CN106148405A - A kind of construction method of Optimization-type HLA-I class transgenic mice - Google Patents
A kind of construction method of Optimization-type HLA-I class transgenic mice Download PDFInfo
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Abstract
The invention discloses the construction method of a kind of Optimization-type HLA-I transgenic mice.The invention provides a kind of method building the HLA-I transgene mouse model that HLA-II antigen strengthens, comprise the steps: to build hTAP bi-transgenic mice；HLA-I transgenic mice and hTAP mice are carried out copulation, and gained double transgenic progeny is the HLA-I transgene mouse model that HLA-II antigen strengthens；Described hTAP transgenic mice is the mice having proceeded to following gene: people source TAP1 gene, people source TAP2 gene, people source PSMB8 gene and people source PSMB9 gene.The HLA-I/hTAP bi-transgenic mice that the present invention breeds out, compared with HLA-I single transgenic mice, strengthens the ability of angtigen presentation.Visible, hTAP transgenic mice contributes to the HLA-II antigen of HLA-I transgenic mice HLA-I and strengthens ctl response.
Description
Technical field
The invention belongs to biological technical field, relate to the construction method of a kind of Optimization-type HLA-I class transgenic mice, especially
Relate to a kind of method that the HLA-I of raising transgenic mice HLA-I molecular antigen offers ability.
Background technology
The angtigen presentation approach of MHC I quasi-molecule includes three phases: the generation of antigen peptide fragment, peptide fragment are transported to endoplasm
The combination of net, peptide fragment and MHC I quasi-molecule and displaying.First, antigen protein in cytoplasm by proteasomal degradation
Become the small peptide of 3-22 residue.These small peptides are through the further pruning of heat shock protein HSP90, by angtigen presentation
Relevant transport protein (transporter associated with antigen presentation, TAP) is optionally transported to
Rough endoplasmic reticulum (rough endoplasmic reticulum, RER) intracavity.In RER, molecular chaperones calcium connection egg
(calnexin, CNX) promotes the folding of free MHC I heavy chain in vain, and raises another molecular chaperones ERp57 rush
Enter the formation of disulfide bond in MHC I quasi-molecule domain；Subsequently, B2M (β 2m) and MHC I heavy chain
In conjunction with, CNX dissociates, and MHC I combines with molecular chaperones calprotectin (calreticulin, CRT).When TAP phase
When pass albumen Tapasin (TAP-associated glyprotein) is transported to TAP near MHC I quasi-molecule, MHC
I quasi-molecule heavy chain/β 2m/ERp57/CRT/Tapasin/TAP together form polypeptide and loads complex
(peptide-loading complex, PLC).After polypeptide is transported RER by TAP, the MHC I class in PLC is divided
Polypeptide is captured by son.MHC I quasi-molecule correctly assembles and combine polypeptide rear stability to be increased, and by from PLC
In discharge, enter secretory pathway arrive cell surface, accept at this CTL monitor thus trigger possible immunity
Response.Therefore, MHC I quasi-molecule antigen processing presentation pathway is the complex process that a polymolecular participates in, Qi Zhongcan
With molecule all will affect the immune response of body.
In view of the importance of MHC I quasi-molecule, at present, multiple people source MHC I quasi-molecule transgenic mice (i.e. HLA
-I transgenic mice) it is built and be used for the screening of the important virus CTL epitope of people, qualification and immunization therapy evaluation,
Such as influenza virus, SARS virus etc., the development and application for vaccine is had laid a good foundation.But, in view of body
Endoantigen processes the complexity with presentation pathway and multifactor property, and the application of independent HLA-I transgenic mice is often made
Become the leakage choosing of epi-position and wrong choosing, affect the correctness of epi-position screening.Document is had to point out, little by HLA-A2 transgenic
Vaccinia virus (VACV) epitope polypeptide screened in Mus only 1/3 can be consistent with human test results (Kotturi
MF,et al.(2009)Of mice and humans:how good are HLA transgenic mice as a model of
human immune responses？Immunome Res 5:3.).
Additionally, it is known that HLA-A3 superfamily (comprising HLA-A11, HLA-A33, HLA-A68 etc.) is in China
Crowd's HLA typing accounts for maximum ratio (about 52.7%) (Sette, A.and J.Sidney (1999) Nine major
HLA class I supertypes account for the vast preponderance of HLA-A and-B polymorphism.
Immunogenetics 50 (3-4): 201-212.), wherein HLA-A11 is distributed the widest in being A3 superfamily, statistics
Analysis also indicates that higher ((2007) In South China such as the beam quick cutting edge of a knife or a sword ground of HLA-A11 gene frequency in China's hepatitis B patient
The preliminary survey of district's chronic hepatitis B patient HLA-A allele distributions and meaning thereof. liver volume 12 4 phase:
240-243), hence setting up the relevant HLA-I transgenic animal model of this family, to carry out epi-position screening significant.
But there are some researches show, this family member's preference combines the polypeptide that C-terminal is basic amino acid, this feature just with
The Preference of mice TAP Binding peptide do not correspond (Falk.K., et al. (1994) Peptide motifs of HLA-A1,
-A11,-A31,and-A33molecules.Immunogenetics 40:238-241.).It is presumed that, in such family
In HLA-I transgenic mice, matched polypeptide will by the efficiency in mice TAP molecular transport to endoplasmic reticulum
It is substantially reduced, thus affects epi-position screening.
Summary content, we are badly in need of building a kind of Optimization-type HLA-I transgenic mice, are allowed to have higher table
Position screening efficiency, the evaluation of screening and vaccine to be preferably applied for CTL epi-position in major disease research.
Summary of the invention
It is an object of the present invention to provide a kind of HLA-I transgene mouse model building HLA-II antigen enhancing
Method.
The method of the HLA-I transgene mouse model that structure HLA-II antigen provided by the present invention strengthens, specifically may be used
Comprise the steps: to build hTAP transgenic mice, by little with described hTAP transgenic for HLA-I transgenic mice
Mus carries out copulation, and the bi-transgenic mice in gained offspring is the HLA-I transgenic mice that HLA-II antigen strengthens
Model；
Described hTAP transgenic mice is the mice having proceeded to following gene: people source TAP1 gene, people source TAP2
Gene, people source PSMB8 gene and people source PSMB9 gene.
In the present invention, described hTAP transgenic mice has also been transferred into two HLA-II quasi-molecule HLA-DOB
Gene and HLA-DMB gene.
Wherein, described bi-transgenic mice is both to have proceeded to the exogenous gene in described HLA-I transgenic mice, also turns
Enter the mice of exogenous gene in described hTAP transgenic mice.
Concrete, that the nucleotides sequence of described people source TAP1 gene is classified as in sequence table sequence 1；Described people source TAP2
The sequence 2 that the nucleotides sequence of gene is classified as in sequence table；The nucleotides sequence of described people source PSMB8 gene is classified as sequence table
In sequence 3；The sequence 4 that the nucleotides sequence of described people source PSMB9 gene is classified as in sequence table.Described HLA-DOB
The sequence 5 that the nucleotides sequence of gene is classified as in sequence table；The nucleotides sequence of described HLA-DMB gene is classified as sequence table
In sequence 6.
Wherein, sequence 1 is made up of 8058 nucleotide；Sequence 2 is made up of 9328 nucleotide；Sequence 3 is by 3455
Individual nucleotide forms；Sequence 4 is made up of 5304 nucleotide；Sequence 5 is made up of 3728 nucleotide；Sequence 6
It is made up of 6403 nucleotide.
More specific, described hTAP transgenic mice is prepared by a method comprising the following steps:
(1) RP11-10A19BAC plasmid is expelled to the sperm by purpose mice 1 and the ovum of purpose mice 2
In the protokaryon of the germ cell formed, zygote transplation enters in replace-conceive dams grows, and obtains F0 for allophenic mice after birth；
Described F0 is PCR checking for allophenic mice and identifies that positive hTAP head builds Mus；
(2) described F0 chimera step (1) obtained is for mice (positive head builds Mus) and described purpose mice 2
Hybridize, identified by PCR and Western blotting analyzes, obtain from F1 generation mice described in having proceeded to
The heterozygote mice of RP11-10A19BAC plasmid, this heterozygote mice is hTAP transgenic mice.
Certainly, described hTAP transgenic mice is alternatively to enter male heterozygote mice with female heterozygote mice
Row one or many hybridizes, and obtains that to have proceeded to the homozygote of described RP11-10A19BAC plasmid little from filial generation
Mus.
In the present invention, described purpose mice 1 is specially B6D2F1 mice, and described purpose mice 2 is specially
C57BL/6 mice, described replace-conceive dams is specially ICR mice.
In the present invention, the GenBank ID of above all of described RP11-10A19BAC plasmid is 207834.
The method building hTAP transgenic mice as above belongs to protection scope of the present invention.
It is a further object to provide a kind of HLA-I transgenic for building HLA-II antigen enhancing little
The test kit of mouse model.
Test kit for building the HLA-I transgene mouse model that HLA-II antigen strengthens provided by the present invention,
Specifically can include described HLA-I transgenic mice, described hTAP transgenic mice and readable carrier；Described can
The side building the HLA-I transgene mouse model that HLA-II antigen strengthens as above is had described in the property read carrier
Method.
A further object of the present invention is to provide a kind of test kit building hTAP transgenic mice.
Test kit for building hTAP transgenic mice provided by the present invention, specifically can include RP11-10A19
BAC plasmid and readable carrier；There is structure hTAP transgenic as above little described in described readable carrier
The method of Mus.
The application in the HLA-II antigen strengthening HLA-I transgenic mice of the described hTAP transgenic mice falls within
Protection scope of the present invention.
In the present invention, described HLA-I transgenic mice can be HLA-A2, HLA-A11, HLA-A1,
The HLA-I transgenic mice that HLA-A24, HLA-B7, HLA-B27, HLA-B4 etc. are currently reported.
In one embodiment of the invention, described HLA-I transgenic mice be specially HLA-A2 transgenic mice or
HLA-A11 transgenic mice.Described HLA-A2 transgenic mice is the C57BL/6J back of the body proceeding to HLA-A2 gene
Scape mice；Described HLA-A11 transgenic mice is the C57BL/6J × BALB/c background proceeding to HLA-A11 gene
Mice.
Described angtigen presentation is offering of the HLA-I restriction epi polypeptide to corresponding transgenic mice.The present invention's
In one embodiment, described HLA-I transgenic mice is specially HLA-A11 transgenic mice；Described HLA-A11
The corresponding angtigen presentation of transgenic mice is to offer HLA-A11 restriction epi polypeptide, and described HLA-A11 limits
Property epitope polypeptide processed is specially HBc141-151, and its aminoacid sequence is STLPETTVVRR (sequence 7).
In the present invention, the HLA-II antigen of described enhancing HLA-I transgenic mice is embodied as described
The enhancing of the ctl response of HLA-I restriction epi polypeptide.
It is demonstrated experimentally that the present invention constructs hTAP transgenic mice, hTAP transgenic mice is turned base with HLA-I
Because mice carries out copulation, breed out HLA-I/hTAP bi-transgenic mice, carry out functional experiment, find and HLA-I
Single transgenic mice is compared, and the ability of angtigen presentation is strengthened by HLA-I/hTAP bi-transgenic mice.Visible, hTAP
Transgenic mice contributes to the angtigen presentation of HLA-I in HLA-I transgenic mice and strengthens ctl response.
Accompanying drawing explanation
Fig. 1 is that PCR identifies people's MHC I class angtigen presentation approach correlation molecule in hTAP transgenic mice.M:
DNA molecular amount standard, each band is descending is followed successively by 500bp, 400bp, 300bp and 200bp.
Fig. 2 is that Western blotting identifies the expression of people's TAP2 albumen in hTAP transgenic mice.
Fig. 3 is in HLA-A2/hTAP bi-transgenic mice, and the expression of HLA-A2 molecule is significantly raised.A:
HLA-A2 expresses streaming figure.A2-hTAP represents HLA-A2/hTAP bi-transgenic mice；A2 represents HLA-A2
Single transgenic mice；WT represents wild type C57BL/6J mice.B:HLA-A2 expression cartogram.
Fig. 4 is in HLA-A11/hTAP bi-transgenic mice, and the expression of HLA-A11 molecule is significantly raised.
A: splenocyte surface HLA-A11 expresses streaming figure (left) and cartogram (right), * * * P < 0.001.B: spleen is thin
Cellular surface H2-KbExpression streaming figure (left) and cartogram (right) thereof.A11-hTAP represents HLA-A11/hTAP
Bi-transgenic mice；A11het represents HLA-A11 single transgenic mice；TAP represents hTAP transgenic mice；
WT represents wild type C57BL/6J mice.
Fig. 5 is that HLA-A11/hTAP bi-transgenic mice is the strongest to the ctl response of HLA-A11 restricted epitope
In HLA-A11 single transgenic mice.A:Elispot detects two kinds of mices ctl response to HBc141-151,
A11-hTAP (n=5), A11het (n=5), peptide concentration 1 μ g/ml；B:Elispot counts statistics block diagram,
* P < 0.05, * * P < 0.01.
Fig. 6 is that in HLA-A11/hTAP bi-transgenic mice, ctl response is obviously enhanced.A:IFN-γ intracellular contaminates
Dispersion point diagram；B:INF-γ+CD8+Cell percentages cartogram,*P < 0.05,**P<0.01。
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
RP11-10A19BAC plasmid, GenBank ID:207834, this plasmid be U.S. CHOIR (Children ' s
Hospital Oakland Research Institute) product.People's MHC I class that RP11-10A19BAC plasmid comprises
Angtigen presentation approach correlation molecule has TAP1, TAP2, PSMB8 and PSMB9, additionally possibly together with two people MHC
II class correlation molecule HLA-DOB, HLA-DMB.
HLA-A2 transgenic mice: seminar give by Institute of Microorganism, Academia Sinica Meng Song east, its heredity back of the body
Scape is C57BL/6J mice.Be recorded in " appointing Yulin etc. the B that (2010) New Type of HLA-A2 limits, c-type HBV is special
The screening of specific CTL epitopes and qualification. Science Bulletin (55) 30:2915～2924 " in a literary composition, the public can be therefrom
Institute of microbiology of academy of science of state obtains.
HLA-A11 transgenic mice: from Taconic company, male model mice numbering 9660-M, female model
Mouse number 9660-F.Its genetic background is C57BL/6J × BALB/c mouse.
C57BL/6J mice: from Beijing HFK Bio-Technology Co., Ltd., phone: 010-60753558.
ICR mice: female, from Beijing Vital River Experimental Animals Technology Co., Ltd..
B6D2F1 mice: male, from Beijing Vital River Experimental Animals Technology Co., Ltd..
PCDNA3.1-HBc (D) plasmid: seminar give by Institute of Microorganism, Academia Sinica Meng Song east.It is recorded in
“Xu Y.,et al(2013)Activation of anti-HBV immune activity by DNA vaccine via
electroporation using heat shock proteins as adjuvant.Sheng Wu Gong Cheng Xue Bao
29 (12): 1765-75 ", in a literary composition, the public can obtain from Institute of Microorganism, Academia Sinica.This plasmid carries hepatitis B
The gene order of virus core antigen, sequence 8 during wherein the gene order of hepatitis B virus core antigen is sequence table.
Embodiment 1, the structure of hTAP transgenic mice
One, F0 is for the acquisition of allophenic mice
1, F0 is for the acquisition of mice
With BAC inject buffer (formula is: 10mM Tris-HCl, 0.1mM EDTA, 100mM NaCl,
10 μMs of spermidines, 30 μMs of spermine, pH7.2) RP11-10A19BAC plasmid is configured to concentration is 1ng/ μ l
Solution.The solution prepared is full of injection needle, injection needle is installed on microinjection microscope,
Carrying out micrurgy under the microscope, (sperm source is in B6D2F1 mice, ovum through germ cell i.e. to promote entry needle
Son derives from C57BL/6 mice) hyalomitome band, utilize the continuous (about 150hPa) of compression pump to inject to protokaryon
DNA in entry needle, if protokaryon expands, shows to inject successfully that (the extremely difficult accurate control of injected slurry volume, with protokaryon volume
Occur substantially to be expanded to directive standard).Germ cell is placed in 37 DEG C of CO2 gas incubator and cultivates 24 hours to 2-
Being transplanted to after cell stage in the female Mus of replace-conceive (ICR mice), the final F0 that obtains is for mice.
2, F0 is prepared for mouse genome
F0 is for mice 14 days clip rat-tail 0.3cm of birth, and adding 400 μ l lysates (is had by south, Shanghai model organism
Limit company provides, catalog number: NMS014) and 40 μ l E.C. 3.4.21.64s (concentration is 10mg/ml), 56 DEG C disappear
Change overnight.Centrifuging and taking supernatant, two volumes dehydrated alcohol precipitates, and 75% washing with alcohol is dissolved in 100 μ l after slightly drying
In pure water, 50 DEG C of baking oven hydrotropies 1 hour.
3, F0 identifies for murine genes type
The each F0 obtained with step 2 for the genome of mice as template, respectively to people source TAP1, TAP2, PSMB8,
(sequence of 6 human source genes is followed successively by sequence 1-6 in sequence table to PSMB9, HLA-DOB and HLA-DMB gene
Shown in) carry out PCR amplification.Primer sequence for each gene is as follows:
For people source TAP1 gene primer to for:
TAP1-F:5 '-GCCTCTTATCGTGTGCTTCT-3 '；
TAP1-R:5 '-CCATCTCCACCCAAGGTC-3 '.
TAP1-F/TAP1-R theoretical amplification product size is 319bp.
For people source TAP2 gene primer to for:
TAP2-F:5 '-CGCCCACAGTGTTAGGT-3 '；
TAP2-R:5 '-GAAGAGGCGTTTGGAATAG-3 '.
TAP2-F/TAP2-R theoretical amplification product size is 234bp.
For people source PSMB8 gene primer to for:
PSMB8-F:5 '-AGTACGTCAGGTATTTAGC-3 '；
PSMB8-R:5 '-GAGGGAGTAGGAGTATATG-3 '.
PSMB8-F/PSMB8-R theoretical amplification product size is 438bp.
For people source PSMB9 gene primer to for:
PSMB9-F:5 '-CACTTAATGTCTCAGTGGGA-3 '；
PSMB9-R:5 '-TATTTCTCTTCCTGTTCCTC-3 '.
PSMB9-F/PSMB9-R theoretical amplification product size is 289bp.
For people source HLA-DOB gene primer to for:
HLA-DOB-F:5 '-TAAACGTGTCTGGTATGTC-3 '；
HLA-DOB-R:5 '-AGTTAAAGATGAATCTGACC-3 '.
HLA-DOB-F/HLA-DOB-R theoretical amplification product size is 347bp.
For people source HLA-DMB gene primer to for:
HLA-DMB-F:5 '-CAAAGGAACATCCAGATGAAG-3 '；
HLA-DMB-R:5 '-TCCCCCATACTCCCTGAAG-3 '.
HLA-DMB-F/HLA-DMB-R theoretical amplification product size is 228bp.
Experiment is arranged with H simultaneously2O is as the blank of template, with C57BL/6J mice (wild type) genome
As the negative control of template, and using human genome as the positive control of template.
Result shows, by germ cell microinjection and transplanting, Recipient mice number is 4, and conceived number is 4, bosom
Pregnant rate 100%；Transplanting ovum number is 30 pieces/, and the mice that is altogether born (F0 generation) number is 24, natality 20%.
Positive 2, positive rate 8.3%.6 human source genes all expand and obtain the F0 of corresponding purpose band and be for mice
RP11-10A19BAC plasmid is injected and transplants successful F0 for allophenic mice.
Two, the acquisition of F1 generation heterozygote mice and genotype identification
1, the acquisition of F1 generation mice
According to step a pair F0 for the genotype identification result of mice, choose positive F0 for allophenic mice with wild
The mice copulation of type C57BL/6J, obtains F1 generation mice (wild type and heterozygous).
2, prepared by F1 generation mouse genome
Carry out with reference to step one 2.
3, F1 generation murine genes type is identified
Carry out with reference to step one 3.
Six human source genes all expand and obtain the F1 generation mice of corresponding purpose band and be and identify positive proceeding to
The F1 generation chimeric mice of RP11-10A19BAC plasmid.The F1 generation that part proceeds to RP11-10A19BAC plasmid is miscellaneous
The PCR qualification result of fit mice (Founder2 and Founder14) is as shown in Figure 1.
4, F1 generation mice expression identification
Take F1 generation Mouse spleen cells as sample extraction albumen, carry out Western for people source TAP2 albumen
Blotting detects.Wherein, used one resist antibody for anti-human source TAP2 albumen (Mus source, MBL Products,
Its catalog number is K0137-3)；Two resist antibody (goat source, Beijing Zhong Shan Golden Bridge biology skill for anti-mouse IgG
Art company limited product, its catalog number is ZDR-5307).Experiment is simultaneously using β-actin as internal reference, and one resists and is
The antibody (its catalog number is DKM9001 for Mus source, three arrow Products) of anti-β-actin；Two resist for anti-mouse
(goat source, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge product, its catalog number is the antibody of IgG
ZDR-5307)。
Experiment arranges C57BL/6J mice (wild type) as comparison simultaneously.
The F1 generation mice having obvious people source TAP2 Protein Detection signal is the expression proceeding to RP11-10A19BAC plasmid
Positive F1 chimeric mice.Part proceed to RP11-10A19BAC plasmid F1 generation heterozygote mice (Founder2 and
Founder14) Western blotting qualification result is as shown in Figure 2.The background of this mice be C57BL/6J ×
B6D2F1, can breed for testing.
In order to confirm F1 generation heterozygote mice further, the present inventor has carried out gene sequencing to it,
Result shows, F1 generation heterozygote mice (Founder2 and Founder14) is really containing sequence 1-6 institute in ordered list
6 human source genes (TAP1, TAP2, PSMB8, PSMB9, HLA-DOB and HLA-DMB) shown.
Three, F10 is for the acquisition of C57BL/6J background hTAP heterozygote mice and genotype identification
The F1 generation heterozygote mice that selecting step two obtains and wild type C57BL/6J carry out copulation, are reflected by genotype
Fixed (step one, 3) and expression identification (step 2 4) and sequencing obtain positive F2 for heterozygote mice.
Again positive F2 is bred with wild type C57BL/6J for heterozygote mice, it is thus achieved that F3 is for heterozygote mice for the positive,
The rest may be inferred enters breeding, and final acquisition F10 is for the positive hTAP heterozygote mice of C57BL/6J background.
Four, the acquisition of C57BL/6J background hTAP homozygote mice and genotype identification
The male F10 that selecting step three obtains is carried out once or many for heterozygote mice with female F10 for heterozygote mice
Secondary copulation, it is thus achieved that mice (wild type, heterozygote, homozygote) is by genotype identification (step 2 3) and expresses
Identify that (step 2 4) obtains positive homozygote mice, can be used for conservation experiment.
The judgement of Mice homozygous:
Meet following I simultaneously) and the mice of II) two conditions be judged to proceed to RP11-10A19BAC plasmid and can table
Reach the hTAP Mice homozygous proceeding to people's source protein:
I) all identify through above step 23 and step 24 and obtain positive findings；
II) all offsprings hybridized with C57BL/6J wild-type mice are through above step 23 and the mirror of step 24
Fixed, the most all can get positive findings.
The optimization function research to HLA-I transgenic mice of embodiment 2, hTAP transgenic mice
One, in HLA-A2/hTAP bi-transgenic mice, HLA-A2 gets a promotion at the expression of cell surface
1, the acquisition of HLA-A2/hTAP bi-transgenic mice
F10 embodiment 1 built is for hTAP transgenic heterozygote mice and HLA-A2 transgenic mice
(C57BL/6 background heterozygote) carries out copulation, identifies filial generation as follows:
(1) genotype identification
The molecule of people source TAP1, TAP2, PSMB8, PSMB9, HLA-DOB and HLA-DMB gene is reflected
Fixed, carry out with reference to embodiment 1 step one 3.The mice of 6 gene PCR result positives is hTAP positive mice,
Otherwise it is hTAP negative mice.
To the primer of the Molecular Identification of HLA-A2 gene to for:
HLA-A2F:5 '-GCCCCGAACCCTCGTC-3 '；
HLA-A2R:5 '-TGAGTGGGCCTTCACTTTC-3 '；
Theoretical amplification clip size is 270bp.The mice of the PCR amplification positive is HLA-A2 positive mice, otherwise is
HLA-A2 negative mice.
(2) protein expression is identified
People source TAP2 albumen is carried out Western blotting detection, carries out with reference to embodiment 1 step 24.People source
TAP2 protein expression for hTAP positive mice, be not expressed as hTAP negative mice.
Detection to the expression of HLA-A2, uses the side that detection mouse peripheral blood onthe surface of monocytes HLA-A2 expresses
Method, the step 2 that colouring method sees below.
(1) according to each filial generation and the qualification result of (2), determine the HLA-A2/hTAP in filial generation
Bi-transgenic mice, HLA-A2 single transgenic mice, hTAP single transgenic mice and wild type WT mice.
2, in HLA-A2/hTAP bi-transgenic mice, HLA-A2 detects at the expression of cell surface
The splenocyte taking HLA-A2/hTAP bi-transgenic mice and HLA-A2 single transgenic mice respectively carries out streaming
Cell surface dyes, and concrete operations are as follows:
(1) put to death mice, cut mice left abdomen and take out spleen, be placed in ferrum screen cloth, contain about 10ml in advance
Grind on the 6cm plate of 2%FBS DMEM culture medium, gained cell suspension is proceeded in 15ml centrifuge tube；
(2) centrifugal: 4 DEG C of 1500 turns/min horizontal centrifugal 5min, abandon supernatant, concussion makes bottom cell upspring gently；
(3) splitting erythrocyte: the 10 × PBS drawing 100 μ l with 200 μ l pipettors is placed on the most stand-by, uses 1ml
Pipettor draws 900 μ l sterilizing ddH2Walk in O addition in 15ml centrifuge tube, rapidly with whirlpool concussion instrument concussion, about
It is rapidly added the ready 100 μ l 10 × PBS in side after 5s, continues concussion mixing 5s, add 2%DMEM extremely
10ml；
(4) centrifugal: 4 DEG C of 1500 turns/min horizontal centrifugal 5min, abandon supernatant, concussion makes bottom cell upspring gently；
(5) clean: add 2%FBS DMEM to 10ml, 4 DEG C of 1500 turns/min horizontal centrifugal 5min, abandon
Supernatant, concussion makes bottom cell upspring gently；
(6) resuspended with filtration: addition 5ml 10%FBS 1640 culture medium is resuspended, is proceeded to by 200 mesh nylon membranes
In new 15ml centrifuge tube, mixing, sucking-off 10 μ l to 1.5ml EP pipe is used for count (cell counting count board, aobvious
Count under micro mirror)；
(7) 1 × 10 is drawn according to count results6Individual splenocyte/only, carry out cell surface molecule dyeing, dividing of dyeing
Son is CD4, CD8, B220 and transgene expression product HLA molecule, and corresponding antibody is respectively anti-mouse
CD4APC (Biolegend company, Catalog:100412), anti-mouse CD8 α PE (Biolegend company,
Catalog:100708), anti-mouse B220PerCP-Cy5.5 (eBioscience, Catalog:45-0452-80),
And anti-HLA-A2 (Biolegend, Catalog:343304).
(8) flow cytometer (Facs Calibur) detection CD4+T cell, CD8+T cell and B cell (B220+)
The expression of surface HLA-A2 molecule.
Every kind of mice randomly selects 6-8 week old 5 and only tests.
Experiment arranges brood hTAP mice and wild type WT mice as comparison simultaneously.
Result shows, the expression of the HLA-A2 on HLA-A2/hTAP bi-transgenic mice splenocyte surface is higher than individually
The HLA-A2 expression of HLA-A2 transgenic mice, and significant difference (Fig. 3).
Two, in HLA-A11/hTAP bi-transgenic mice, the expression of HLA-A11 is significantly improved
1, the acquisition of HLA-A11/hTAP bi-transgenic mice
F10 embodiment 1 built is for hTAP transgenic heterozygote mice and HLA-A11 transgenic mice (heterozygosis
Son) carry out copulation, by step 23 in embodiment 1 and the qualification of step 24, respectively obtain HLA-A11/hTAP
Bi-transgenic mice, HLA-A11 single transgenic mice, hTAP mice and wild type WT mice.
2, in HLA-A11/hTAP bi-transgenic mice, HLA-A11 detects at the expression of cell surface
A. application Flow cytometry HLA-A11/hTAP bi-transgenic mice and HLA-A11 single transgenic mice
The expression of splenocyte surface HLA-A11 molecule, concrete operations are as follows:
(1) splenocyte acquisition methods is with (1) in abovementioned steps 1-(6).
(2) 1 × 10 is drawn according to count results6Individual splenocyte/only, carry out cell surface molecule dyeing, dividing of dyeing
Son is CD4, CD8, B220 and transgene expression product HLA molecule, and corresponding antibody is respectively anti-mouse
CD4APC (Biolegend company, Catalog:100412), anti-mouse CD8 α PE (Biolegend company,
Catalog:100708), anti-mouse B220PerCP-Cy5.5 (eBioscience, Catalog:45-0452-80),
And anti-HLA ABC FITC (Beckman, Catalog:PN IM1838U).
(3) flow cytometer (Facs Calibur) detection CD4+T cell, CD8+T cell and B cell (B220+)
The expression of surface HLA-A11 molecule.
Every kind of mice randomly selects 6-8 week old 4-6 and only tests.
Experiment arranges brood hTAP mice and wild type WT mice as comparison simultaneously.
Result shows, compared with independent HLA-A11 transgenic mice, HLA-A11/hTAP bi-transgenic mice
HLA-A11 expression has obtained significant rise (A in Fig. 4).This rise imply that at bi-transgenic mice
In, owing to obtaining the auxiliary of people source TAP molecule etc., HLA-A11 is attached to the probability of appropriate epitope and is greatly increased,
Thus antigen is stablized submission to the ability of cell surface and also can been significantly enhanced.
B. the present inventor have detected this rise effect further and is directed to HLA-A11/hTAP double transgenic
HLA-A11 molecule or mice self MHC I quasi-molecule H2-K in miceb.Concrete operations are as follows:
(1) splenocyte acquisition methods is with (1) in abovementioned steps 1-(6).
(2) 1 × 10 is drawn according to count results6Individual splenocyte/only, carry out cell surface molecule dyeing, dividing of dyeing
Son is CD4, CD8, B220 and transgene expression product HLA molecule, and corresponding antibody is respectively anti-mouse
(Biolegend is public for CD4APC (Biolegend company, Catalog:100412), anti-mouse CD8 α FITC
Department, Catalog:100705), anti-mouse B220PerCP-Cy5.5 (eBioscience, Catalog:45-0452-80),
And anti-mouse H2-Kb(Biolegend, Catalog:116507).
(3) flow cytometer (Facs Calibur) detection CD4+T cell, CD8+T cell and B cell (B220+)
Surface H2-KbThe expression of molecule.
Every kind of mice randomly selects 4-6 and only tests.
Experiment arranges brood hTAP mice and wild type WT mice as comparison simultaneously.
Result shows, this rise effect mainly for HLA-A11 molecule in HLA-A11/hTAP bi-transgenic mice,
Mice self MHC I quasi-molecule H2-KbThere is not significant up-regulated (B in Fig. 4).
Three, compared with HLA-A11 single transgenic mice, HLA-A11 in HLA-A11/hTAP bi-transgenic mice
The CTL activity of restricted epitope is obviously enhanced
In order to verify the hTAP transgenic mice optimization function to HLA transgenic mice, the invention of the present invention further
People's method by DNA immunization, compares HLA-A11/hTAP bi-transgenic mice little with HLA-A11 single transgene
The Mus ctl response to same epitope.
HLA-A11/hTAP bi-transgenic mice and HLA-A11 transgenic mice only respectively randomly select 6-8 week old 5-7,
Mice is carried out twice intramuscular injection hepatitis B virus core antigen carrier for expression of eukaryon pCDNA3.1-HBc (D) plasmid (every
Secondary injection volume is only 100 μ g/, 14 days, the interval of double injection), two kinds of mices of detection in the 11st day after final injection
For HLA-A11 restricted epitope HBc141-151 in HBc cAg (STLPETTVVRR, sequence 7)
Ctl response.Concrete operations are as follows:
(1) splenocyte acquisition methods is with (1) in abovementioned steps 1-(6), carries out following IFN-γ Elispot afterwards
Detection and intracellular IFN-γ dye, and detect ctl response.
(2) IFN-γ Elispot detection: mice IFN-γ Elispot detection kit is from BD company, article No.
(551083), concrete operation step presses the operation of test kit description.Process is as follows: the first step, uses anti-mouse IFN-
Gamma antibodies (test kit comprises) is coated Elispot 96 orifice plate 8～16 hours；Second step, obtains splenocyte same day,
Treat gaging hole adds the 10%FBS 1640 culture medium 100 μ l containing specific stimulus object (polypeptide) accordingly, add
Containing 1 × 106The 10%FBS 1640 culture medium 100 μ l of individual sample to be tested cell, forms the cultivating system of 200 μ l, carefully
Born of the same parents are layered on flat plate bottom equably；3rd step, puts into CO by the Tissue Culture Plate being added with stimulus object2Incubator is cultivated
18～20 hours, in stimulating course, the CTL cell of polypeptid specificity will be stimulated continuous release by corresponding polypeptide
IFN-γ, the IFN-γ of secretion will be in cell position by IFN-γ antibody capture the most coated on Elispot plate；The
Four steps, discard cellular liquid, wash plate, add the anti-IFN-γ antibody of Biotin labelling, the cell position of secretion of gamma-IFN
Put by the anti-IFN-γ antibody labeling of Biotin labelling；5th step, chromogenic reaction, test kit AEC Substrate Set
(BD company, article No. 551951), the position of secretion of gamma-IFN will form a certain size punctation, 1 speckle
Represent specific antigenic specificity CD8+T cell, the number of speckle embodies the immunoreactive intensity of CTL.
(3) polypeptide stimulates and the dyeing of intracellular IFN-γ: the first step, 96 hole U-shaped floor cells culture plate (Costar, goods
Number: Costar-3799) treat that adding the 10%FBS 1640 containing particular stimulation thing (polypeptide) in gaging hole cultivates accordingly
Base 100 μ l, adds containing 1 × 106The 10%FBS 1640 culture medium 100 μ l of individual sample to be tested cell, forms 200 μ l
Cultivating system, be simultaneously introduced Monensin (Biolegend, article No.: 420701) during stimulus object preparation, make final
Cultivating system Monensin concentration becomes 1 × (stock solution is 1000 ×)；Second step, 37 DEG C of CO2Incubator cultivates 4～6
Hour；3rd step, cell surface dye, use antibody be anti-mouse CD4PerCP (three arrow companies, Catalog:
M10043-32C), anti-mouse CD8 α PE (Biolegend company, Catalog:100708)；4th step, born of the same parents
Interior IFN-γ dyes, and the antibody of use is anti-mouse IFN-γ APC (eBioscience, Catalog:17-7311-82)；
5th step, flow cytometer (Facs Calibur) detection CD8+The IFN-γ expression of T cell, with negative control
(being not added with polypeptide to stimulate) is compared, CD8+IFN-γ+Cell be the CTL cell of antigenic specificity, CD8+IFN-γ+
The percentage ratio of cell the highest, illustrate that corresponding ctl response is the strongest.
Elispot testing result is as it is shown in figure 5, the ctl response intensity in HLA-A11/hTAP bi-transgenic mice is bright
Aobvious more than HLA-A11 single transgenic mice, and significant difference.This shows that hTAP transgenic mice not only increases
The expression of HLA-A11 in HLA-A11 transgenic mice, also enhances HLA-A11 simultaneously and carries restricted epitope
In, promote internal ctl response.
Polypeptide stimulates with intracellular IFN-γ coloration result as shown in Figure 6, compared with HLA-A11 single transgenic mice,
After HBc141-151 polypeptide stimulates, the dyeing of intracellular IFN-γ shows that HLA-A11/hTAP bi-transgenic mice is expressed more
IFN-γ, activates out higher ctl response, and significant difference.
Claims (10)
1. the method building the HLA-I transgene mouse model that HLA-II antigen strengthens, comprises the steps:
Build hTAP transgenic mice；Described hTAP transgenic mice and HLA-I transgenic mice are carried out copulation, institute
Obtain the bi-transgenic mice in offspring and be the HLA-I transgene mouse model that HLA-II antigen strengthens；
Described hTAP transgenic mice is the mice having proceeded to following gene: people source TAP1 gene, people source TAP2
Gene, people source PSMB8 gene, people source PSMB9 gene.
Method the most according to claim 1, it is characterised in that: described hTAP transgenic mice is also transferred into
People source HLA-DOB gene and people source HLA-DMB gene.
Method the most according to claim 1 and 2, the nucleotides sequence of described people source TAP1 gene is classified as sequence table
In sequence 1；The sequence 2 that the nucleotides sequence of described people source TAP2 gene is classified as in sequence table；Described people source PSMB8
The sequence 3 that the nucleotides sequence of gene is classified as in sequence table；The nucleotides sequence of described people source PSMB9 gene is classified as sequence table
In sequence 4.
Method the most according to claim 2, it is characterised in that: the nucleotides sequence of described HLA-DOB gene
The sequence 5 being classified as in sequence table；The sequence 6 that the nucleotides sequence of described HLA-DMB gene is classified as in sequence table.
5. according to described method arbitrary in claim 1-4, it is characterised in that: described hTAP transgenic mice is
It is prepared by a method comprising the following steps:
(1) RP11-10A19BAC plasmid is expelled to the sperm by purpose mice 1 and the ovum of purpose mice 2
In the protokaryon of the germ cell formed, the described zygote transplation after processing enters in replace-conceive dams grows, and obtains after birth
F0 is for allophenic mice；
(2) the described F0 that step (1) obtains is hybridized with described purpose mice 2 for allophenic mice, from
F1 generation mice obtains the heterozygote mice having proceeded to described RP11-10A19BAC plasmid, is described hTAP and turns
DNA murine.
Method the most according to claim 5, it is characterised in that: in the process, also include male
Described heterozygote mice and female described heterozygote mice carry out one or many hybridization, obtain and turn from filial generation
Having entered the step of the homozygote mice of described RP11-10A19BAC plasmid, described homozygote mice is also described hTAP
Transgenic mice.
7. according to the method described in claim 5 or 6, it is characterised in that: described purpose mice 1 is B6D2F1
Mice, described purpose mice 2 is C57BL/6 mice, and described replace-conceive dams is ICR mice.
8. the method building hTAP transgenic mice, for claim 5-7 arbitrary described in hTAP turn base
Preparation method because of mice.
9. for building a test kit for the HLA-I transgene mouse model that HLA-II antigen strengthens, including
HLA-I transgenic mice, the hTAP transgenic mice utilizing method structure described in claim 8 and readable load
Body A；Have the right described in described readable carrier A arbitrary described method in requirement 1-4；Or
A kind of test kit building hTAP transgenic mice, including RP11-10A19BAC plasmid and readable load
Body B；Have the right described in described readable carrier B method described in requirement 8.
10.hTAP the application that transgenic mice is in the HLA-II antigen strengthening HLA-I transgenic mice；Described
HTAP transgenic mice is the mice building gained according to method described in claim 8.
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|---|---|---|---|---|
| WO2001023577A2 (en) * | 1999-09-30 | 2001-04-05 | Institut Pasteur | Hybrid or chimeric polynucleotides, proteins, and compositions comprising hepatitis b virus sequences |
| CN101626780A (en) * | 2007-01-19 | 2010-01-13 | 不列颠哥伦比亚大学 | HAT acetylation promoters and its compositions purposes in promoting immunogenicity |
| CN104046593A (en) * | 2013-03-14 | 2014-09-17 | 浙江大学 | Human cell with low immunogenicity and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001023577A2 (en) * | 1999-09-30 | 2001-04-05 | Institut Pasteur | Hybrid or chimeric polynucleotides, proteins, and compositions comprising hepatitis b virus sequences |
| CN101626780A (en) * | 2007-01-19 | 2010-01-13 | 不列颠哥伦比亚大学 | HAT acetylation promoters and its compositions purposes in promoting immunogenicity |
| CN104046593A (en) * | 2013-03-14 | 2014-09-17 | 浙江大学 | Human cell with low immunogenicity and preparation method thereof |
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| Title |
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| MANHUANG ET AL.: "improved transgenic mouse model for studying hla class antigen presentation", 《SCIENTIFIC REPORTS》 * |
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