses in patients.

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A tetramer of a hepatitis B virus-specific peptide segment, characterized in that: comprising the peptide segment; The sequence of the peptide segment is shown in SEQ ID NO.1; The preparation method of the tetramer is: (1) mixing the peptide fragment with dimethyl sulfoxide to obtain a peptide solution with a concentration of 8 to 12 mg / mL; (2) Mix the peptide solution with Quick Switch Tm , peptide exchange factors, and incubate in dark for 3.5-4.5 hours to obtain a protein complex; (3) mixing the protein complex with streptavidin-phycoerythrin to obtain a tetramer; The peptide solution and Quick Switch Tm The volume ratio of the mixture is 1:45-55; The volume ratio of the peptide solution to the peptide exchange factor mixture is 1:1-2; The peptide exchange factor is sodium azide buffer; The concentration of the sodium azide buffer is 0.06-0.08 wt %; The molar concentration ratio of the protein complex to streptavidin-phycoerythrin is 4-5:

1.

2. A kit for detecting hepatitis B virus-specific CD8+T cells, characterized in that: The kit comprises the following components: The tetramer of claim 1, a 96-well culture plate, a negative control, a positive control, a culture solution, a washing solution, CD3 labeled with Percp-Cy5.5, and CD8 labeled with APC; The preparation method of the negative control is as follows: extracting peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulating the peripheral blood mononuclear cells with a stimulator 2, adding interleukin-2 on the third day of stimulation culture, and continuing the stimulation culture for 3 to 5 days to obtain a negative control; The stimulant 2 includes the following components: 1640 medium, interleukin-12, and bovine serum albumin; The mass volume ratio of interleukin-12 to 1640 culture medium is 1 μg: 9-11 mL; The mass volume ratio of the bovine serum albumin to the 1640 culture medium is 1 g: 9-11 mL; The volume ratio of the stimulator 2 to the peripheral blood mononuclear cells is 1:0.8-1.2; The concentration of peripheral blood mononuclear cells is 2 to 4×10 6 Pieces / mL; The final concentration of interleukin-2 is 50 IU / mL; The preparation method of the positive control is as follows: extracting peripheral blood mononuclear cells from a blood sample of a hepatitis B patient, stimulating the peripheral blood mononuclear cells with a stimulator 3, adding interleukin-2 on the third day of stimulation culture, and continuing the stimulation culture for 3 to 5 days to obtain a positive control; The stimulant 3 comprises the following components: 1640 medium, interleukin-12, bovine serum albumin, and phytohemagglutinin; The mass volume ratio of interleukin-12 to 1640 culture medium is 1 μg: 9-11 mL; the mass volume ratio of bovine serum albumin to 1640 culture medium is 1 g: 9-11 mL; the mass volume ratio of phytohemagglutinin to 1640 culture medium is 25 μg: 9-11 mL; the volume ratio of stimulator 3 to peripheral blood mononuclear cells is 1: 0.8-1.2; The concentration of peripheral blood mononuclear cells is 2 to 4×10 6 Pieces / mL; The final concentration of interleukin-2 is 50 IU / mL; The culture medium is 1640 culture medium containing fetal bovine serum; The final concentration of the fetal bovine serum is 9-11wt%; The washing solution is phosphate buffered saline.

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