阅读说明：本技术 一种全人源化抗乙肝病毒单克隆抗体、制备方法及其应用 (Fully humanized anti-hepatitis B virus monoclonal antibody, preparation method and application thereof ) 是由 宋相伟 王雪丽 张丽辉 辛树权 于 2020-06-01 设计创作，主要内容包括：本发明公开了一种全人源化抗乙肝病毒单克隆抗体、制备方法及其应用,属于生物医学及基因工程抗体技术领域,包括重链可变区和轻链可变区；所述重链可变区的氨基酸序列如SEQ ID NO.2所示；所述轻链可变区的氨基酸序列如SEQ ID NO:4所示；该抗体的编码DNA,包括重链可变区和轻链可变区；所述重链可变区的编码DNA序列如SEQ ID NO.1所示；所述轻链可变区的编码DNA序列如SEQ ID NO:3所示。本发明通过EBV病毒将B细胞永生化,结合有限稀释法筛选天然全人源乙肝病毒单克隆抗体的方法,该筛选方法无需大型昂贵仪器,简单易行,适于在常规生物实验室进行开展。(The invention discloses a fully humanized anti-hepatitis B virus monoclonal antibody, a preparation method and application thereof, belonging to the technical field of biomedicine and genetic engineering antibodies and comprising a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4; the encoding DNA of the antibody comprises a heavy chain variable region and a light chain variable region; the coding DNA sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the coding DNA sequence of the light chain variable region is shown in SEQ ID NO. 3. The method for screening the natural fully human hepatitis B virus monoclonal antibody by the EBV virus immortalizing the B cell and combining the limiting dilution method does not need large expensive instruments, is simple and easy to implement and is suitable for being developed in a conventional biological laboratory.)

1. A fully humanized anti-hepatitis B virus monoclonal antibody is characterized by comprising a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown as SEQID NO. 4.

2. The DNA encoding the fully humanized anti-hepatitis B virus monoclonal antibody of claim 1, which comprises a heavy chain variable region and a light chain variable region; the coding DNA sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the coding DNA sequence of the light chain variable region is shown in SEQ ID NO. 3.

3. The method for preparing the fully humanized anti-hepatitis B virus monoclonal antibody according to claim 1, comprising the following steps:

(1) and separating peripheral blood lymphocytes: selecting healthy people who have been injected with hepatitis B vaccine for approximately three to six months and have hepatitis B surface antigen, e antigen negativity and hepatitis B surface antibody positivity as B cell providers; extracting 30 ml of peripheral blood of a provider, separating peripheral blood lymphocytes (PBMC) by using lymphocyte separating medium (Histopaque-1077), and re-suspending the cells by using Duchen Phosphate Buffer Solution (DPBS);

(2) and B cell separation: treating the DPBS Cell suspension with a Dead Cell Removal kit (MACS) to remove Dead cells; centrifuging for 5 minutes at 300 r/min, and discarding the supernatant; b cells were isolated using the B cell isolation kit (MACS).

(3) B cell immortalization: preparing a B cell infection culture medium; resuspend B cells with this medium to a concentration of 100 cells/mL and inoculate in a 96-well plate, 200 μ Ι _ suspension/well; mitomycin C inactivated PBMC was also used as feeder cells in 5% CO₂Cell cloning can occur after 10 days of culture at 37 ℃;

(4) screening positive B cells secreting HBV antibody: coating with hepatitis B virus surface antigen S, and detecting supernatant of B cell culture by ELISA method;

(5) screening monoclonal B cells: screening monoclonal cells from the cells with positive supernatant by using a limiting dilution method, transferring the cells into a 96-well plate, and continuously culturing for 7-10 days;

(6) detecting the supernatant obtained in the culture step (5) by using an ELISA method again;

(7) and obtaining the variable region gene of the fully human HBV antibody: b cells with positive supernatant detection are subjected to RNA extraction, cDNA synthesis and sequencing to obtain gene sequences of variable region codes of heavy chains and light chains of the antibodies;

(8) and purifying the fully human HBV antibody protein: the obtained variable region coding sequence is subjected to expression vector construction, is transfected into CHO cells for expression, and is subjected to antibody purification by using proterin A, so that the fully human anti-hepatitis B virus monoclonal antibody is obtained after purification.

4. The method for preparing a fully humanized anti-hepatitis B virus monoclonal antibody according to claim 3, wherein the B cell infection medium in step (3) is 100mL of complete medium: 0.5mLCpG2006:1mL EBV.

5. The use of the fully humanized anti-hepatitis b virus monoclonal antibody of claim 1 in the preparation of a medicament or diagnostic agent for the prevention or treatment of hepatitis b virus-related liver disease.

Technical Field

The invention belongs to the technical field of biomedicine and genetic engineering antibodies, and particularly relates to a fully humanized anti-hepatitis B virus monoclonal antibody, a preparation method and application thereof.

Background

Statistically, about 20 million people worldwide are infected with Hepatitis B Virus (HBV), about 3 to 5 million people suffer from chronic HBV infection, and 100 to 150 million patients worldwide die from cirrhosis, liver failure and liver cancer caused by acute or chronic HBV infection every year. Of these, 25% -40% eventually die of cirrhosis and liver cancer (HCC). The World Health Organization (WHO) reported that hepatitis b accounted for the seventh of the top 10 disease causes of death worldwide.

Hepatitis b treatment can be divided into two main categories: one is antiviral therapy and the other is immunotherapy. Antiviral therapeutic agents mainly include interferon-alpha (IFN α) and nucleoside drugs such as interferon-alpha, peginterferon-alpha, lamivudine, adefovir dipivoxil, entecavir and telbivudine. The drugs have the problems of long treatment period, low efficiency, easy generation of drug resistance of viruses and the like. Therefore, researchers at home and abroad are also researching the treatment of hepatitis B by using an immune method, wherein the monoclonal antibody treatment method is one of immunotherapy. The early application of immunization for treating Hepatitis B mainly utilizes blood-derived Hepatitis B Immunoglobulin (HBIG). HBIG is an immunoglobulin against the S protein of hepatitis B virus surface antigen isolated from human serum or plasma. According to literature reports, HBIG can inhibit virus particles from infecting host cells by blocking the combination of hepatitis B virus S protein and hepatocyte surface receptors; or blocking viral infection by inhibiting the release of S protein and viral particles from the cell. The traditional method for preparing HBIG is to screen out high-titer blood plasma from blood of normal people and obtain the HBIG through low-temperature ethanol purification. Although the obtaining method is very effective, the quality control is complicated because the obtained good immunoglobulin is particularly dependent on high-titer plasma, and the immunoglobulin is a blood product and is directly derived from human blood, so that the method has potential risk of blood-borne disease infection. Therefore, hepatitis B immunoglobulin immunotherapy has certain limitations in clinical application.

In recent years, antibody drugs have become popular drugs in the global drug market with their high specificity. The monoclonal antibody as one kind of antibody has three unique action mechanisms, including targeting effect, blocking effect and signal conducting effect, and is used mainly in treating tumor, autoimmune disease and infectious disease, especially cancer. Monoclonal antibodies (monoclonal antibodies) are the most important biotechnological products after vaccines and recombinant proteins, and are the strategic advances in the fields of biotechnology and biomedical industry in the 21 st century, because monoclonal antibodies have been successfully applied to the treatment of tumors, autoimmune diseases, infectious diseases, transplant rejection, and the like. The monoclonal antibody technology develops for 35 years and goes through four stages of murine, chimeric, humanized and fully human monoclonal antibodies. All sequences of the fully human antibody are human sequences, and the fully human antibody has the characteristics of small side effect, long half-life period, remarkable treatment effect and the like, and gradually becomes the mainstream direction of antibody drug development.

Compared with the immunotherapy antibody of hepatitis B immunoglobulin directly separated by blood, the non-serum source fully human monoclonal antibody has safer, more accurate and more effective treatment prospect. The monoclonal antibody with therapeutic action developed by using unique biological characteristics of monoclonal antibody medicine can bring the medicine for curing hepatitis B into a new era.

Disclosure of Invention

Aiming at the defects of the drugs for treating hepatitis B in the prior art, the invention aims to provide a fully human anti-hepatitis B virus monoclonal antibody with neutralizing activity, and lays a foundation for the development of drugs for treating hepatitis B.

The invention is realized by the following technical scheme:

a fully humanized anti-hepatitis B virus monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 2; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.

The coding DNA of the fully humanized anti-hepatitis B virus monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the coding DNA sequence of the heavy chain variable region is shown in SEQ ID NO. 1; the coding DNA sequence of the light chain variable region is shown as SEQID NO. 3.

A method for preparing a fully humanized anti-hepatitis B virus monoclonal antibody comprises the following steps:

(1) separating peripheral blood lymphocyte, selecting healthy people injected with hepatitis B vaccine for about three to six months and having hepatitis B surface antigen, e antigen negative and hepatitis B surface antibody positive as B cell provider; extracting 30 ml of peripheral blood of a provider, separating peripheral blood lymphocytes (PBMC) by using lymphocyte separating medium (Histopaque-1077), and re-suspending the cells by using Duchen Phosphate Buffer Solution (DPBS);

(2) and B cell separation: treating the DPBS Cell suspension with a Dead Cell Removal kit (MACS) to remove Dead cells; centrifuging for 5 minutes at 300 r/min, and discarding the supernatant; b cells were isolated using the B cell isolation kit (MACS).

(3) B cell immortalization: preparing a B cell infection culture medium (100mL of complete culture medium: 0.5mLCpG2006:1 mLEBV); resuspend B cells with this medium to a concentration of 100 cells/mL and inoculate in a 96-well plate, 200 μ Ι _ suspension/well; mitomycin C inactivated PBMC was also used as feeder cells in 5% CO₂Cell clones appeared after about 10 days of culture at 37 ℃.

(4) Screening positive B cells secreting HBV antibody: hepatitis B virus surface antigen S was used for coating, and supernatant from B cell culture was examined by ELISA.

(5) Screening monoclonal B cells: and (4) screening monoclonal cells of the cells with positive supernatant by using a limiting dilution method, transferring the cells into a 96-well plate, and continuously culturing for 7-10 days.

(6) And (5) detecting the supernatant obtained in the step (5) by ELISA again.

(7) And obtaining the variable region gene of the fully human HBV antibody: and (3) carrying out RNA extraction, cDNA synthesis and sequencing on the B cells with positive supernatant detection to obtain gene sequences of variable region codes of the heavy chain and the light chain of the antibody.

(8) And purifying the fully human HBV antibody protein: the obtained variable region coding sequence is subjected to expression vector construction, is transfected into CHO cells for expression, and is subjected to antibody purification by using proterin A, so that the fully human anti-hepatitis B virus monoclonal antibody is obtained after purification.

The invention also provides the application of the fully humanized anti-hepatitis B virus monoclonal antibody in preparing a medicament or a diagnostic reagent for preventing or treating hepatitis B virus related liver diseases.

Compared with the prior art, the invention has the following advantages:

the invention provides a method for screening natural fully human hepatitis B virus monoclonal antibody by EBV virus B cell immortalization and combining with a limiting dilution method, the screening method does not need large expensive instruments, is simple and easy to implement, and is suitable for being developed in conventional biological laboratories.

Drawings

FIG. 1 is a SDS-PAGE detection image of Protein A purified fully human HBV antibody;

FIG. 2 is a diagram showing morphological changes of HBV neutralizing cells of fully human monoclonal antibody;

in the figure: a is normal HepRG cells; b is HBV infected HepRG cell; c is HBV monoclonal antibody 0.3 mug/mL; d is HBV monoclonal antibody 3 mug/mL;

FIG. 3 is a graph showing the measurement of HBV nucleic acid content in the neutralizing activity of HBV fully human antibody;

in the figure: con is normal HepRG cells; m: HepRG cells added with HBV; s1, 0.3 mu g/mLHBV monoclonal antibody; s2, 3 mu g/mL HBV monoclonal antibody.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.