Hepatitis B PreS1 antigen as well as preparation method and application thereof

Abstract

The invention relates to the technical field of immunological detection, in particular to a hepatitis B PreS1 antigen as well as a preparation method and application thereof. The preparation method comprises the following steps: firstly, selecting an active PreS1 polypeptide sequence, coupling the active PreS1 polypeptide sequence to a hepatitis B surface antigen HBsAg molecule through a cross-linking agent to obtain a hepatitis B PreS1 antigen with hepatitis B surface antigen activity and PreS1 antigen activity, the hepatitis B PreS1 antigen can be used as a calibrator and a positive control in a hepatitis B PreS1 antigen detection kit, the kit uses an anti-PreS1 antibody as a coating, and the detection sensitivity is high. And an anti-HBsAg antibody is used as an enzyme label. The artificial coupling hepatitis B PreS1 antigen prepared by the invention can effectively replace a PreS1 real positive sample used in the market at present, has no biological safety risk, is stable in source, has various indexes such as reactivity, vehicle-mounted stability and thermal stability superior to those of the real positive sample, and has relatively high innovativeness and applicability.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological detection, and particularly relates to hepatitis B PreS1 antigen, a preparation method thereof and an application thereof. Background Art

[0002] Hepatitis B virus serum markers (HBVM) as a conventional item for hepatitis B virus diagnosis can no longer meet the clinical needs. HBV-DNA, viral surface antigen (HBsAg), and hepatitis B virus preS1 (PreS1) antigen are all used as evidence of HBV infection and replication in the human body. In recent years, the detection of preS1 antigen can better reflect the HBV replication situation than HBeAg. Therefore, the preS1 antigen, as an index of HBV virus infection and replication, is more sensitive than HBeAg and has the value of independent detection, playing an important supplementary role in the detection of the "five items (two pairs and a half)" markers of HBV.

[0003] The outer membrane proteins of hepatitis B virus (HBV) include three components: S, preS2, and preS1. The preS1 (PreS1) antigen is a pre-protein encoded by the preS1 region of the hepatitis B virus genome, with strong immunogenicity and being an important component of hepatitis B surface antigen (HBsAg). The preS1 (PreS1) antigen protein plays an important role in the process of the virus invading hepatocytes. The most important mediating site for the virus to attach to hepatocytes is the amino acid (AA) 21-47 fragment of the preS1 protein. As long as this section of the mutated virus is intact, it is infectious. The protein containing preS1 mainly exists in Dane particles and tubular particles. The preS1 protein plays a very important role in virus infection, assembly, replication, and stimulating the body to produce an immune response. The hepatitis B virus (HBV) preS1 antigen (Pres1-Ag) is located on the outer membrane protein of the hepatitis B virus. It exists on the surface of intact HBV particles and is closely related to the infection and replication of HBV. Therefore, it is of great significance to develop a detection method for hepatitis B virus preS1 antigen.

[0004] The detection of hepatitis B virus preS1 antigen is a scientific research achievement completed by the Shanghai Institute of Biochemistry, Chinese Academy of Sciences. It passed the national certification in 2000 and was approved by the State Food and Drug Administration for clinical use in December of the same year. Tertiary hospitals in the vast majority of regions across the country have carried out the detection of S1 antigen. The official launch of the hepatitis B virus preS1 antigen detection kit into the clinic and its combined application with the hepatitis B "two pairs and a half" detection in nearly a hundred hospitals in Beijing, Sichuan, Zhejiang, Henan, Shenzhen, Hainan and other places in recent years have fully demonstrated the important value of this reagent in the clinical diagnosis and treatment guidance of viral hepatitis B. The detection of preS1 antigen (Pre-S1Ag) is an important supplement and enhancement to the hepatitis B "two pairs and a half", especially the determination of e antigen and HBV-DNA.

[0005] However, the calibrators and positive controls currently used in hepatitis B virus pre-S1 antigen detection reagents are both prepared by adding high-value true pre-S1 positives, which have many problems. For example, they have limited sources, are difficult to screen, carry storage risks, and pose significant biosafety risks. Moreover, during use, they have low titers and poor stability, seriously affecting the popularization and use of hepatitis B virus pre-S1 antigen detection methods. Summary of the Invention

[0006] In view of this, the present invention provides a hepatitis B pre-S1 antigen, its preparation method, and its application. The present invention first selects an active pre-S1 polypeptide sequence and couples it to the hepatitis B surface antigen HBsAg molecule through a cross-linking agent to obtain a hepatitis B pre-S1 antigen that simultaneously has the activity of hepatitis B surface antigen and pre-S1 antigen. It can be used as a calibrator and positive control in a hepatitis B pre-S1 antigen detection kit. The kit uses an anti-pre-S1 antibody as the coating and an anti-HBsAg antibody as the enzyme label. The artificially conjugated hepatitis B pre-S1 antigen prepared by the present invention can effectively replace the currently used pre-S1 true positive samples in the market. It has no biosafety risks, has a stable source, and its various performance indicators such as reactivity, vehicle stability, and thermal stability are all superior to true positive samples, showing high innovation and applicability.

[0007] To achieve the above invention objectives, the present invention provides the following technical solutions:

[0008] The present invention provides a hepatitis B pre-S1 antigen, which includes hepatitis B surface antigen HBsAg, a cross-linking agent, and a pre-S1 polypeptide; the hepatitis B surface antigen HBsAg is connected to the pre-S1 polypeptide through the cross-linking agent; the amino acid sequence of the pre-S1 polypeptide is as shown in SEQ ID NO:1.

[0009] The present invention also provides a preparation method of the hepatitis B pre-S1 antigen, including: taking the cross-linking agent to activate the amino group in the hepatitis B surface antigen HBsAg, followed by dialysis to obtain the activated hepatitis B surface antigen HBsAg; taking the pre-S1 polypeptide and coupling it with the activated hepatitis B surface antigen HBsAg, followed by dialysis to obtain the hepatitis B pre-S1 antigen.

[0010] In some specific embodiments of the present invention, the cross-linking agent includes SMCC or EDC.

[0011] In some specific embodiments of the present invention, the use form of SMCC includes sulfo-SMCC.

[0012] In some specific embodiments of the present invention, the molar ratio of the cross-linking agent to the hepatitis B surface antigen HBsAg is (20~40):1.

[0013] In some specific embodiments of the present invention, the molar ratio of the PreS1 polypeptide to the hepatitis B surface antigen HBsAg is (10-20):1.

[0014] In some specific embodiments of the present invention, the initial concentration of the hepatitis B surface antigen HBsAg is 5 mg / mL.

[0015] In some specific embodiments of the present invention, the concentration of Sulfo-SMCC is 5 mg / mL.

[0016] In some specific embodiments of the present invention, the concentration of the activated hepatitis B surface antigen HBsAg is 4 mg / mL.

[0017] In some specific embodiments of the present invention, the concentration of the PreS1 polypeptide is 1 mg / mL.

[0018] In some specific embodiments of the present invention, the activation conditions include reacting in a light-protected environment at 25°C;

[0019] The temperature of the coupling is 25°C.

[0020] In some specific embodiments of the present invention, the activation time is 5 h.

[0021] In some specific embodiments of the present invention, the coupling time is 14 h.

[0022] In some specific embodiments of the present invention, the pore size of the dialysis membrane is 50 KD.

[0023] In some specific embodiments of the present invention, the volume ratio of the dialysis fluid to the sample during dialysis is ≥50:1; the number of dialysis times is 5 times.

[0024] In some specific embodiments of the present invention, the dialysis fluid includes 0.01 M PBS pH 7.2 buffer or 0.067 M PBS pH 6.7 buffer.

[0025] In some specific embodiments of the present invention, the hepatitis B PreS1 antigen is mixed with an equal volume of glycerol and stored at low temperature in the dark.

[0026] The present invention also provides the following applications as calibration products or positive control products:

[0027] (I), the hepatitis B PreS1 antigen; and / or

[0028] (II), the hepatitis B PreS1 antigen prepared by the preparation method.

[0029] The present invention also provides the use of any of the following items in the preparation of a hepatitis B PreS1 antigen detection kit:

[0030] (I), the hepatitis B PreS1 antigen described above; and / or

[0031] (II), the hepatitis B PreS1 antigen prepared by the preparation method described above.

[0032] The present invention also provides a product, including any of the following items:

[0033] (I), the hepatitis B PreS1 antigen described above; and / or

[0034] (II), the hepatitis B PreS1 antigen prepared by the preparation method described above.

[0035] The present invention includes, but is not limited to, providing the following beneficial effects:

[0036] The present invention first selects an active PreS1 polypeptide sequence, couples it to the hepatitis B surface antigen HBsAg molecule through a cross-linking agent, and obtains a hepatitis B PreS1 antigen that simultaneously has the activity of the hepatitis B surface antigen and the PreS1 antigen activity. It can be used as a calibrator and a positive control in a hepatitis B PreS1 antigen detection kit. This kit uses an anti-PreS1 antibody as the coating and an anti-HBsAg antibody as the enzyme label. The artificially conjugated hepatitis B PreS1 antigen prepared by the present invention can effectively replace the currently used PreS1 true positive samples in the market, has no biosafety risk, has a stable source, and various performance indicators such as reactivity, vehicle stability, and thermal stability are all superior to the true positive samples, and has high innovation and applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0038] Figure 1 Shows the synthesis process route of the hepatitis B PreS1 antigen. DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention discloses a hepatitis B PreS1 antigen, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications described in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0040] The object of the present invention is to provide a method for preparing an artificial conjugate of hepatitis B PreS1 antigen to replace the currently used PreS1 true positive samples in the market, which has no biosafety risk, stable source, and effectively promotes the application of hepatitis B PreS1 detection kits. An active PreS1 polypeptide sequence is selected, and according to the amino acid composition of the polypeptide sequence, different crosslinking agents are selected to be coupled into the hepatitis B surface antigen HBsAg molecule, resulting in a hepatitis B PreS1 antigen that simultaneously has the activity of hepatitis B surface antigen and PreS1 antigen, which can be used as a calibrator and positive control in the hepatitis B PreS1 antigen detection kit. The artificial hepatitis B PreS1 antigen prepared by the present invention can effectively replace the currently used PreS1 true positive samples in the market, has no biosafety risk, stable source, low cost, and its performance is superior to the currently used PreS1 true positive high value.

[0041] First, a PreS1 polypeptide with relatively high activity is screened out through ELISA experiments. According to the amino acid composition in the polypeptide sequence, EDC or SMCC coupling agents are selected to be linked into the HBsAg molecule. In the method of the present invention, the crosslinking agent selected is SMCC, which activates the amino group in the HBsAg molecule, introduces a maleimide group, and couples with the cysteine in the polypeptide molecule.

[0042] The PreS1 polypeptide sequence selected in the present invention is: CAFGANSNNPDWDFNP (SEQ ID NO:1). Because its cysteine C contains a mercapto group, it can be coupled using SMCC.

[0043] After the polypeptide sequence is selected, the method for preparing the artificial hepatitis B PreS1 antigen of the present invention is as follows:

[0044] First, the HBsAg antigen is dialyzed into PBS, and the actual concentration is detected for standby; weigh sulfo-SMCC (purchased from Thermo Fisher, product number: 22322), dissolve it in PBS to 1 mg / mL, add it to the HBsAg antigen according to a certain molar ratio, and react in a water bath at 25 °C in the dark for 5 h to activate the amino group in the HBsAg antigen molecule to transform into maleimide. This active functional group can selectively couple with the mercapto group in the polypeptide molecule. After the reaction is completed, dialyze it into PBS to remove the excess sulfo-SMCC. The dialysis ratio is not less than 1 / 50, and dialysis is performed five times; open the dialysis bag, transfer the HBsAg activated by SMCC to a glass bottle and record the volume, and detect the concentration for use.

[0045] Take 1 mg / vial of polypeptide (synthesized by Sangon Biotech), dissolve it in PBS to 1 mg / mL, and add it to the HBsAg antigen solution activated by SMCC according to the molar ratio of PreS1 polypeptide to HBsAg antigen of 10 / 1 to 20 / 1. React overnight in a water bath at 25°C. Utilize the cysteine sulfhydryl group in the polypeptide molecule to achieve site-specific coupling with SMCC. After the reaction, dialyze it with a 50KD dialysis bag into PBS to remove the excess polypeptide. The dialysis ratio is not less than 1 / 50, and dialysis is performed five times to thoroughly dialyze the polypeptide. Unfold the dialysis bag, transfer the PreS1 polypeptide-HBsAg conjugate to a glass bottle, record the volume, detect the concentration, add an equal volume of glycerol, and store it at -20°C.

[0046] In the present invention, by designing a highly active PreS1 polypeptide and for the first time through an artificial coupling method, the polypeptide is coupled into the hepatitis B surface antigen HBsAg molecule to obtain a hepatitis B PreS1 antigen that simultaneously has the activities of hepatitis B surface antigen and PreS1 antigen, which can be used as a calibrator and positive control in a hepatitis B PreS1 antigen detection kit. Its process flow chart is as Figure 1 shown.

[0047] Specifically, its advantages are reflected in the following aspects:

[0048] 1. The raw materials are sufficient in source, low in cost, and there is no risk of out-of-stock. The PreS1 polypeptide is synthesized by Sangon Biotech and has a moderate price. The HBsAg antigen can be recombinantly expressed, and the technology is relatively mature.

[0049] 2. The coupling process is simple, the conditions are mild. The designed polypeptide contains cysteine, and there is a sulfhydryl group available for site-specific coupling. After the HBsAg antigen is activated by SMCC, the finished product can be obtained by one-step coupling. The coupling process is carried out under neutral conditions and has almost no influence on the protein configuration.

[0050] 3. There is no biological risk during the preparation and use process. Currently, the calibrator and positive control used in the existing PreS1 antigen detection kit are both added with a high value of true positive PreS1, which has a relatively large biosafety risk and greater risks in use and storage.

[0051] 4. The finished product has good stability. By adding a high value of true positive PreS1 as the calibrator and positive control used in the PreS1 antigen detection kit, there are relatively serious stability problems. After being tested for 3 days in a vehicle and 7 days at 37°C, the decline rate is as high as 27 - 60%, which seriously affects the use and promotion of the PreS1 antigen detection kit. However, in the present invention, through artificial coupling, two stable components, the PreS1 polypeptide and HBsAg, are combined together by chemical bonds, and the obtained hepatitis B PreS1 antigen has good stability. After experimental assessment, the decline rate after being tested for 3 days in a vehicle and 7 days at 37°C is only less than 5%.

[0052] Unless otherwise specified, the raw materials and reagents used in the hepatitis B PreS1 antigen, its preparation method and application provided by the present invention can be purchased from the market.

[0053] The present invention will be further illustrated below with reference to the examples:

[0054] Preparation Example 1 Preparation of Artificially Coupled Hepatitis B PreS1 Antigen

[0055] First step, activation of HBsAg

[0056] Dialyze 5 mg / mL of HBsAg into 0.01 M PBS pH 7.2 with a dialysis ratio of not less than 1 / 50 for four times; open the dialysis bag in a transparent glass bottle, detect the concentration by the A280 1 Abs method, dilute it to 4 mg / mL, and record the mass and volume; take out Sulfo-SMCC (purchased from thermofisher, A39268), dissolve it in 0.01 M PBS pH 7.2 to form a 1 mg / mL solution after restoring to room temperature, add Sulfo-SMCC to the HBsAg solution according to the molar ratio of Sulfo-SMCC / HBsAg of 40 / 1, couple at 25°C in a water bath in the dark for 5 h, dialyze with 0.01 M PBS pH 7.2 buffer at 4°C for 16 h, and change the solution 4 times during dialysis to completely remove the residual Sulfo-SMCC. After dialysis, accurately measure the concentration of HBsAg-SMCC after dialysis by the A280 1 Abs method, and record the volume and concentration for standby.

[0057] Second step, preparation of PreS1 polypeptide solution

[0058] Take out 1 mg / branch of PreS1 polypeptide (purchased from Sangon Biotech), dissolve it in 0.01 M PBS pH 7.2 to form a 1 mg / mL solution after restoring to room temperature, mix well until it is colorless and transparent without floccules for standby.

[0059] Third step, cross-linking reaction of PreS1 polypeptide and HBsAg

[0060] Mix in a transparent glass bottle according to the molar ratio of PreS1 polypeptide to SMCC-activated HBsAg (HBsAg-SMCC) of 10 / 1, and couple at 25°C in a water bath for 14 h.

[0061] Fourth step, post-treatment and preservation

[0062] After the reaction, dialysis was carried out using a 50KD dialysis bag into 0.067M PBS pH6.7 buffer solution at 4°C for 16h. The solution was changed 4 times during dialysis to completely remove the residual PreS1 polypeptide. After dialysis, the concentration of HBsAg-SMCC-Peptide was accurately measured using the A280 1Abs method. The measurement was repeated three times, and the average value was recorded along with the volume. An equal volume of glycerol was added, mixed well, and stored at -20°C in the dark to obtain the hepatitis B PreS1 antigen.

[0063] Preparation Example 2 Preparation of Artificially Coupled Hepatitis B PreS1 Antigen

[0064] The first step, activation of HBsAg

[0065] Dialyze 5mg / mL of HBsAg into 0.01M PBS pH7.2 with a dialysis ratio of not less than 1 / 50, and dialyze four times; open the dialysis bag in a transparent glass bottle, detect the concentration using the A280 1Abs method, and dilute it to 4mg / mL, recording the mass and volume; take out Sulfo-SMCC (purchased from thermofisher, A39268), dissolve it in 0.01M PBS pH7.2 to form a 1mg / mL solution after restoring to room temperature, add Sulfo-SMCC to the HBsAg solution according to the molar ratio of Sulfo-SMCC / HBsAg of 20 / 1, couple at 25°C in a water bath in the dark for 5h, and dialyze with 0.01M PBS pH7.2 buffer solution at 4°C for 16h. The solution was changed 4 times during dialysis to completely remove the residual Sulfo-SMCC. After dialysis, accurately measure the concentration of HBsAg-SMCC after dialysis using the A280 1Abs method, record the volume and concentration, and set aside for use.

[0066] The second step, preparation of PreS1 polypeptide solution

[0067] Take out 1mg / vial of PreS1 polypeptide (purchased from Sangon Biotech), dissolve it in 0.01M PBS pH7.2 to form a 1mg / mL solution after restoring to room temperature, mix well until it is colorless and transparent without flocculants, and set aside for use.

[0068] The third step, cross-linking reaction of PreS1 polypeptide and HBsAg

[0069] Mix in a transparent glass bottle according to the molar ratio of PreS1 polypeptide to SMCC-activated HBsAg (HBsAg-SMCC) of 20 / 1, and couple in a water bath at 25°C for 14h.

[0070] The fourth step, post-treatment and storage

[0071] After the reaction, dialysis was carried out using a 50KD dialysis bag until the final 0.067M PBS pH6.7 buffer solution, and dialysis was carried out at 4°C for 16h. The solution was changed 4 times during dialysis to completely remove the residual PreS1 polypeptide. After dialysis, the concentration of HBsAg-SMCC-Peptide was accurately measured using the A280 1Abs method, detected three times, the average value was taken and the volume was recorded, and an equal volume of glycerol was added, mixed well, and stored at -20°C in the dark to obtain hepatitis B PreS1 antigen.

[0072] Example

[0073] Using the AutoLumo A2000 Plus hepatitis B virus pre-S1 antigen detection kit from Antu Biological, batch number: 20230929, the performance of the hepatitis B PreS1 antigen prepared in Preparation Examples 1 and 2 was detected. This method was compared with the addition of real positive high-value stability data of PreS1. After the hepatitis B PreS1 antigen and real positive high-value of PreS1 prepared in Preparation Examples 1 and 2 were respectively formulated into working solutions, the stability was evaluated after being carried in a vehicle for 3 days and at 37°C for 7 days. The results are shown in Table 1.

[0074] The results showed that by adding the real positive high-value of PreS1 as the calibration product and positive control used in the PreS1 antigen detection kit, there were relatively serious stability problems. After being carried in a vehicle for 3 days and at 37°C for 7 days, the reduction rate was 27 - 60%, which seriously affected the use and promotion of the PreS1 antigen detection kit. However, in the present invention, through artificial coupling, two stable components, PreS1 polypeptide and HBsAg, were combined together using chemical bonds, and the obtained hepatitis B PreS1 antigen had good stability. After experimental evaluation, the reduction rate after being carried in a vehicle for 3 days and at 37°C for 7 days was only less than 5%.

[0075] Table 1 Comparison of the stability data of the hepatitis B PreS1 antigen prepared in the present invention with the addition of real positive high-value of PreS1

[0076]

[0077] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Hepatitis B PreS1 antigen, characterized in that It comprises hepatitis B surface antigen HBsAg, a coupling agent and a PreS1 polypeptide; the hepatitis B surface antigen HBsAg is connected to the PreS1 polypeptide via the coupling agent; the amino acid sequence of the PreS1 polypeptide is shown in SEQ ID NO:

1.

2. The method for preparing hepatitis B PreS1 antigen according to claim 1, characterized in that: include: Taking the coupling agent to activate the amino group in the hepatitis B surface antigen HBsAg, dialyzing, and obtaining the activated hepatitis B surface antigen HBsAg; The PreS1 polypeptide is coupled with the activated hepatitis B surface antigen HBsAg, and dialyzed to obtain the hepatitis B PreS1 antigen.

3. The preparation method according to claim 2, characterized in that: The coupling agent includes SMCC or EDC.

4. The preparation method according to claim 3, characterized in that: The use form of SMCC includes sulfo-SMCC.

5. The preparation method according to any one of claims 2 to 4, characterized in that: The molar ratio of the coupling agent to the hepatitis B surface antigen HBsAg includes (20~40):

1.

6. The preparation method according to any one of claims 2 to 5, characterized in that: The molar ratio of the PreS1 polypeptide to the hepatitis B surface antigen HBsAg includes (10~20):

1.

7. The preparation method according to any one of claims 2 to 6, characterized in that: The activation conditions include reacting at 25°C in a dark environment; The coupling temperature includes 25°C.

8. Use of any of the following as calibrators or positive controls: (I) The hepatitis B PreS1 antigen as claimed in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 7.

9. Use of any of the following in the preparation of a hepatitis B PreS1 antigen detection kit: (I) The hepatitis B PreS1 antigen as claimed in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 7.

10. A product, characterized in that Include any of the following: (I) The hepatitis B PreS1 antigen as claimed in claim 1; and / or (II) Hepatitis B PreS1 antigen prepared by the preparation method according to any one of claims 2 to 7.

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