CN116649294A - Construction of hepatitis B surface antigen specific B cell receptor gene knock-in mouse model - Google Patents

Construction of hepatitis B surface antigen specific B cell receptor gene knock-in mouse model Download PDF

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CN116649294A
CN116649294A CN202310054808.7A CN202310054808A CN116649294A CN 116649294 A CN116649294 A CN 116649294A CN 202310054808 A CN202310054808 A CN 202310054808A CN 116649294 A CN116649294 A CN 116649294A
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hbsag
specific
hepatitis
mouse
cells
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李建华
李晓芳
白露
袁正宏
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Fudan University
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Abstract

The application discloses construction of 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 the HBsAg and differentiate into a hair center B cell and a plasma cell. Therefore, the model can be used for basic research of hepatitis B virus humoral immune response and tolerance mechanism, and can also be used for application research of developing medicines and vaccines for breaking HBsAg immune tolerance. In a word, the establishment of the model lays a solid foundation for the development of a functional curative immune means for hepatitis B.

Description

Construction of hepatitis B surface antigen specific B cell receptor gene knock-in mouse model
Technical Field
The application relates to the field of biological medicine, in particular to construction of a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model.
Background
Hepatitis B virus (hepatitis B virus, HBV) causes hepatitis B (hepatitis B for short), and the late infection is easy to develop into liver cirrhosis and liver cancer, and is one of important infectious diseases which endanger public health. After artificial immunization, the long-term existence of the HBsAg antibody Anti-HBs is the main protective force, and the existence of the HBV surface antigen HBsAg has an important effect on HBV immune tolerance through research, and the appearance of the Anti-HBs after hepatitis B treatment has higher correlation with good health after healing. However, it is not clear how humoral immune responses against HBsAg are generated in acute hepatitis b infection and why tolerance occurs in chronic hepatitis b.
As key cells of the humoral immune system, B cells specifically recognize and bind antigens mainly through BCR to elicit a humoral immune response. The advent of BCR gene knockout mice solves the barrier that the number of specific B cells in the body is too small to be studied in depth, and at present, BCR gene knockout mice specific for HBsAg are not available.
Regarding how the humoral immunity of hepatitis B controls hepatitis B infection, how the immune tolerance of patients with chronic hepatitis B is formed and whether targets are likely to be found in the patients for hepatitis B treatment are all urgent to be clarified. Based on this, the present inventors have provided a method for establishing a mouse with the gene knockin of HBsAg BCR for in-depth investigation of generation and tolerance of humoral immunity of hepatitis B virus, etc.
Disclosure of Invention
The application provides a method for establishing a HBsAg BCR gene knock-in mouse, which is further used for establishing a platform for generating humoral immunity of hepatitis B virus and researching tolerance.
To achieve the purpose, the application provides the following technical scheme:
in a first aspect of the present application, there is provided a method for constructing a hepatitis B surface antigen-specific B cell receptor gene knock-in mouse model, comprising the steps of:
s1, constructing a recombinant plasmid of 129G1 monoclonal antibody specific for HBsAg, and naming the recombinant plasmid as an HBsAg BCR gene;
s2, injecting the HBsAg BCR gene, cas9 and gRNA into the mouse embryo by using a CRISPR-Cas9 method, and enabling the HBsAg BCR gene to replace the original Ighd4-1-Ighj4 region positioned on chromosome 12 through gRNA guidance.
Preferably, the HBsAg BCR gene comprises: a light chain VJ fragment and a VDJ fragment of a heavy chain specific for HBsAg, a kappa light chain fragment huck of a human antibody and a porcine enterovirus Porcine teschovirus a fragment P2A;
the VJ fragment and VDJ fragment are inserted in tandem, with the VJ fragment of the light chain followed by one hucκ and P2A.
In the application, the sequence of the HBsAg specific antibody 129G1 (namely BCR) is taken as the sequence of the gene knock-in BCR, the light chain and the heavy chain fragment of the 129G1 antibody are expressed in series, and the Ighd4-1-Ighj4 region of the C57BL/6 mouse is replaced, so that the simultaneous knock-in of the light chain and the heavy chain of the HBsAg BCR is completed in one step; namely, the mice can obtain mice which express the BCR light chain and heavy chain simultaneously without the need of tedious propagation.
Preferably, the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO.1.
SEQ ID NO.1：
1TCGACA GTATGC AGAGGG CTGTAT CCACTG GAGAGG ATGAAG TCACTG AGTTGG AAAACA61GAACAG GACAGG CACCTA ACAAGT GGTTGC TATAGC CCACTG TTACCC TTTTAC ATGTAT121AGGCTC AGGATA AGCAGT GATACT GTGAGG TTTATG TGTGAG AACATC ACAGTA TAAACA181CATCTC AATAGA GGTCTT AGAGAT CAGCAC AATTAG TGAGAA GTCATA AACAGT AGATAC241TATAAG GCATAG GCTCAG CTACCT AGGGTC AGGTAT CTGTGT AAATCT GATTGT GTATCA301GGTTTA GATCAA TATGAC TTAGGG AGGCGA GTCATA TGCAAA TCTAAG AAGACT TTAGAG361AAGAAA TCTGAG GCTCAC CTCACA TAACAG CAAGAG AGTGTC CGGTTA GTCTCA AGGAAG421ACTGAG ACACAG TCTTAG ATATCA CCATGG GTTGGT CCTGCA TTATAC TGTTCC TTGTGG481CTACAG CGACGG GAGTTC ACAGCC AAATCG TTCTCA CCCAGT CTCCAG CAATCA TGTCTG541CATCTC CAGGGG AGAAGG TCACCA TGACCT GCAGTG CCAGTT CAAGTG TAAATT ACGTGC601ACTGGT ACCAGC AGAAGT CTGGCA CCTCCC CCAAAA GATGGA TTTATG ACACAT CCAAAC661TGGCTT CTGGAG TCCCTG TTCGCT TCAGTG GCAGTG GGTCTG GGACCT CTTATT CTCTCA721CAATCA GCAGCA TGGAGG CTGAAG ATGCTG CCACTT ATTACT GCCAGC AGTGGA CTAGTT781TCCCGT ACACGT TTGGAG CTGGGA CCAAGC TGGAGC TAAAGC GGCGTA CGGTCG CTGCAC841CATCTG TCTTCA TCTTCC CGCCAT CTGATG AACAAT TGAAAT CTGGAA CTGCCT CTGTTG901TGTGCC TGCTGA ATAACT TCTATC CCAGAG AGGCCA AAGTCC AGTGGA AAGTGG ATAACG961CCCTCC AATCGG GAAACT CCCAGG AGAGTG TCACAG AACAGG ACTCCA AGGACT CCACCT1021ACAGCC TCTCCT CCACCC TGACGC TGTCCA AAGCGG ACTACG AGAAAC ACAAAG TCTACG1081CCTGCG AAGTCA CCCATC AGGGCC TGTCCT CGCCCG TCACAA AGTCCT TCAACA GGGGAG1141AGTGTG GAAGCG GCGCCA CTAATT TCAGTC TTCTGA AACAGG CCGGAG ACGTGG AGGAGA1201ATCCTG GACCCA TGGGAT GGTCAT GTATCA TCCTTT TTCTAG TAGCAA CTGCAA CCGGTG1261TACATT CCCAGG TGCAGC TACAGC AGTCTG GGCCTC AGCTGA TTAGGC CTGGGG CTTCAG1321TAAAGA TCTCCT GCAAGC CTTCTG GTTACT CATTCT CCGACT ACTGGA TGCACT GGGTGA1381AGCAGA GGCCTG GACAAG GTCTTG AGTGGA TTGGCA TGATTG ATCCTT CCGATA GTGAAA1441CTAGGT TAAATC AGGACT TCAAGG ACAAGG CCACAT TGACTG TAGACA AAGTCT CCAGCA1501CAGCCT ACATGC AACTCA GCAGCC CGACAT CTGAGG ACTCTG CGGTCT ATTATT GTGCAA1561GAAACT ATAGGT ACGACC ACTTTG CTTTGG ACTCCT GGGGTC AAGGAA CCTCAG TCACCG1621TCTCCT CAGGTA AGCTGG CTTTTT TCTTTC TGCACA TTCCAT TCTGAA ACGGGA TCGATT1681GCATAT CGATT
Nucleotide sequence of the deleted Ighd4-1-Ighj4 region (SEQ ID NO. 4): 1TCGAGA ACTTTA GCGACT GTTTTG AGAGAA ATCATT GGTCCC TGACTC AAGAGA TGACTG61GCAGAT TGGGGA TCAGAA TACCCA TACTCT GTGGCT AGTGTG AGGTTT AAGCCT CAGAGT121CCCTGT GGTCTC TGACTG GTGCAA GGTTTT GACTAA GCGGAG CACCAC AGTGCT AACTGG181GACCAC GGTGAC ACGTGG CTCAAC AAAAAC CTTCTG TTTGGA GCTCTC CAGGGG CAGCCT241GAGCTA TGAGGA AGTAGA GAGGCT TGAGAA ATCTGA GGAAGA AAAGAG TAGATC TGAGAG301GAAAGG TAGCTT TCTGGA GGTCAG GAGACA GTGCAG AGAAGA ACGAGT TACTGT GGACAG361GTCTTA GATGGG GAAAGA ATGAGC AAATGC AAGCAT CAGAAG GGTGGA TGCAAT GTCCTG421CCAAGG ACTTAC CAAGAG GATCCC CGGACA GAGCAG GCAGGT GGAGTT GACTGA GAGGAC481AGGATA GGTGCA GGTCCC TCTCTT GTTTCC TTTCTC CTTCTC CTGTTT CCTTCT TCTCTT541GTCACA GGTCTC ACTATG CTAGCC AAGGCT AGCCTG AAAGAT TACCAT CCTACA GATGGG601CCCATC CAGTTG AATTAA GGTGGA GATCTC TCCAAA CATCTG AGTTTC TGAGGC TTGGAT661GCCACT GGGGAC GCCAAG GGACTT TGGGAT GGGTTT GGTTGG CCCCAG ATGAAG GGCTAC721TTCACT GGGTCT ATAATT ACTCTG ATGTCT AGGACC AGGGGG CTCAGG TCACTC AGGTCA781GGTGAG TCCTGC ATCTGG GGACTG TGGGGT TCAGGT GGCCTA AGGCAG GATGTG GAGAGA841GTTTTA GTATAG GAACAG AGGCAG AACAGA GACTGT GCTACT GGTACT TCGATG TCTGGG901GCACAG GGACCA CGGTCA CCGTCT CCTCAG GTAAGC TGGCTT TTTTCT TTCTGC ACATTC961CATTCT GAAACG GGAAAA GATATT CTCAGA TCTCCC CATGTC AGGCCA TCTGCC ACACTC1021TGCATG CTGCAG AAGCTT TTCTGT AAGGAT AGGGTC TTCACT CCCAGG AAAAGA GGCAGT1081CAGAGG CTAGCT GCCTGT GGAACA GTGACA ATCATG GAAAAT AGGCAT TTACAT TGTTAG1141GCTACA TGGGTA GATGGG TTTTTG TACACC CACTAA AGGGGT CTATGA TAGTGT GACTAC1201TTTGAC TACTGG GGCCAA GGCACC ACTCTC ACAGTC TCCTCA GGTGAG TCCTTA CAACCT1261CTCTCT TCTATT CAGCTT AAATAG ATTTTA CTGCAT TTGTTG GGGGGG AAATGT GTGTAT1321CTGAAT TTCAGG TCATGA AGGACT AGGGAC ACCTTG GGAGTC AGAAAG GGTCAT TGGGAG1381CCCTGG CTGACG CAGACA GACATC CTCAGC TCCCAT ACTTCA TGGCCA GAGATT TATAGG1441GATCCT GGCCAG CATTGC CGCTAG GTCCCT CTCTTC TATGCT TTCTTT GTCCCT CACTGG1501CCTCCA TCTGAG ATCATC CTGGAG CCCTAG CCAAGG ATCATT TATTGT CAGGGG TCTAAT1561CATTGT TGTCAC AATGTG CCTGGT TTGCTT ACTGGG GCCAAG GGACTC TGGTCA CTGTCT1621CTGCAG GTGAGT CCTAAC TTCTCC CATTCT AAATGC ATGTTG GGGGGA TTCTGG GCCTTC1681AGGACC AAGATT CTCTGC AAACGG GAATCA AGATTC AACCCC TTTGTC CCAAAG TTGAGA1741CATGGG TCTGGG TCAGGG ACTCTC TGCCTG CTGGTC TGTGGT GACATT AGAACT GAAGTA1801TGATGA AGGATC TGCCAG AACTGA AGCTTG AAGTCT GAGGCA GAATCT TGTCCA GGGTCT1861ATCGGA CTCTTG TGAGAA TTAGGG GCTGAC AGTTGA TGGTGA CAATTT CAGGGT CAGTGA1921CTGTCT GGTTTC TCTGAG GTGAGG CTGGAA TATAGG TCACCT TGAAGA CTAAAG AGGGGT1981CCAGGG GCTTCT GCACAG GCAGGG AACAGA ATGTGG AACAAT GACTTG AATGGT TGATTC2041TTGTGT GACACC AGGAAT TGGCAT AATGTC TGAGTT GCCCAG GGGTGA TTCTAG TCAGAC2101TCTGGG GTTTTT GTCGGG TATAGA GGAAAA ATCCAC TATTGT GATTAC TATGCT ATGGAC2161TACTGG GGTCAA GGAACC TCAGTC ACCGTC TCCTCA GGTAAG AATGGC CTCTCC AGGTCT2221TTATTT TTAACC TTTGTT ATGGAG TTTTCT GAGCAT TGCAGA CTAATC TTGGAT ATTTGT2281CCCTGA GGGAGC CGGCTG AGAGAA GTTGGG AAATAA ACTGTC TAGGGA TC
Preferably, the nucleotide sequence of the gRNA is SEQ ID NO.2 or SEQ ID NO.3.
SEQ ID NO.2
GTTGGGAAATAAACTGTCTAGGG
SEQ ID NO.3
GCTAAAGTTCTCGAGCCTGTGGG
Preferably, the mice are of the C57BL/6 strain.
In a second aspect of the application, there is provided a hepatitis B surface antigen specific B cell receptor gene knock-in mouse model constructed using the method of the application.
Preferably, 70-90% of B cells in peripheral blood specifically recognize HBsAg and differentiate into hair center B cells and plasma cells.
The construction method of the hepatitis B surface antigen specific B cell receptor gene knock-in mouse model comprises the following steps:
s1, constructing a recombinant plasmid of an antibody 129G1 of the HBsAg, and naming the recombinant plasmid as an HBsAg BCR gene;
s2, injecting the HBsAg BCR gene, cas9 and gRNA into the mouse embryo by using a CRISPR-Cas9 method, and enabling the HBsAg BCR gene to replace the original Ighd4-1-Ighj4 region positioned on chromosome 12 through gRNA guidance.
Preferably, the HBsAg BCR gene comprises: a light chain VJ fragment and a VDJ fragment of a heavy chain specific for HBsAg, a kappa light chain fragment huck of a human antibody and a porcine enterovirus Porcine teschovirus a fragment P2A;
the VJ fragment and VDJ fragment are inserted in tandem, with the VJ fragment of the light chain followed by one hucκ and P2A.
Preferably, the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO.1.
Preferably, the nucleotide sequence of the gRNA is SEQ ID NO.2.
Preferably, the mice are of the C57BL/6 strain.
In a third aspect, the application provides the use of a mouse model according to the application in the study of the mechanism of HBsAg specific B-cell immune response and/or the mechanism of HBsAg specific B-cell immune tolerance.
Preferably, the study of the mechanism of HBsAg-specific B-cell immune response includes: the adoptive transfer of B cells from the mouse model to wild-type mice in numbers approaching physiological conditions can strongly proliferate, activate and differentiate into center of growth B cells and plasma cells in HBV acute mouse model.
Preferably, the study of the HBsAg-specific B cell immune tolerance mechanism comprises: the B cells from the mouse model are transferred to wild-type mice in an amount approaching physiological state, and can be activated, proliferated, differentiated and migrated in HBV chronic mouse model.
Compared with the prior art, the application has the beneficial effects and remarkable progress that: the application constructs a gene knock-in mouse for expressing HBsAg specific BCR based on a mouse of a C57BL/6 strain by a homologous recombination method. The gene level, antigen-antibody binding level and the proliferation, activation, differentiation and other aspects of the specificity of the HBsAg of the gene knocked-in mouse B cells are verified. The 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 of hepatitis B virus humoral immune response and tolerance mechanism, and application research of developing medicaments, vaccines and the like for breaking HBsAg immune tolerance.
Drawings
In order to more clearly illustrate the technical solution of the present application, a brief description will be given below of the drawings that are required to be used for the embodiments of the present application.
It is obvious that the drawings in the following description are only drawings of some embodiments of the present application, and that other drawings may be obtained from these drawings without inventive faculty for a person skilled in the art, but these other drawings also fall within the drawings required for the embodiments of the present application.
FIG. 1 is a schematic diagram of the construction strategy of the HBsAg BCR gene knock-in mice in example 1 of the present application;
FIG. 2A is a schematic diagram of the Southern Blot identification of the sites of cleavage in example 2 of the present application;
FIG. 2B is a Southern Blot confirmation of the insertion of a knock-in fragment of example 2 of the present application;
FIG. 2C is a schematic diagram of a targeting segment of a PCR identification primer in example 2 of the present application;
FIG. 2D is a diagram showing the PCR assay result in example 2 of the present application;
FIG. 3A is a schematic flow chart of HBsAg BCR staining in example 3 of the present application;
FIG. 3B is a sample of HBsAg BCR gene knock-in mice HBsAg specific BCR staining verification in example 3 of the present application;
FIG. 4A is a schematic diagram showing in vivo differentiation detection of B cells of mice knocked in with the HBsAg BCR gene in example 4 of the present application;
FIG. 4B shows the differentiation of WT B cells and HBsAg BCR gene-knocked-in B cells into hair center B cells after immunization with pHBV1.3 in example 4 of the present application;
FIG. 4C shows the differentiation of WT B cells and HBsAg BCR gene-knocked-in B cells into plasma cells after immunization with pHBV1.3 in example 4 of the present application;
FIG. 4D shows the differentiation and localization of the HBsAg BCR gene knocked-in B cells in the spleen of WT mice in accordance with example 4 of the present application, scale bar: 500 μm.
Detailed Description
In order to make the purposes, technical solutions, beneficial effects and significant improvements of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application.
It is apparent that all of the described embodiments are only some, but not all, embodiments of the application; all other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
It is to be understood that:
the specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art according to the specific circumstances.
It should also be noted that the following embodiments may be combined with each other, and the same or similar concepts or processes may not be described in detail in some embodiments.
The following describes the technical scheme of the present application in detail by using specific examples.
EXAMPLE 1 construction of HBsAg BCR Gene knock-in mouse model
The application utilizes CRISPR-Cas9 method to inject Igh and Igkappa sequences of antibody 129G1 of pre-recombined HBsAg into mouse embryo, and replaces original Ighd4-1-Ighj4 region located on chromosome 12 by gRNA guidance of targeting Igh, thus completing simultaneous knocking-in of light chain and heavy chain of HBsAg BCR in one step, the specific manufacturing principle is shown in figure 1, and the replacement fragment and knocking-in fragment sequences are SEQ ID NO.1 and SEQ ID NO.4 respectively.
To construct the targeting vector, the homology arms will be generated by PCR using BAC clones RP24-154H17 and RP23-5I20 from the C57BL/6 library as templates. Cas9 and gRNA were co-injected with targeting vector into fertilized eggs (gRNA sequences SEQ ID No.2 and SEQ ID No. 3) to generate knock-in mice. The specific knock-in mice were then identified by sequencing and PCR. The above construction was completed by the Guangzhou racing biotechnology company.
As shown in fig. 1, a light chain VJ fragment specific for HBsAg and a VDJ fragment of heavy chain are inserted in tandem, wherein the VJ fragment of light chain is followed by a kappa light chain fragment (huck) carrying a human antibody, which can be used as a tag to detect B cells specifically expressing HBsAg-specific BCR; the porcine enterovirus Porcine teschovirus A (P2A) fragment is a self-cleaving fragment that serves to split the light and heavy chains.
EXAMPLE 2 Gene level verification of HBsAg BCR Gene knockout mice
This example demonstrates the insertion of a targeting fragment by using Southern Blot and PCR on the gene level in the HBsAg BCR gene knock-in mice constructed in example 1.
Experimental grouping: normal wild mice (WT), HBsAg BCR gene knock-in Mice (MT) prepared in example 1.
The experimental method comprises the following steps:
southern Blot method and results
2.1, preparing DNA to be tested to obtain genome DNA of the WT group mice and MT group mice. Lysing the cells with an appropriate chemical reagent, digesting most of the proteins and RNA with proteases and RNases; protein was removed by extraction with an organic reagent (phenol/chloroform).
And (5) preparing a southern blot probe. The WT group mouse DNA obtained in the step 3.1 is respectively amplified by using Southern Blot primers as follows to obtain 5 'end and 3' hybridization fragments which can form base complementary pairing with a DNA sample to be detected, the fragments are respectively cloned to a pUC19 vector (containing a T7 in vitro transcription promoter), and the Rogowski in vitro transcription and digoxin marking are carried out by using a Rogowski in vitro transcription probe preparation kit (Cat. No. 12039672910), and the split charging probe is kept at the temperature of minus 80 ℃ for standby.
Primers for 5’Probe:
5’Probe forward primer(SEQ ID NO.5):5’-TACAGAGCAGAATCCCAGCCAAGAG-3’
5’Probe reverse primer(SEQ ID NO.6):5’-TCAGCAGTAGGTGCTTAGGGAGCAT-3’
Primers for 3’Probe:
3’Probe forward primer(SEQ ID NO.7):5’-GAATCTGTGTGATGGTGTTGGTGGA-3’
3’Probe reverse primer(SEQ ID NO.8):5’-GAGGCTAGATGCCTTTCTCCCTTGA-3’
2.2 cleavage by Mfel restriction endonuclease. Taking the sample DNA solution obtained in the step 3.1, adding the sample DNA solution into a 0.5ml Eppendorf tube, adding 2 μl of 10 Xrestriction enzyme buffer, adding 6-10U of corresponding restriction enzyme, adding sterilized double distilled water to the total volume of 20 μl, preserving the temperature at 37 ℃ for enzymolysis for 2 hours, heating at 65 ℃ for 5 minutes or stopping the reaction with a proper amount of 0.5 mol/LEDTNA 2.
As a result, as shown in FIG. 2A, the WT mouse could obtain 7.69KB and the MT mouse could obtain a fragment having a size of 11.12 kb.
2.3, separating DNA samples by agarose gel electrophoresis. And 2. Mu.l of the DNA enzymatic hydrolysate obtained in the step 2.2 is added with 10. Mu.l of a sample buffer (containing bromophenol blue indicator and glycerol), and the mixture is subjected to horizontal electrophoresis on a 0.8% agarose gel (containing 0.5. Mu.g/ml ethidium bromide) at a voltage of <5V/cm for about 2 hours.
The electrophoretically separated DNA sample is alkali denatured and transferred to a solid support (nylon membrane). The agarose gel after electrophoresis is denatured in alkaline denaturation solution (0.5M NaOH,1.5M NaCl) for 45min, rinsed with double distilled water for 3 times, treated with neutralization solution (1 MTris-HCl,1.5M NaCl,pH7.4) for 45min, and then a transfer system is installed according to a downstream capillary transfer method, and the transfer film is used for 8 hours.
The DNA immobilized on the membrane was annealed and hybridized with the digoxin-labeled probe. The nylon membrane containing the sample DNA obtained above was baked at 80℃for 2 hours, then placed in a hybridization tube, 5ml of a hybridization solution was added thereto for prehybridization at 42℃for 30 minutes, and then the prepared southern blot probe (10 minutes after denaturation at 65℃before probe addition) was added thereto for 5 minutes in an ice bath, followed by hybridization for about 8 hours.
The membrane was incubated with alkaline phosphatase-labeled digoxin antibody. Discarding the hybridization solution in the hybridization tube, adding a low-stringency washing solution at 25 ℃ for washing for 5 minutes, repeating the washing for 15 minutes at 68 ℃ and repeating the washing for one time, adding a blocking solution at 25 ℃ for blocking for 1 hour, and then adding a digoxin antibody at 25 ℃ for incubation for 30 minutes.
Alkaline phosphatase substrate was added to develop color. Discarding the antibody incubation buffer solution in the hybridization tube, adding a washing buffer solution, washing for 15 minutes at 25 ℃, repeating for 5 minutes by adding a detection buffer solution, repeating for one time, taking out the membrane, placing the membrane on oilpaper, dripping a luminescent solution, and developing and detecting.
The results are shown in the left panel of FIG. 2B, where WT represents the WT mouse group and 3, 5, 27 and 28 represent the MT mouse group. MT mice obtained both fragments of 11.12kb and 7.69kb, whereas WT mice had fragments of only 11.12kb in size.
2.4 Southern Blot experiments were performed using the same procedure as steps 2.2 and 2.3, using the other restriction enzyme BamHI.
The results are shown in the right panel of FIG. 2B, where WT represents the WT mouse group and 3, 5, 27 and 28 represent the MT mouse group. MT mice obtained both fragments of 12.85kb and 10.16kb, whereas WT mice had fragments of only 10.16kb in size.
PCR method and results
2.5, preparing DNA to be detected, and obtaining the genome DNA of the WT group mice and the MT group mice by adopting the method of the step 2.1.
2.6 primers were designed based on WT alleles and alternative sequences, the primer design principle being shown in FIG. 2C, using a common front primer F1 and separate rear primers R1 and R2.
2.7, PCR extension is carried out on the DNA obtained in the step 2.5 by adopting the primer designed in the step 2.6.
The results are shown in FIG. 2D, where WT represents the WT mouse group and KI1 and KI2 represent the MT mouse group. The WT and MT groups can be amplified to give fragments of 443bp and 285bp, respectively. Example 1 proved successful in directing Cas9 enzymatic cleavage by gRNA, substituting the Ighd4-1-Ighj4 sequence of C57BL/6 mice for the coding sequence of monoclonal antibody 129G1 for HBsAg.
Wherein, the liquid crystal display device comprises a liquid crystal display device,
F1(SEQ ID NO.9):5’-CTGATAGGCACCCAAGTACACTA-3’
R1(SEQ ID NO.10):5’-GCTATAGCAACCACTTGTTAGGTG-3’
R2(SEQ ID NO.11):5’-CTTCCTCATAGCTCAGGCTGC-3’。
EXAMPLE 3 antigen-antibody binding assay validation mice
As shown in FIG. 3A, the binding (binding) of the HBsAg BCR gene knock-in mouse B cells constructed in example 1 to HBsAg was confirmed by fluorescent-labeled HBsAg (HBsAg-Alexa 647) staining.
3.1, crosslinking the HBsAg and Alexa647 fluorescent dye to obtain HBsAg-Alexa647;
1) HBsAg was dissolved to 10mg/mL (10X) using PBS;
2)84mg NaHCO 3 added to 1mL ddH 2 In O, 1mol/L NaHCO is prepared 3 A solution;
3) HBsAg (10X) was diluted to 1X using PBS to a final volume of 100uL;
4) 10uL of 1M NaHCO is taken 3 The solution was added to 100uL HBsAg (1X);
5) The above solution was added dropwise to Alexa Fluor 647 dye solution. And (3) injection: every drop needs to be shaken
6) Incubate for 2h at room temperature in the dark. And (3) injection: shaking once every 15min, and disabling the vortex or the pipetting gun for blowing;
7) Equilibrate column 30min before incubation is completed: breaking off the bottom seal of the column, placing the column in a 15mL centrifuge tube with a collecting tube, and carrying out 5min air separation on the column for 1 time at 4 ℃ and washing for 4 times by 500 uLPBS;
8) Adding the incubated solution into a column, replacing a column bottom collecting pipe, and centrifuging at 4 ℃ for 350g of 5 min;
9) The antibodies were transferred to brown antibody tubes and stored at 4 ℃.
3.2, HBsAg BCR Gene knock-in B cells and HBsAg-Alexa647 co-incubated, enabling cells binding to HBsAg to carry Alexa647 dye;
1) Collecting the orbital blood into an anticoagulant tube;
2) Adding erythrocyte lysate, and after re-suspending, cracking red on ice for 10min;
3) Centrifuging at 4deg.C for 5min at 500g, discarding supernatant, resuspending the precipitate with PBS+2% FBS, centrifuging at 4deg.C for 5min at 500g, discarding supernatant;
4) Compounding antibody mix, according to 1:200 adding B220 and HBsAg-Alexa flow to PBS+2% FBS buffer
647, blowing and uniformly mixing;
5) Resuspending the cell pellet with antibody mix and staining in the dark on ice for 30min;
6) PBS+2% FBS buffer is added for 2 times, 500g is centrifuged for 5min, and finally the mixture is resuspended by PBS+2% FBS buffer and put on the machine.
3.3, detecting through a stream.
As a result, as shown in FIG. 3B, 80-90% of B cells, which were knocked into the peripheral blood of mice by fluorescence-labeled HBsAg staining analysis, showed binding of HBsAg, and all of these cells expressed HuCκ at the same time.
In conclusion, the results of this example obtained a HBsAg BCR gene knock-in mouse whose B cells had the property of binding specifically to HBsAg.
Example 4 response-layer verification of specific HBsAg antigen mice
The present example is a functional verification of B cells of mice knocked in by HBsAg BCR gene, in particular, a verification of the ability of the mice to differentiate into effector cells and produce antibodies after receiving specific antigen stimulation in vivo.
4.1, as shown in FIG. 4A, the majority of B cells in the body of the HBsAg BCR gene knock-in mice are HBsAg antigen-specific B cells, and in order to make the experiment more physiological, the HBsAg BCR gene was knocked inB cell negative selection purification to 1X 10 6 cell/adoptive transfer to WT mice alone for experiments. After adoptive transfer for 1 day, 20 mug of pHBV1.3 plasmid is injected into the tail vein at high pressure (more HBsAg can be detected in peripheral blood after 1 day of plasmid injection), and after 4.5 days of immunization, mice are sacrificed to obtain spleen single cell suspension for dyeing germinal center B cells (GC B) and Plasma Cells (PC);
4.2, the cells which are transferred in succession are separated by the HuCκ mark, the differentiation condition is analyzed,
as a result, as shown in FIG. 4B, each spleen was resuspended at 1ml during the experiment, 50ul of lysed erythrocytes were taken per well and stained, wherein 2-3 x 10≡5 HuCκ+ cells were obtained, i.e., only 4-6 fold increase in number had been obtained in the spleen after 4.5 days of immunization, indicating that significant proliferation of the HBsAg BCR gene knock-in B cells adoptively transferred to WT mice occurred.
4.3, analyzing the differentiation condition of GCB and PC in B cells;
results as shown in fig. 4B and 4C, after preliminary removal of dead, adherent and dead cells by CD3, CD8, CD11B, CD11C, huck tag was used directly to distinguish huck-WT mouse background B cells from huck+ HBsAg BCR gene knock-in (HBsAg-binding) B cells, as can be seen by comparison of background WT cells, with GC B and PC being about 10-fold and 40-fold higher compared to WT B cells, respectively, because WT B cells contain multiple unrelated B cells that recognize other antigens but are unable to recognize HBsAg; from the quantitative point of view, huCκ+ was present in about 2/3 of all PCs formed, and the numbers of GC B cells HuCκ+ and HuCκ -were comparable.
4.4, analysis by immunohistochemical staining;
as a result, as shown in FIG. 4D, the HBsAg BCR gene knockin B cells differentiated to form germinal center B cells (GL-7+) constituting germinal center and plasma cells (CD138+) located in the outer follicular region.
In summary, acute infection of phbv1.3 in WT mice with HBsAg BCR gene knock-in B cells strongly proliferated and differentiated into center B cells and plasma cells, which can form antibodies of the IgG2a class by class switching.
The experiment proves that the application successfully constructs HBV surface antigen specific HBsAg BCR gene knock-in mice, 70-90% of B cells of which can specifically recognize HBsAg. The antigen-specific B cells can be activated and differentiated into corresponding effector cells in vitro and in vivo by antigen stimulation, and can also be subjected to class switching. By mating with HBV transgenic mice, the phenomenon of HBsAg-specific B cell tolerance is obtained, and a phenotype similar to depletion can be induced in an AAV-HBV chronic mouse model. Therefore, the designed and constructed HBsAg BCR gene knock-in mice can be used as an important tool for researching various biological processes including antibody generation, tolerance, formation of exhaustion and the like in chronic HBV infection humoral immunity.
In the description of the above specification:
the terms "this embodiment," "an embodiment of the application," "as shown in … …," "further improved embodiments," and the like, mean that a particular feature, structure, material, or characteristic described in the embodiment or example is included in at least one embodiment or example of the application; in this specification, a schematic representation of the above terms is not necessarily directed to the same embodiment or example, and the particular features, structures, materials, or characteristics described, etc. may be combined or combined in any suitable manner in any one or more embodiments or examples; furthermore, various embodiments or examples, as well as features of various embodiments or examples, described in this specification may be combined or combined by one of ordinary skill in the art without undue experimentation.
Finally, it should be noted that:
the above embodiments are only for illustrating the technical solution of the present application, and are not limiting thereof;
although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some or all of the technical features thereof, without departing from the spirit of the technical solutions of the embodiments of the present application, and that insubstantial improvements and modifications or substitutions by one skilled in the art from the disclosure herein are within the scope of the application as claimed.

Claims (10)

1. The construction method of the hepatitis B surface antigen specific B cell receptor gene knock-in mouse model is characterized by comprising the following steps:
s1, constructing a recombinant plasmid of 129G1 monoclonal antibody specific for HBsAg, and naming the recombinant plasmid as an HBsAg BCR gene;
s2, injecting the HBsAg BCR gene, cas9 and gRNA into the mouse embryo by using a CRISPR-Cas9 method, and enabling the HBsAg BCR gene to replace the original Ighd4-1-Ighj4 region positioned on chromosome 12 through gRNA guidance.
2. The method for constructing a hepatitis B surface antigen-specific B cell receptor gene knock-in mouse model according to claim 1, wherein the HBsAg BCR gene comprises: a light chain VJ fragment and a VDJ fragment of a heavy chain specific for HBsAg, a kappa light chain fragment huck of a human antibody and a porcine enterovirus Porcine teschovirus a fragment P2A;
the VJ fragment and VDJ fragment are inserted in tandem, with the VJ fragment of the light chain followed by one hucκ and P2A.
3. The method for constructing a model of a hepatitis B surface antigen specific B cell receptor gene knock-in mouse according to claim 2, wherein the nucleotide sequence of the HBsAg BCR gene is SEQ ID NO.1.
4. The method for constructing a model of a hepatitis B surface antigen specific B cell receptor gene knock-in mouse according to claim 1, wherein the nucleotide sequence of the gRNA is SEQ ID NO.2 or SEQ ID NO.3.
5. The method for constructing a model of a hepatitis B surface antigen-specific B cell receptor gene knock-in mouse according to claim 2, wherein the mouse is a C57BL/6 strain mouse.
6. A mouse model of hepatitis B surface antigen specific B cell receptor gene knock-in, wherein the mouse model is constructed using the method of any one of claims 1-5.
7. The mouse model of claim 6, wherein 70-90% of B cells in peripheral blood specifically recognize HBsAg and differentiate into hair center B cells and plasma cells.
8. Use of a mouse model according to claim 6 or 7 for the study of HBsAg specific B cell immune response mechanisms and/or HBsAg specific B cell immune tolerance mechanisms.
9. The use according to claim 8, wherein the investigation of the HBsAg-specific B-cell immune response mechanism comprises: the adoptive transfer of B cells from the mouse model to wild-type mice in numbers approaching physiological conditions can strongly proliferate, activate and differentiate into center of growth B cells and plasma cells in HBV acute mouse model.
10. The use according to claim 8, wherein the study of HBsAg-specific B-cell immune tolerance mechanisms comprises: the B cells from the mouse model are transferred to wild-type mice in an amount approaching physiological state, and can be activated, proliferated, differentiated and migrated in HBV chronic mouse model.
CN202310054808.7A 2023-02-03 2023-02-03 Construction of hepatitis B surface antigen specific B cell receptor gene knock-in mouse model Pending CN116649294A (en)

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