CN110642943A

CN110642943A - Nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen and application thereof

Info

Publication number: CN110642943A
Application number: CN201910664688.6A
Authority: CN
Inventors: 张军方
Original assignee: Shenzhen Winner Biotechnology Co Ltd
Current assignee: Shenzhen Winner Biotechnology Co Ltd
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2020-01-03

Abstract

The invention discloses a nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS 1). The invention obtains the nano antibody of anti-HBV-preS1 by screening the alpaca immunized by the HBV-preS 1. The invention also relates to fragments of the antibodies and complete antibodies fused with Fc, the antibodies are used for treating hepatitis B virus and liver cancer, and a method for preparing the antibodies.

Description

Nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen and application thereof

Technical Field

The invention relates to the technical field of antibody medicines, in particular to a nano antibody sequence of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS 1). The invention is used for diagnosing or treating hepatitis B and liver cancer.

Background

The invention relates to a nano antibody of anti-hepatitis B virus surface antigen pre-S1 antigen (HBV-preS1), which is used for diagnosing or treating hepatitis B and liver cancer.

After more than thirty years of development, therapeutic monoclonal antibody drugs have made great progress in the fields of tumors and autoimmune diseases, and are ready to become one of the important means for clinical treatment of such diseases. Development of therapeutic antibodies against HBV is expected to provide a new therapeutic strategy for chronic HBV infection.

Infection with Hepatitis B Virus (HBV) is a serious public health problem that faces globally. Statistics show that more than 20 million people worldwide are infected with HBV, of which about 3.5 million people are infected with Chronic HBV (CHB). Hepatitis b causes a serious economic burden on our country because of the extremely high infection rate of HBV infection and the mortality rate of liver cancer developed by patients with chronic hepatitis b ranked second among the total mortality rates of cancer in our country. Infection with HBV varies greatly from country to country and from region to region, with the majority of hepatitis b carriers concentrating in asia, africa and the western pacific. Therefore, hepatitis B virus infection and a series of liver diseases caused by hepatitis B virus infection, liver cirrhosis, liver cancer and the like become one of the public health problems all over the world.

Therefore, HBV infection, especially chronic HBV infection, and liver diseases such as chronic hepatitis, liver cirrhosis and primary liver cancer cause a heavy economic burden on patients. The currently approved drugs for clinical treatment of chronic hepatitis B include interferon and nucleoside analogs, which have positive effects on inhibiting virus replication and delaying disease progression, but still have the problems of long treatment period, low response rate, serious side effects (interferons) and potential drug resistance (nucleoside analogs), and the like, so that the development of anti-HBV drugs based on new targets or new mechanisms is urgently needed.

Small circular DNA (3182-3221) with HBV genome size of 3.2kb base pair (bp) is one of the smallest known eukaryotic cell DNA viruses. The HBV genome has 4 Open Reading Frames (ORFs): S-ORF, P-ORF, C-ORF, X-ORF. The S-ORF encodes three envelope proteins, SHBs (S), MHBs (preS2+ S) and LHBs (preS1+ preS2+ S). The C-ORF has a pre-C region (preC), a secreted protein (HBeAg) that plays an important role in the development of immune tolerance to chronic infections, and a C gene region that constitutes the viral Capsid and that envelops the viral genome, this segment of the HBV genome being the most conserved and targeted epitope for immune attack. The gene product HBx protein of X-ORF has transcription function of a transcription activation enhancer and a promoter, is a multifunctional trans-regulatory factor, and has important relation with virus infection, transcription, replication, apoptosis and HCC occurrence. The P-ORF is the longest ORF in the HBV genome, responsible for transcription of DNA polymerase. In addition, the respective open reading frames of HBV are highly overlapping, having a very compact structure.

The HBV genome contains 4 genes, 4 different mRNAs are transcribed and can encode and synthesize 9 different virus proteins, including outer membrane proteins (L-HBsAg, M-HBsAg and S-HBsAg) and structural proteins of nucleocapsid (HBcAg) of the assembled virus; 3P proteins (terminal protein, DNAp and RNase H) and HBx protein which regulates the replication function of the virus, and HBeAg protein encoded by another pc/C gene is also a functional protein.

Hepatitis B virus outer membrane protein (HBsAg), also known as Australian antigen, was first discovered by Blumberg in 1965 and was widely used to quantify the amount of HBsAg in serum to predict the level of HBV infection in patients. The HBV outer membrane protein is synthesized by S-ORF code, comprising 3 outer membrane components of L-HBsAg, M-HBsAg and S-HBsAg, 3 proteins have respective initial codes, but the termination codes of S-HBsAg are used together.

The core protein of HBV is encoded by the C-ORF (nucleotein), and has the form 2: one is the structural component of the virus nucleocapsid, HBcAg; the other is soluble, secreted HBeAg. The C gene of HBV has 2 related ORFs, in which two initiation codons are distinguished as Pre-C (Pre-C) and C region, but have a common stop codon. The Pre-C/P mRNA synthesizes the nucleocapsid protein HBcAg of P21, the Pre-C-mRNA generates P25Pre-C protein, and the protein becomes HBeAg after a series of treatments. HBcAg can be spontaneously assembled into a Capsid structure in different cell lines, such as bacteria, yeast, mammalian cells and the like, without other HBV related proteins or elements, and can be used as a vector of a genetic engineering vaccine.

According to the infection 2011 epidemic situation distribution report published by the ministry of health, the method comprises the following steps: 1.5 hundred million people break through in liver disease patients in China, wherein the chronic hepatitis B patients reach 4000-. At present, about 3000 thousands of liver cirrhosis patients in China exist, 4.7% of liver cirrhosis patients are finally transformed into liver cancer, about 30 thousands of people die of liver cancer every year, and China becomes a large country for liver disease occurrence.

The most major risk factor for the development of liver cancer is chronic HBV infection. In recent years, it has been discovered that viruses may play a direct role as mutagens, and researchers have discovered that viral DNA sequences are integrated into potential oncogenes from an increasing number of HCC cases. Several other factors are also associated with the risk of HCC development in patients with chronic hepatitis b, including alcohol, metabolic and environmental factors (e.g., aflatoxin), among others.

At present, human beings mainly take prophylactic vaccines as a strategy for treating HBV, but for patients with chronic hepatitis B, effective antiviral drugs are required to be found. Seven drugs have been approved by FDA for treating chronic hepatitis b in the past 20 years, and the first two drugs for treating hepatitis b are Interferon (Interferon-alfa-2b) and Lamivudine (Lamivudine), which have been well used in the last 90 th century. At the beginning of this century, five drugs were approved by the FDA for the treatment of hepatitis b: adefovir dipivoxil (adefovir dipivoxil), interferon (alfa-2 apoeg interferon alfa-2a), tenofovir disoproxil fumarate (tenofovir), entecavir (entecavir), telbivudine (telbivudine).

Single domain antibodies (SDAb), also known as nanobodies or heavy chain antibodies (hcAb), were originally isolated from camelids (alpacas) as a mutant form of antibody with a very small molecular weight of only 12-15kDa, but only about 1/12 the molecular weight of conventional antibodies. A single domain antibody, consisting of only heavy chains and no light chains, differs from the constitutive structure of conventional antibodies in that the Fab region is only a single domain, linked to the Fc region by a hinge region. The single-domain antibody has stable conformation and good water solubility, and can still be combined with antigen with high affinity in gastric juice and viscera. The single domain antibody has small molecular weight, so that the single domain antibody can recognize certain unique antigen epitopes, for example, the single domain antibody can enter the active site of enzyme or enter the crack of a bacterial or virus surface receptor, and the single domain antibody can be used for simulating drugs to design agonists, enzyme inhibitors or antagonists and the like of small molecule receptors.

Disclosure of Invention

The invention uses HBV-preS1 antigen to immunize alpaca, collects PBMC after separation and immunization, extracts total RNA and obtains cDNA, obtains antibody library by phage display technology, and further uses HBV-preS1 to screen and obtain nano antibody specifically reacting with the antibody. The nano antibody prepared by the invention aiming at HBV surface antigen pre-S1 antigen and CDR1, CDR2 and CDR3 sequences of the antibody composition are disclosed, the nano antibody can specifically react with HBV-preS1, can effectively reduce the content of HBV virus in a cell model and a mouse in vivo model, has an obvious effect of treating hepatitis B, and can block the conversion of chronic hepatitis B to liver cancer.

According to a first aspect of the present invention, there is provided a nanobody against HBV-preS1, which is obtained by immunizing alpaca with HBV-preS1, and screening using phage display technology.

Further, the molecule is a nanobody against hepatitis b virus.

Further, the above molecule is a nanobody against HBV-preS 1.

Further, the above molecules are screened by phage display technology.

According to a second aspect of the invention, the invention provides a molecule comprising a CDR1, CDR2, CDR3 sequence.

Further, the sequence of the molecular CDR1 is SEQ ID NO: 1 to SEQ ID NO: 12 all sequences.

Further, the sequence of the molecular CDR2 is SEQ ID NO: 13 to SEQ ID NO: 21 all sequences.

Further, the sequence of the molecular CDR3 is SEQ ID NO: 22 to SEQ ID NO: 30 all sequences.

According to the third aspect of the present invention, the molecule provided by the present invention has a significant therapeutic effect on hepatitis b and liver cancer.

Further, the above molecule has a therapeutic effect on hepatitis B virus.

Further, the above molecule can reduce HBsAg levels.

Further, the above molecules can reduce HBeAg levels.

Furthermore, the molecule can have a therapeutic effect on liver cancer.

Drawings

FIG. 1 is an electrophoretogram of PCR amplification products in one embodiment of the present invention (M: DNA marker 2000; lanes 1 and 2: amplification products);

FIG. 2 is a diagram showing a double-cut electrophoresis of pHEN1 and VHH in FIG. 2.4 in one embodiment of the present invention (M: DNA marker 2000; lane 1: pHEN 1; lane 2: pHEN1 after sfil/notI cleavage; lane 3: VHH after sfil/notI cleavage);

FIG. 3 shows colony PCR verified library insertion rates in one embodiment of the invention (M: DNA marker 2000; lanes 1-48: randomly picked clones);

FIG. 4 is a sequence of the selected nanobody;

FIG. 5 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the serum HBsAg level in HBV transgenic mice compared to the Control nanobody (Control VHH);

FIG. 6 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the serum HBeAg level in the HBV transgenic mouse compared to the Control nanobody (Control VHH);

FIG. 7 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention can significantly reduce the HBV DNA level in the HBV transgenic mouse compared to the Control nanobody (Control VHH);

FIG. 8 shows that the Anti-HBV surface antigen pre-S1 antigen nanobody (Anti-HBV-preS1VHH) screened according to an embodiment of the present invention significantly inhibits tumor growth of hepatoma carcinoma cells integrated with HBV genome in mice compared to the Control nanobody (Control VHH);

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.

Example 1: preparation of HBV-PRES1 antigen

The immunogen used by the experiment aiming at alpaca is hepatitis B surface antigen pre-S1 antigen HBV-preS1, an E.coli prokaryotic expression system is adopted, thalli are crushed and centrifuged, and a nickel column is used for separation and purification. The purified protein was quantitatively analyzed on a 12% polyacrylamide gel. And then subpackaging, freeze-drying and storing the purified protein at-80 ℃ (so as to avoid protein degradation caused by repeated freeze thawing).

Example 2: alpaca immunity and blood sampling

The Alpaca is immunized four times, HBV-preS1 is used for immunizing the Alpaca (Alpaca), the injection position is the cervical lymph node of the Alpaca, half of samples are respectively injected into the left lymph node and the right lymph node of the Alpaca, and the whole process of low-temperature preservation of protein and adjuvant is required to be carried out during the operation process, so that the protein degradation caused by repeated freeze thawing is avoided.

The primary immunization dose was 200ug mixed with complete Freund's adjuvant, boosting at week 2, 100ug HBV-PRES1 mixed with incomplete Freund's adjuvant, boosting at week 6, 100ug HBV-PRES1 mixed with incomplete Freund's adjuvant, boosting at week 10, and 100ug HBV-PRES1 mixed with incomplete Freund's adjuvant. 10ml of blood is taken before each immunization, EDTA is used for anticoagulation, and plasma and PBMC are respectively separated.

After the fourth immunization, 40ml of blood was taken from the alpaca, and each time, blood was taken from the jugular vein. The experiment selects 10ml anticoagulation blood collection tubes, if the blood sample needs to be placed for more than 2 hours and then the next operation is carried out, the blood sample placed in each tube should not exceed 5 ml; if the blood sample is processed further within 2 hours in time, no more than 8ml of blood sample is placed in each tube. When blood is drawn into the anticoagulation tube, the blood is required to be evenly mixed by immediately reversing for a plurality of times so as to avoid the coagulation reaction caused by uneven mixing.

Example 3: isolation of alpaca lymphocytes PBMC

Fresh blood samples should be stored at 18-20 deg.C and lymphocyte separation should be completed as soon as possible within 2-6 hours (preferably within 2 hours) after blood withdrawal. The procedure for lymphocyte isolation was as follows: 1) the Histopaque-1077 lymphocyte isolate was removed from the refrigerator at 4 ℃ one day in advance and allowed to stand at room temperature. 2) 5ml of the Histopaque-1077 lymphocyte isolate at room temperature was taken into a 15ml centrifuge tube, and 8ml of whole blood was carefully and slowly added above the surface of the Histopaque-1077 lymphocyte isolate. 3)400g, 20 ℃, the speed of rise is 0, the speed of fall is 0, and the centrifugation is carried out for 30 minutes. The centrifuged sample should be divided into four layers, which are serum, lymphocytes, and dextran and erythrocytes in the lymphocyte separation medium from top to bottom. The monocytes in the second turbid layer were carefully aspirated to avoid aspiration into the underlying liquid. The aspirated mononuclear cells were transferred to a new 15ml centrifuge tube, and the supernatant serum was dispensed into a 1.5ml centrifuge tube and stored at-80 ℃ for a long period of time. 4) 10ml of room temperature isotonic Phosphate Buffer (PBS) was added, mixed by gentle inversion, and centrifuged at 250g at 20 ℃ for 10 minutes. 5) The supernatant was removed and the pellet was retained. 5ml of PBS at room temperature was added, the cells were resuspended, gently mixed, 250g, and centrifuged at 20 ℃ for 10 minutes. 6) The supernatant was removed and the pellet was retained, removing as much of the PBS as possible, which would otherwise cause cloudy pellets in the lysate in subsequent steps. 7) Adding the frozen stock solution to suspend the cells, and freezing and storing the cells for later use by liquid nitrogen.

Example 4: library construction

Total RNA extraction

1. Extraction of Total RNA

A. 50mL of alpaca peripheral blood is collected and is separated by lymphocyte separating medium to obtain lymphocytes. B. Total RNA was extracted using Trizol. C. The result of detecting RNA 6711 by an ultraviolet spectrophotometer is as follows: OD_260/280＝1.99，OD_260/230When the RNA is 1.43, the RNA is not obviously degraded and has a reasonable purity; total RNA concentration was 809.3 ng/. mu.L. Two bands, 28S and 18S, were visualized by agarose gel electrophoresis.

2.RT-PCR

The reverse transcription system is as follows: oligo dT (2.5. mu.M) 1. mu.L, dNTP (10mM each) 1. mu.L, Total RNA 2.428. mu.g, H₂O Up to 10. mu.L. Mixing, keeping at 65 deg.C for 5min, and rapidly ice-cooling;

step

2, 10. mu.L of reaction solution in Step1, 5. mu.L of PrimeScript Buffer 4. mu.L, 0.5. mu.L of RNaseinhibitor (40U/. mu.L), 0.5. mu.L of PrimeScript RTase (200U/. mu.L), RNase free H₂O Up to 20. mu.L. After mixing, reverse transcription is carried out according to the following conditions: 30min at 42 ℃; 50 ℃ for 15 min; 70 ℃ for 15min.

VHH gene amplification (nested PCR)

Amplifying the VHH gene by adopting nested PCR, purifying and concentrating a PCR product by using a DNA purification kit, recovering a band of 750bp by using a DNA product gel recovery kit, and quantifying by using an ultraviolet spectrophotometer; and recovering the PCR product by using a DNA product gel recovery kit, and quantifying by using an ultraviolet spectrophotometer. The VHH gene is amplified by adopting nested PCR, the result is shown in figure 1, the target gene VHH with about 500bp is finally obtained, and the VHH is recovered by adopting a PCR product gel recovery kit.

Example 5: library transformation

The target gene VHH and the vector pHEN1 were subjected to double digestion with SfiI and Not1, and the result is shown in FIG. 2, after the digested VHH and pHEN1 were ligated with T4DNA ligase, they were transformed into TG1 electroporation competent cells, and a VHH gene library was constructed. And co-transforming for 15 times, mixing, and uniformly coating in 6 pieces of culture dishes with phi 150 mm. At the same time, 0.1. mu.L, 0.01. mu.L, 0.001. mu.L and 0.0001. mu.L of the mixed transformation liquid were uniformly applied to a culture dish of phi 90mm for library volume calculation, and the library volume was calculated to be 1.395X 10⁸cfu. Randomly selecting 48 colonies from the culture dish for calculating library capacity, performing colony PCR, and calculating target gene insertion rate of the library, wherein the library insertion rate is 100%, and the actual library capacity of the library is 1.395 × 10⁸cfu. In addition, 36 colonies were randomly selected, amplified, sequenced, analyzed for sequence diversity, translated into amino acid sequences, sequence aligned and functionally partitioned with prediction, the results are shown in FIG. 3.

Example 6: library rescue

Inoculating 10-100 times of live cells in the gene library, culturing, rescuing with M13K07 phage after logarithmic phase, centrifugally collecting phage, purifying with PEG-NaCl to obtain phage display library with titer of 3.4 × 10¹³cfu/mL. Can be directly used for affinity screening of subsequent specific phage.

Example 7: specific Nanobody screening

The method selects an avidin magnetic bead method for screening, the expressed and purified HBV-PRES1 is marked by biotin, the marked antigen is mixed with a phage library, and then avidin magnetic beads are added. Since the screened phages were amplified after the first round of screening, theoretically, phages with the correct Flag antibodies were enriched and after the second round of screening more positive than the first round should be obtained. Next, we amplify the phage eluted in the second round of screening, add helper phage again for rescue, and then continue the third round of screening. A bacteriophage. And obtaining the phage with high titer by 3 rounds of screening.

We used ELISA experiments to further select different monoclonals from the phage obtained from the magnetic bead screening for qualitative screening. We coated the antigen on an enzyme-linked plate, and incubated with a solution of nanobodies to bind to the antigen. Subsequently, Anti-M13p III monoclonal antibody of murine origin was used to bind to VHH-p III fusion protein in nanobodies, and goat Anti-mouse antibody labeled with Alkaline Phosphatase (AP) was used as secondary antibody and p NPP was used as substrate for color development. Color development result of ELISA experiment, and light absorption value data of reaction system measured by microplate reader at wavelength of 450mm

Example 8: sequencing of the screened Nanobody

The bacterial liquid corresponding to the well with obvious color reaction in the ELISA experiment is sent to the detection, and Sanger sequencing service is provided by Guangzhou Egyptian biotechnology, Inc. The resulting sequences were screened as in FIG. 4.

Example 9: the nano antibody obtained by screening can obviously reduce the HBsAg level of the serum of HBV transgenic mice

The screened anti-HBV-preS1 nano antibody is injected into HBV transgenic mice through tail vein, and anti-GFP nano antibody is used as a control group. The serum HBsAg and HBeAg levels of mice and the HBV-preS1 and HBV replication levels in liver are detected, and the result shows that the anti-HBV-preS1 nano antibody can obviously reduce the serum HBsAg level of mice compared with the anti-HBV-preS1 nano antibody treatment group, as shown in figure 5.

Example 10: the nano antibody obtained by screening can obviously reduce the serum HBeAg level of HBV transgenic mice

The screened anti-HBV-preS1 nano antibody is injected into HBV transgenic mice through tail vein, and anti-GFP nano antibody is used as a control group. The serum HBsAg and HBeAg levels of mice and the HBV-preS1 and HBV replication levels in liver are detected, and the result shows that the anti-HBV-preS1 nano antibody can obviously reduce the serum HBeAg level of mice compared with the anti-HBV-preS1 nano antibody treatment group of the control group, as shown in figure 6.

Example 11: the nano antibody obtained by screening can obviously reduce the DNA level of HBV in the serum of HBV transgenic mice

The screened anti-HBV-preS1 nano antibody is injected into HBV transgenic mice through tail vein, and anti-GFP nano antibody is used as a control group. The results of the detection of mouse serum HBsAg, HBeAg level and liver HBV-preS1, HBV replication level show that the anti-HBV-preS1 nano antibody can significantly reduce the mouse serum HBV DNA level compared with the control group in the anti-HBV-preS1 nano antibody treatment group, as shown in FIG. 7.

Example 12: the screened nano antibody has the treatment effect on liver cancer

The mouse liver cancer cell Hepa1-6 cell integrated with HBV genome is inoculated to the subcutaneous of mouse to make in vivo liver cancer tumorigenesis model. The screened anti-HBV-preS1 nano antibodies are injected into HBV transgenic mice through tail veins, and anti-GFP nano antibodies are used as a control group. The results of the detection of the tumor volume of the liver cancer cells show that the anti-HBV-preS1 nano antibody treatment group can obviously inhibit the growth of the liver cancer cells compared with the control group, the tumor volume is obviously smaller than that of the control group, and the anti-HBV-preS1 nano antibody treatment group has obvious effect on the treatment of the liver cancer, as shown in figure 8.

SEQ ID NO：1

Anti-HBV-prS1 VHH CDR1

GFIFSIYV

SEQ ID NO：2

Anti-HBV-prS1 VHH CDR1

GFISSIYD

SEQ ID NO：3

Anti-HBV-prS1 VHH CDR1

GFTFSSYS

SEQ ID NO：4

Anti-HBV-prS1 VHH CDR1

GSIFSRYT

SEQ ID NO：5

Anti-HBV-prS1 VHH CDR1

GVMFSIYD

SEQ ID NO：6

Anti-HBV-prS1 VHH CDR1

GFTLDYYG

SEQ ID NO：7

Anti-HBV-prS1 VHH CDR1

GSVFSRYT

SEQ ID NO：8

Anti-HBV-prS1 VHH CDR1

GFTFSTYS

SEQ ID NO：9

Anti-HBV-prS1 VHH CDR1

GNIFSRYT

SEQ ID NO：10

Anti-HBV-prS1 VHH CDR1

GRIFSRYT

SEQ ID NO：11

Anti-HBV-prS1 VHH CDR1

GSIFSRYV

SEQ ID NO：12

Anti-HBV-prS1 VHH CDR1

GFILSIYT

SEQ ID NO：13

Anti-HBV-prS1 VHH CDR2

INNIGTT

SEQ ID NO：14

Anti-HBV-prS1 VHH CDR2

IGRGGRYT

SEQ ID NO：15

Anti-HBV-prS1 VHH CDR2

IVSNVGGG

SEQ ID NO：16

Anti-HBV-prS1 VHH CDR2

ITSGVST

SEQ ID NO：17

Anti-HBV-prS1 VHH CDR2

IGRGGGT

SEQ ID NO：18

Anti-HBV-prS1 VHH CDR2

TNNIGTT

SEQ ID NO：19

Anti-HBV-prS1 VHH CDR2

ITSGGST

SEQ ID NO：20

Anti-HBV-prS1 VHH CDR2

ISGVGGRT

SEQ ID NO：21

Anti-HBV-prS1 VHH CDR2

ISSSGGST

SEQ ID NO：22

Anti-HBV-prS1 VHH CDR3

SCKAYQTLEDGLGPGVLEY

SEQ ID NO：23

Anti-HBV-prS1 VHH CDR3

RYYRSDDDFPEYAQDY

SEQ ID NO：24

Anti-HBV-prS1 VHH CDR3

RYYRSDDDFPEHAYEY

SEQ ID NO：25

Anti-HBV-prS1 VHH CDR3

KAYQTLEDGLGPGVLEY

SEQ ID NO：26

Anti-HBV-prS1 VHH CDR3

RYYRSDDDFPEYAYDY

SEQ ID NO：27

Anti-HBV-prS1 VHH CDR3

AARKNNYYCSGYLSSGGARHDY

SEQ ID NO：28

Anti-HBV-prS1 VHH CDR3

VYYCNADFGLGL

SEQ ID NO：29

Anti-HBV-prS1 VHH CDR3

NALFGIDGL

SEQ ID NO：30

Anti-HBV-prS1 VHH CDR3

HADFGLDGL

Claims

1. A nano antibody and antibody fragment for resisting the pre-S1 antigen (HBV-preS1) of the surface antigen of hepatitis B virus is disclosed, which contains the amino acid sequence regions of CDR1, CDR2 and CDR 3.

2. The molecule of claim 1, wherein said antibody is an antibody against hepatitis b virus surface antigen pre S1 antigen (HBV-preS 1).

3. The molecule according to claim 1, characterized in that said antibody is a nanobody, also called single domain antibody or heavy chain antibody.

4. The molecule of claim 1, wherein the antibody fragment can be combined with an Fc to form a complete antibody.

5. The molecule of claim 1, wherein the antibody fragment can be combined with other antibodies to form a bi-or multi-specific antibody.

6. The molecule of claim 1, wherein said nanobody is an antibody drug conjugate comprised of a toxin, radionuclide combination.

7. The molecule of claim 1, wherein said antibody is useful for the diagnosis and treatment of hepatitis b virus.

8. The molecule of claim 1, wherein said antibody is useful for the diagnosis and treatment of liver cancer.

9. The molecule of claim 1, comprising the sequences of antibody CDR1, CDR2, CDR 3.

10. The antibody CDR1 sequence may be SEQ ID NO: 1 to SEQ ID NO: 12 or a sequence of any one of the above.

11. The antibody CDR2 sequence may be SEQ ID NO: 13 to SEQ ID NO: 21 any one of the sequences.

12. The antibody CDR3 sequence may be SEQ ID NO: 22 to SEQ ID NO: 30 or a sequence thereof.

13. The antibody comprises a sequence combination of CDR1, CDR2, CDR 3.

CDR1 sequence is SEQ ID NO: 1 to SEQ ID NO: 12, the amino acid sequences of which are: GFIFSIYV, GFISSIYD, GFTFSSYS, GSIFSRYT, GMFSIYD, GFTLDYYG, GSVFSRYT, GFTFSTYS, GNIFSRYT, GRIFSRYT, GSIFSRYV, GFILSIYT.

CDR2 sequence is SEQ ID NO: 13 to SEQ ID NO: 21, the amino acid sequences of which are: INNIGTT, IGRGGRYT, IVSNVGGG, ITSGVST, IGRGGGT, TNNIGTT, ITSGGST, ISGVGGTT, ISSSGGST.

CDR3 sequence is SEQ ID NO: 55 to SEQ ID NO: 80, the amino acid sequences of which are: SCKAYQTLEDGLGPGVLEY, RYYRSDDDFPEYAQDY, RYYRSDDDFPEHAYEY, KAYQTLEDGLGPGVLEY, RYYRSDDDFPEYAYDY, AARKNNYYCSGYLSSGGARHDY, VYYCNADFGLGL, NALFGIDGL, HADFGLDGL are provided.