Construction of hepatitis b surface antigen-specific b cell receptor gene knock-in mouse model

Abstract

The application discloses a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model, and establishes a hepatitis B virus surface antigen (HBsAg) specific B cell receptor (BCR) gene knock-in mouse model, wherein 70-90% of B cells of the mouse can specifically recognize HBsAg, and differentiate into germinal center B cells and plasma cells. Therefore, the model can be used for basic research on hepatitis B antiviral humoral immune response and tolerance mechanism, and can also be used for application research on development of drugs and vaccines for breaking HBsAg immune tolerance. In summary, the establishment of the model lays a solid foundation for development of a functional cure immune means for hepatitis B.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model. BACKGROUND

[0002] Hepatitis B virus (HBV) causes hepatitis B (hepatitis B for short), which is prone to develop into cirrhosis and liver cancer in the late stage of infection, and is one of the important infectious diseases that endanger public health. After artificial immunization, the long-term existence of Anti-HBs, the main protective force of HBsAg, is the main protective force, and the existence of HBV surface antigen HBsAg plays an important role in HBV immune tolerance. The appearance of Anti-HBs after hepatitis B treatment is generally highly correlated with good prognosis. However, in acute hepatitis B infection, how the humoral immune response to HBsAg is generated and why tolerance occurs in slow hepatitis B are not very clear.

[0003] As a key cell of the humoral immune system, B cells mainly recognize and bind antigens specifically through BCR to initiate a humoral immune response. The emergence of BCR gene knock-in mice solves the barrier of too few specific B cells in vivo and makes it difficult to conduct in-depth research. At present, there is no BCR gene knock-in mouse specific to HBsAg.

[0004] How hepatitis B humoral immunity controls hepatitis B infection, how immune tolerance is formed in patients with slow hepatitis B, and whether it is possible to find a target for hepatitis B treatment are all urgent to be clarified. Based on this, the inventors of the present application provide a method for establishing an HBsAg BCR gene knock-in mouse, which is used for in-depth research on the generation and tolerance of hepatitis B virus humoral immunity. SUMMARY

[0005] The present application provides a method for establishing an HBsAg BCR gene knock-in mouse, which is further used to establish a platform for studying the generation and tolerance of hepatitis B virus humoral immunity.

[0006] To achieve this purpose, the present application provides the following technical solutions:

[0007] In a first aspect of the present application, a method for constructing a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model is provided, comprising the following steps:

[0008] S1, construct a recombinant plasmid of 129G1 monoclonal antibody specific to HBsAg, named HBsAg BCR gene;

[0009] S2, a method of using CRISPR-Cas9, injecting HBsAg BCR gene, Cas9 and gRNA into mouse embryo, replacing the original Ighd4-1-Ighj4 region on chromosome 12 by gRNA guidance.

[0010] Preferably, the HBsAg BCR gene comprises: light chain VJ fragment and heavy chain VDJ fragment specific to HBsAg, human antibody kappa light chain fragment HuCκ and porcine enterovirus Porcine teschovirus 2A fragment P2A.

[0011] The VJ fragment and the VDJ fragment are inserted in series, wherein the light chain VJ fragment is followed by a HuCκ and a P2A.

[0012] In the present application, the sequence of HBsAg specific antibody 129G1 (i.e. BCR) is used as the sequence of gene knock-in BCR, the light chain and heavy chain fragments of 129G1 antibody are expressed in series, and the Ighd4-1-Ighj4 region of C57BL / 6 mouse itself is replaced, so that the simultaneous knock-in of the light chain and the heavy chain of HBsAg BCR is completed in one step; that is, the mouse does not need to go through tedious breeding to obtain the mouse expressing BCR light chain and heavy chain simultaneously.

[0013] Preferably, the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO. 1.

[0014] SEQ ID NO. 1:

[0015] 1 TCGACA GTATGC AGAGGG CTGTAT CCACTG GAGAGG ATGAAG TCACTG AGTTGGAAAACA61 GAACAG GACAGG CACCTA ACAAGT GGTTGC TATAGC CCACTG TTACCC TTTTACATGTAT121 AGGCTC AGGATA AGCAGT GATACT GTGAGG TTTATG TGTGAG AACATC ACAGTATAAACA181 CATCTC AATAGA GGTCTT AGAGAT CAGCAC AATTAG TGAGAA GTCATA AACAGTAGATAC241 TATAAG GCATAG GCTCAG CTACCT AGGGTC AGGTAT CTGTGT AAATCT GATTGTGTATCA301 GGTTTA GATCAA TATGAC TTAGGG AGGCGA GTCATA TGCAAA TCTAAG AAGACTTTAGAG361 AAGAAA TCTGAG GCTCAC CTCACA TAACAG CAAGAG AGTGTC CGGTTA GTCTCAAGGAAG421 ACTGAG ACACAG TCTTAG ATATCA CCATGG GTTGGT CCTGCA TTATAC TGTTCCTTGTGG481 CTACAG CGACGG GAGTTC ACAGCC AAATCG TTCTCA CCCAGT CTCCAG CAATCATGTCTG541 CATCTC CAGGGG AGAAGG TCACCA TGACCT GCAGTG CCAGTT CAAGTG TAAATTACGTGC601 ACTGGT ACCAGC AGAAGT CTGGCA CCTCCC CCAAAA GATGGA TTTATG ACACATCCAAAC661 TGGCTT CTGGAG TCCCTG TTCGCT TCAGTG GCAGTG GGTCTG GGACCT CTTATTCTCTCA721 CAATCA GCAGCA TGGAGG CTGAAG ATGCTG CCACTT ATTACT GCCAGC AGTGGACTAGTT781 TCCCGT ACACGT TTGGAG CTGGGA CCAAGC TGGAGC TAAAGC GGCGTA CGGTCGCTGCAC841 CATCTGTCTTCA TCTTCC CGCCAT CTGATG AACAAT TGAAAT CTGGAA CTGCCTCTGTTG901TGTGCC TGCTGA ATAACT TCTATC CCAGAG AGGCCA AAGTCC AGTGGA AAGTGGATAACG961CCCTCC AATCGG GAAACT CCCAGG AGAGTG TCACAG AACAGG ACTCCA AGGACTCCACCT1021ACAGCC TCTCCT CCACCC TGACGC TGTCCA AAGCGG ACTACG AGAAAC ACAAAGTCTACG1081CCTGCG AAGTCA CCCATC AGGGCC TGTCCT CGCCCG TCACAA AGTCCT TCAACAGGGGAG1141AGTGTG GAAGCG GCGCCA CTAATT TCAGTC TTCTGA AACAGG CCGGAG ACGTGGAGGAGA1201ATCCTG GACCCA TGGGAT GGTCAT GTATCA TCCTTT TTCTAG TAGCAA CTGCAACCGGTG1261TACATT CCCAGG TGCAGC TACAGC AGTCTG GGCCTC AGCTGA TTAGGC CTGGGGCTTCAG1321TAAAGA TCTCCT GCAAGC CTTCTG GTTACT CATTCT CCGACT ACTGGA TGCACTGGGTGA1381AGCAGA GGCCTG GACAAG GTCTTG AGTGGA TTGGCA TGATTG ATCCTT CCGATAGTGAAA1441CTAGGT TAAATC AGGACT TCAAGG ACAAGG CCACAT TGACTG TAGACA AAGTCTCCAGCA1501CAGCCT ACATGC AACTCA GCAGCC CGACAT CTGAGG ACTCTG CGGTCT ATTATTGTGCAA1561GAAACT ATAGGT ACGACC ACTTTG CTTTGG ACTCCT GGGGTC AAGGAA CCTCAGTCACCG1621TCTCCT CAGGTA AGCTGG CTTTTT TCTTTC TGCACA TTCCAT TCTGAAACGGGATCGATT1681GCATAT CGATT

[0016] Nucleotide sequence of the deleted Ighd4-1-Ighj4 region (SEQ ID NO. 4): 1 TCGAGA ACTTTAGCGACT GTTTTG AGAGAA ATCATT GGTCCC TGACTC AAGAGA TGACTG 61 GCAGAT TGGGGA TCAGAATACCCA TACTCT GTGGCT AGTGTG AGGTTT AAGCCT CAGAGT 121 CCCTGT GGTCTC TGACTGGTGCAA GGTTTT GACTAA GCGGAG CACCAC AGTGCT AACTGG 181 GACCAC GGTGAC ACGTGGCTCAAC AAAAAC CTTCTG TTTGGA GCTCTC CAGGGG CAGCCT 241 GAGCTA TGAGGA AGTAGAGAGGCT TGAGAA ATCTGA GGAAGA AAAGAG TAGATC TGAGAG 301 GAAAGG TAGCTT TCTGGAGGTCAG GAGACA GTGCAG AGAAGA ACGAGT TACTGT GGACAG 361 GTCTTA GATGGG GAAAGAATGAGC AAATGC AAGCAT CAGAAG GGTGGA TGCAAT GTCCTG 421 CCAAGG ACTTAC CAAGAGGATCCC CGGACA GAGCAG GCAGGT GGAGTT GACTGA GAGGAC 481 AGGATA GGTGCA GGTCCCTCTCTT GTTTCC TTTCTC CTTCTC CTGTTT CCTTCT TCTCTT 541 GTCACA GGTCTC ACTATGCTAGCC AAGGCT AGCCTG AAAGAT TACCAT CCTACA GATGGG 601 CCCATC CAGTTG AATTAAGGTGGA GATCTC TCCAAA CATCTG AGTTTC TGAGGC TTGGAT 661 GCCACT GGGGAC GCCAAGGGACTT TGGGAT GGGTTT GGTTGG CCCCAG ATGAAG GGCTAC 721 TTCACT GGGTCT ATAATTACTCTG ATGTCT AGGACC AGGGGG CTCAGG TCACTC AGGTCA 781 GGTGAG TCCTGC ATCTGGGGACTG TGGGGTTCAGGT GGCCTA AGGCAG GATGTG GAGAGA 841 GTTTTA GTATAG GAACAGAGGCAG AACAGA GACTGT GCTACT GGTACT TCGATG TCTGGG 901 GCACAG GGACCA CGGTCACCGTCT CCTCAG GTAAGC TGGCTT TTTTCT TTCTGC ACATTC 961 CATTCT GAAACG GGAAAAGATATT CTCAGA TCTCCC CATGTC AGGCCA TCTGCC ACACTC 1021 TGCATG CTGCAG AAGCTTTTCTGT AAGGAT AGGGTC TTCACT CCCAGG AAAAGA GGCAGT 1081 CAGAGG CTAGCT GCCTGTGGAACA GTGACA ATCATG GAAAAT AGGCAT TTACAT TGTTAG 1141 GCTACA TGGGTA GATGGGTTTTTG TACACC CACTAA AGGGGT CTATGA TAGTGT GACTAC 1201 TTTGAC TACTGG GGCCAAGGCACC ACTCTC ACAGTC TCCTCA GGTGAG TCCTTA CAACCT 1261 CTCTCT TCTATT CAGCTTAAATAG ATTTTA CTGCAT TTGTTG GGGGGG AAATGT GTGTAT 1321 CTGAAT TTCAGG TCATGAAGGACT AGGGAC ACCTTG GGAGTC AGAAAG GGTCAT TGGGAG 1381 CCCTGG CTGACG CAGACAGACATC CTCAGC TCCCAT ACTTCA TGGCCA GAGATT TATAGG 1441 GATCCT GGCCAG CATTGCCGCTAG GTCCCT CTCTTC TATGCT TTCTTT GTCCCT CACTGG 1501 CCTCCA TCTGAG ATCATCCTGGAG CCCTAG CCAAGG ATCATT TATTGT CAGGGG TCTAAT 1561 CATTGT TGTCAC AATGTGCCTGGT TTGCTT ACTGGG GCCAAG GGACTC TGGTCA CTGTCT 1621 CTGCAG GTGAGT CCTAACTTCTCCCATTCT AAATGC ATGTTG GGGGGA TTCTGG GCCTTC1681 AGGACC AAGATT CTCTGCAAACGG GAATCA AGATTC AACCCC TTTGTC CCAAAG TTGAGA1741 CATGGG TCTGGG TCAGGGACTCTC TGCCTG CTGGTC TGTGGT GACATT AGAACT GAAGTA1801 TGATGA AGGATC TGCCAGAACTGA AGCTTG AAGTCT GAGGCA GAATCT TGTCCA GGGTCT1861 ATCGGA CTCTTG TGAGAATTAGGG GCTGAC AGTTGA TGGTGA CAATTT CAGGGT CAGTGA1921 CTGTCT GGTTTC TCTGAGGTGAGG CTGGAA TATAGG TCACCT TGAAGA CTAAAG AGGGGT1981 CCAGGG GCTTCT GCACAGGCAGGG AACAGA ATGTGG AACAAT GACTTG AATGGT TGATTC2041 TTGTGT GACACC AGGAATTGGCAT AATGTC TGAGTT GCCCAG GGGTGA TTCTAG TCAGAC2101 TCTGGG GTTTTT GTCGGGTATAGA GGAAAA ATCCAC TATTGT GATTAC TATGCT ATGGAC2161 TACTGG GGTCAA GGAACCTCAGTC ACCGTC TCCTCA GGTAAG AATGGC CTCTCC AGGTCT2221 TTATTT TTAACC TTTGTTATGGAG TTTTCT GAGCAT TGCAGA CTAATC TTGGAT ATTTGT2281 CCCTGA GGGAGC CGGCTGAGAGAA GTTGGG AAATAA ACTGTC TAGGGA TC

[0017] Preferably, the nucleotide sequence of the gRNA is SEQ ID NO. 2 or SEQ ID NO. 3.

[0018] SEQ ID NO. 2

[0019] GTTGGGAAATAAACTGTCTAGGG

[0020] SEQ ID NO. 3

[0021] GCTAAAGTTCTCGAGCCTGTGGG

[0022] Preferably, the mouse is a C57BL / 6 strain mouse.

[0023] In a second aspect of the present application, a mouse model of hepatitis B surface antigen specific B cell receptor gene knock-in is provided, which is constructed by the method of the present application.

[0024] Preferably, 70-90% of the B cells in the peripheral blood of the mouse can specifically recognize HBsAg and differentiate into germinal center B cells and plasma cells.

[0025] The method for constructing the mouse model of hepatitis B surface antigen specific B cell receptor gene knock-in comprises the following steps:

[0026] S1, constructing a recombinant plasmid of antibody 129G1 of HBsAg, named HBsAg BCR gene;

[0027] S2, using the method of CRISPR-Cas9, injecting the HBsAg BCR gene, Cas9 and gRNA into the mouse embryo, and replacing the original Ighd4-1-Ighj4 region located on chromosome 12 by gRNA guidance.

[0028] Preferably, the HBsAg BCR gene comprises: a light chain VJ fragment and a heavy chain VDJ fragment specific to HBsAg, a human antibody kappa light chain fragment HuCκ, and a porcine enterovirus Porcine teschovirus 2A fragment P2A.

[0029] The VJ fragment and the VDJ fragment are inserted in series, wherein the VJ fragment of the light chain carries a HuCκ and a P2A.

[0030] Preferably, the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO. 1.

[0031] Preferably, the nucleotide sequence of the gRNA is SEQ ID NO. 2.

[0032] Preferably, the mouse is a C57BL / 6 strain mouse.

[0033] In a third aspect of the present application, the mouse model of the present application is applied in the research of HBsAg specific B cell immune response mechanism and / or HBsAg specific B cell immune tolerance mechanism.

[0034] Preferably, the study of the HBsAg-specific B cell immune response mechanism comprises: B cells from the mouse model are adoptively transferred to wild-type mice in a quantity close to a physiological state, and can proliferate, activate and differentiate into germinal center B cells and plasma cells in the HBV acute mouse model.

[0035] Preferably, the study of the HBsAg-specific B cell immune tolerance mechanism comprises: B cells from the mouse model are adoptively transferred to wild-type mice in a quantity close to a physiological state, and can proliferate, activate and differentiate, and migrate in the HBV chronic mouse model.

[0036] Compared with the prior art, the present application has the beneficial effects and significant progress that: the present application constructs a gene knock-in mouse expressing HBsAg-specific BCR based on C57BL / 6 strain mice by a homologous recombination method. The present application verifies the gene level, the antigen-antibody binding level and the proliferation, activation and differentiation of the B cells of the gene knock-in mouse against HBsAg. The present application establishes a hepatitis B virus surface antigen (HBsAg)-specific B cell receptor (BCR) gene knock-in mouse model, which can be used for basic research on hepatitis B antiviral humoral immune response and tolerance mechanism, and for application research on developing drugs and vaccines for breaking HBsAg immune tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions of the present application, the drawings required for the embodiments of the present application will be briefly introduced as follows.

[0038] Obviously, the drawings in the following description are only the drawings of part of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor, but these other drawings also belong to the drawings required for the embodiments of the present application.

[0039] Figure 1 Figure A is a construction strategy mode diagram of the HBsAg BCR gene knock-in mouse in embodiment 1 of the present application;

[0040] Figure 2 Figure A is a construction strategy mode diagram of the HBsAg BCR gene knock-in mouse in embodiment 1 of the present application;

[0041] Figure 2 Figure B is a Southern Blot confirmation of the insertion of the knock-in fragment in embodiment 2 of the present application;

[0042] Figure 2 Figure C is a schematic diagram of the primer targeting segment identified by PCR in embodiment 2 of the present application;

[0043] Figure 2D is the schematic diagram of PCR identification result in Example 2 of the application;

[0044] Figure 3 A is the schematic diagram of HBsAg BCR flow staining in Example 3 of the application;

[0045] Figure 3 B is the verification of HBsAg BCR gene knock-in mouse HBsAg specific BCR staining in Example 3 of the application;

[0046] Figure 4 A is the schematic diagram of in vivo differentiation detection of B cells of HBsAg BCR gene knock-in mouse in Example 4 of the application;

[0047] Figure 4 B is the differentiation of WT B cells and HBsAg BCR gene knock-in B cells into germinal center B cells after receiving pHBV1.3 immunization in Example 4 of the application;

[0048] Figure 4 C is the differentiation of WT B cells and HBsAg BCR gene knock-in B cells into plasma cells after receiving pHBV1.3 immunization in Example 4 of the application;

[0049] Figure 4 D is the differentiation and localization of HBsAg BCR gene knock-in B cells in the spleen of WT mice in Example 4 of the application, scale: 500 μm. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme, beneficial effects and significant progress of the embodiments of the application clearer, below, the technical scheme in the embodiments of the application will be clearly and completely described in combination with the embodiments of the application.

[0051] Obviously, all the described embodiments are only part of the embodiments of the application, not all the embodiments; all other embodiments obtained by those of ordinary skill in the art based on the embodiments in the application without creative labor belong to the protection scope of the application.

[0052] It should be understood that:

[0053] For those of ordinary skill in the art, the specific meaning of the above terms in the application can be understood according to specific circumstances.

[0054] It should also be noted that the following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments.

[0055] The technical scheme of the application will be described in detail below with specific embodiments.

[0056] Construction of HBsAg BCR gene knock-in mouse model

[0057] The present application uses the method of CRISPR-Cas9 to inject the pre-recombined antibody 129G1 of HBsAg into the mouse embryo, and the Igh and Igκ sequences of the antibody 129G1 of HBsAg are replaced by the gRNA targeting Igh to guide it to replace the original Ighd4-1-Ighj4 region on chromosome 12, so that the simultaneous knock-in of the light chain and heavy chain of the HBsAg BCR is completed in one step. The specific production principle is shown in Figure 1 The replacement fragment and the knock-in fragment sequence are SEQ ID NO. 1 and SEQ ID NO. 4, respectively.

[0058] In order to construct the targeting vector, the BAC clones RP24-154H17 and RP23-5I20 from the C57BL / 6 library are used as templates to generate homologous arms by PCR. Cas9 and gRNA are co-injected into fertilized eggs (gRNA sequences are SEQ ID NO. 2 and SEQ ID NO. 3) with the targeting vector to generate gene knock-in mice. Then specific gene knock-in mice are identified by sequencing and PCR methods. The above construction is entrusted to Guangzhou Saiye Biotechnology Co., Ltd.

[0059] As shown in Figure 1 , the light chain VJ fragment and the heavy chain VDJ fragment specific for HBsAg are inserted in series, and the light chain VJ fragment carries a κ light chain fragment (HuCκ) of a human antibody after the insertion, which can be used as a tag to detect B cells that specifically express HBsAg specific BCR; the porcine enterovirus Porcine teschovirus 2A (P2A) fragment is a self-cleaving fragment that serves to separate the light chain and the heavy chain.

[0060] Example 2: Verification of HBsAg BCR gene knock-in mice at the gene level

[0061] This embodiment is to verify the insertion of the targeting fragment at the gene level by using the Southern Blot and PCR methods on the HBsAg BCR gene knock-in mice constructed in Example 1.

[0062] Experimental grouping: normal wild-type mice (WT), HBsAg BCR gene knock-in mice (MT) prepared in Example 1.

[0063] Experimental method:

[0064] Southern Blot method and results

[0065] 2.1, Preparation of genomic DNA of WT group mice and MT group mice. The cells were lysed with appropriate chemical reagents, and most of the proteins and RNA were digested with protease and RNase; and the proteins were removed by organic reagent (phenol / chloroform) extraction method.

[0066] Southern blot probe was prepared. The WT group mouse DNA obtained in step 3.1 was subjected to PCR amplification using the following Southern Blot primers, respectively, to obtain 5' end and 3' hybridization fragments that can form base complementary pairing with the DNA sample to be detected, and the fragments were cloned into pUC19 vector (containing T7 in vitro transcription promoter), and in vitro transcription and digoxin labeling were performed using Roche in vitro transcription probe preparation kit (Cat. No. 12039672910), and the probe was stored at -80°C for standby use.

[0067] Primers for 5' Probe:

[0068] 5' Probe forward primer (SEQ ID NO. 5): 5'-TACAGAGCAGAATCCCAGCCAAGAG-3'

[0069] 5' Probe reverse primer (SEQ ID NO. 6): 5'-TCAGCAGTAGGTGCTTAGGGAGCAT-3'

[0070] Primers for 3' Probe:

[0071] 3' Probe forward primer (SEQ ID NO. 7): 5'-GAATCTGTGTGATGGTGTTGGTGGA-3'

[0072] 3' Probe reverse primer (SEQ ID NO. 8): 5'-GAGGCTAGATGCCTTTCTCCCTTGA-3'

[0073] 2.2, Cleavage by Mfel restriction endonuclease. The sample DNA solution obtained in step 3.1 was taken into a 0.5 ml Eppendorf tube, 2 μl of 10x restriction endonuclease buffer, 6-10 U of the corresponding restriction endonuclease were added, and sterilized double distilled water was added to a total volume of 20 μl, and incubated at 37°C for 2 hours, and heated at 65°C for 5 minutes or terminated by adding an appropriate amount of 0.5 mol / L EDTA Na2.

[0074] The results are as follows Figure 2As shown in A, the WT mice can obtain 7.69 KB, and the MT mice can obtain fragments with a size of 11.12 kb.

[0075] 2.3, Agarose gel electrophoresis separates DNA samples. Take 2 μl of the DNA enzyme solution obtained in step 2.2 and add 10 μl of sample buffer (containing bromophenol blue indicator and glycerol) to the 0.8% agarose gel (containing 0.5 μg / ml ethidium bromide) for horizontal electrophoresis, voltage < 5 V / cm, time about 2 hours.

[0076] The electrophoretically separated DNA sample is alkali denatured and transferred to a solid support (nylon membrane). The agarose gel after electrophoresis is denatured in an alkali denaturation solution (0.5 M NaOH, 1.5 M NaCl) for 45 min, rinsed with double distilled water for 3 times, treated with neutralization solution (1 M Tris-HCl, 1.5 M NaCl, pH 7.4) for 45 min, and the transfer system is installed according to the downward capillary transfer method, and the membrane is transferred for 8 hours.

[0077] The DNA fixed on the membrane is annealed and hybridized with digoxin-labeled probes. The nylon membrane containing the sample DNA obtained above is baked at 80°C for 2 hours, placed in a hybridization tube, 5 ml of hybridization solution is added and pre-hybridized at 42°C for 30 min, and then the above prepared southern blot probe is added (the probe is denatured at 65°C for 10 min and then immediately placed in ice water for 5 min), and hybridized for about 8 hours.

[0078] The membrane is incubated with alkaline phosphatase-labeled digoxin antibody. The hybridization solution in the above hybridization tube is discarded, low stringent washing solution is added and washed at 25°C for 5 min, repeated once, high stringent washing solution is added and washed at 68°C for 15 min, repeated once, blocking solution is added and blocked at 25°C for 1 hour, then digoxin antibody is added and incubated at 25°C for 30 min.

[0079] Color development by adding alkaline phosphatase substrate. The antibody incubation buffer in the above hybridization tube is discarded, washing buffer is added and washed at 25°C for 15 min, repeated once, detection buffer is added and washed for 5 min, repeated once, the membrane is taken out and placed on oil paper, and luminescent solution is added for development and detection.

[0080] The results are as follows Figure 2 As shown in the left graph of B, WT represents the WT mouse group, and 3, 5, 27 and 28 represent the MT mouse group. The MT mice can obtain two fragments of 11.12 kb and 7.69 kb, while the WT mice only have a fragment of 11.12 kb.

[0081] 2.4, Southern Blot experiment is performed with another restriction endonuclease BamHI using the same method as steps 2.2 and 2.3.

[0082] The results are as follows Figure 2As shown in Figure B on the right, WT represents the WT mouse group, and 3, 5, 27, and 28 represent the MT mouse groups. MT mice can obtain two fragments, 12.85kb and 10.16kb, while WT mice only have a 10.16kb fragment.

[0083] PCR methods and results

[0084] 2.5. Prepare the DNA to be tested. Genomic DNA of WT group mice and MT group mice was obtained by using the method in step 2.1.

[0085] 2.6 Design primers based on WT alleles and substitution sequences. The primer design principle is as follows: Figure 2 As shown in C, a common front primer F1 and separate back primers R1 and R2 are used.

[0086] 2.7. Perform PCR amplification on the DNA obtained in step 2.5 using the primers designed in step 2.6.

[0087] The results are as follows Figure 2 As shown in Figure D, WT represents the WT mouse group, and KI1 and KI2 represent the MT mouse groups. Fragments of 443 bp and 285 bp in size were amplified in the WT and MT groups, respectively. This demonstrates that Example 1 successfully replaced the Ighd4-1-Ighj4 sequence of C57BL / 6 mice with the coding sequence of the HBsAg monoclonal antibody 129G1 via gRNA-guided Cas9 enzyme cleavage.

[0088] in,

[0089] F1(SEQ ID NO.9):5'-CTGATAGGCACCCAAGTACACTA-3'

[0090] R1(SEQ ID NO.10):5'-GCTATAGCAACCACTTGTTAGGTG-3'

[0091] R2 (SEQ ID NO. 11): 5'-CTTCCTCATAGCTCAGGCTGC-3'.

[0092] Example 3: Antigen-antibody binding layer verification in mice

[0093] like Figure 3 As shown in A, this embodiment verifies the binding of HBsAg to HBsAg in the HBsAg BCR gene knock-in mouse B cells constructed in Example 1 using fluorescently labeled HBsAg (HBsAg-Alexa647) staining.

[0094] 3.1, Cross-link HBsAg and Alexa647 fluorescent dye to get HBsAg-Alexa647;

[0095] 1) Dissolve HBsAg to 10mg / mL (10X) using PBS;

[0096] 2) 84mg NaHCO3 is added to 1mL ddH2O to make 1mol / L NaHCO3 solution;

[0097] 3) Dilute HBsAg (10X) to 1X using PBS to make the final volume to 100uL;

[0098] 4) Take 10uL 1M NaHCO3 solution and add to 100uL HBsAg (1X);

[0099] 5) Dropwise add the above solution to Alexa Fluor 647 dye solution. Note: Shake after each drop

[0100] 6) Incubate at room temperature for 2h in the dark. Note: Shake every 15min, do not vortex or pipette blow;

[0101] 7) 30min before the end of incubation, equilibrate the column: break the bottom of the column, place it in a 15mL centrifuge tube with a collection tube, 4°C 350g for 5min, empty the column once, wash 4 times with 500uL PBS;

[0102] 8) Add the incubated solution to the column, change the collection tube at the bottom of the column, centrifuge at 4°C 350g for 5min;

[0103] 9) Transfer the antibody to a brown antibody tube and store at 4°C.

[0104] 3.2, HBsAg BCR gene knock-in B cells and HBsAg-Alexa647 co-incubation can make cells that can bind to HBsAg carry Alexa647 dye;

[0105] 1) Collect blood from the orbit into an anticoagulant tube;

[0106] 2) Add red blood cell lysis solution, resuspend and lyse red blood cells on ice for 10min;

[0107] 3) 4°C, 500g centrifuge for 5min, discard the supernatant, resuspend the precipitate with PBS+2% FBS, 4°C, 500g centrifuge for 5min, discard the supernatant;

[0108] 4) Prepare antibody mix, add B220 and HBsAg-Alexa flour to PBS+2% FBS buffer according to 1:200

[0109] 647, blow and mix evenly;

[0110] 5) Resuspend the cell pellet with antibody mix, stain on ice for 30 min in the dark;

[0111] 6) Wash twice with PBS+2%FBS buffer, 500g centrifuge for 5 min, finally resuspend with PBS+2%FBS buffer, and put on the machine.

[0112] 3.3, Detection by flow cytometry.

[0113] Results are shown in Figure 3 B, through fluorescence-labeled HBsAg staining analysis, 80-90% of B cells in the peripheral blood of HBsAg BCR gene knock-in mice showed specific binding to HBsAg, and these cells all expressed the knock-in tag expression HuCκ.

[0114] In summary, the results of this example obtained HBsAg BCR gene knock-in mice, and the B cells of the mice have the property of specific binding to HBsAg.

[0115] Example 4 Verification of mice at the level of specific HBsAg antigen response

[0116] This example is a verification of the function of B cells of HBsAg BCR gene knock-in mice, specifically a verification of their ability to differentiate into effector cells and produce antibodies after in vivo stimulation by specific antigens.

[0117] 4.1, As shown in Figure 4 A, most B cells in HBsAg BCR gene knock-in mice are HBsAg antigen-specific B cells. To make the experiment more in line with physiological conditions, HBsAg BCR gene knock-in B cells were negatively selected and purified to 1x10 6 cells per mouse and transferred to WT mice for experiments. One day after adoptive transfer, 20 μg of pHBV1.3 plasmid was injected into the tail vein of the mice under high pressure (it was detected that a large amount of HBsAg could be detected in the peripheral blood one day after plasmid injection), and the mice were sacrificed 4.5 days after immunization to obtain spleen single cell suspension for germinal center B cell (GC B) and plasma cell (PC) staining;

[0118] 4.2, The transferred cells were separated by HuCκ labeling, and their differentiation was analyzed,

[0119] Results are shown in Figure 4As shown in B, each spleen was resuspended with 1 ml during the experiment, and 50 ul of lysed red blood cells were taken from each well for staining, and 2-3*10^5 HuCκ+ cells were obtained, i.e. only 4-6 times increase in quantity in the spleen after 4.5 days of immunization, indicating that the HBsAg BCR gene knock-in B cells transferred to WT mice have undergone significant proliferation.

[0120] 4.3, analyze the differentiation of GCB and PC in B cells;

[0121] The results are shown in Figure 4 As shown in B and 4C, after removing dead cells, adhesions and dead cells, and preliminarily removing non-B cell lines by CD3, CD8, CD11b, CD11c, HuCκ was used to distinguish the background B cells of HuCκ- WT mice and HuCκ+ HBsAg BCR gene knock-in (HBsAg-binding) B cells. As compared with the background WT cells, the proportion of GC B and PC in the HBsAg BCR gene knock-in B cells was about 10 times and 40 times higher, respectively, because the WT B cells contained a variety of irrelevant B cells that recognized other antigens but not HBsAg; in terms of quantity, HuCκ+ accounted for about 2 / 3 of all PC, and the number of HuCκ+ and HuCκ- GC B cells was comparable.

[0122] 4.4, analysis by immunohistochemical staining;

[0123] The results are shown in Figure 4 As shown in D, the HBsAg BCR gene knock-in B cells differentiated into germinal center B cells (GL-7+) that constitute the germinal center and plasma cells (CD138+) located in the outer follicle region.

[0124] In summary, the HBsAg BCR gene knock-in B cells can strongly proliferate and differentiate into germinal center B cells and plasma cells after pHBV1.3 acute infection in WT mice, and the latter can form IgG2a class antibodies through class switching.

[0125] The above experiment proves that the HBsAg BCR gene knock-in mouse specific to HBV surface antigen is successfully constructed, and 70-90% of B cells of the mouse can specifically recognize HBsAg. The antigen-specific B cells can be activated and differentiated into corresponding effector cells in vivo and in vitro, and can further undergo class switching. By mating with HBV transgenic mice, the phenomenon of HBsAg-specific B cell tolerance is obtained, and a similar depletion phenotype can be induced in the AAV-HBV chronic mouse model. Therefore, the HBsAg BCR gene knock-in mouse designed and constructed by us can be used as an important tool for studying various biological processes of humoral immunity in chronic HBV infection, including the production of antibodies, tolerance and the formation of depletion.

[0126] In the description of the above specification:

[0127] The description of the terms "the present embodiment", "the embodiment of the present invention", "as shown", "further", "further improved technical solutions" and the like means that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in the present specification, the illustrative description of the above terms is not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described can be combined or combined in any one or more embodiments or examples in a suitable manner; in addition, the person skilled in the art can combine or combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0128] Finally, it should be noted that:

[0129] The above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto;

[0130] Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features, and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and the non-essential improvements and adjustments or replacements made by the person skilled in the art according to the content of the specification are all within the scope of the present application.

Claims

1. A method for constructing a mouse model of knock-in of a hepatitis B surface antigen-specific B cell receptor gene, characterized by, Comprise the following steps: S1, construct a recombinant plasmid of 129G1 monoclonal antibody specific to HBsAg, named HBsAg BCR gene; the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO. 1; S2, using the method of CRISPR-Cas9, injecting the HBsAg BCR gene, Cas9 and gRNA into mouse embryos, replacing the original Ighd4-1-Ighj4 region located on chromosome 12 by gRNA guidance.

2. The method for constructing a hepatitis B surface antigen-specific B cell receptor knock-in mouse model according to claim 1, wherein the mouse is a BALB / c mouse. The HBsAg BCR gene comprises: light chain VJ fragment and heavy chain VDJ fragment specific to HBsAg, human antibody kappa light chain fragment HuCk and porcine enterovirus Porcine teschovirus 2A fragment P2A; The VJ fragment and VDJ fragment are inserted in series, wherein the VJ fragment of the light chain carries a HuCk and P2A.

3. The method for constructing a hepatitis B surface antigen-specific B cell receptor knock-in mouse model according to claim 1, wherein the mouse is a BALB / c mouse. The nucleotide sequence of the gRNA is SEQ ID NO. 2 or SEQ ID NO.

3.

4. The method for constructing a hepatitis B surface antigen-specific B cell receptor knock-in mouse model according to claim 2, wherein the mouse is a BALB / c mouse. The mouse is a mouse of C57BL / 6 strain.

5. The application of the mouse model constructed by the construction method of claim 1 in the research of HBsAg specific B cell immune response mechanism and / or HBsAg specific B cell immune tolerance mechanism.

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