CN106148405B - 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
- Publication number
- CN106148405B CN106148405B CN201510136784.5A CN201510136784A CN106148405B CN 106148405 B CN106148405 B CN 106148405B CN 201510136784 A CN201510136784 A CN 201510136784A CN 106148405 B CN106148405 B CN 106148405B
- Authority
- CN
- China
- Prior art keywords
- hla
- transgenic mice
- mouse
- htap
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of construction methods of Optimization-type HLA-I transgenic mice.The present invention provides a kind of methods of the HLA-I transgene mouse model of building HLA-II antigen enhancing, include the following steps: to construct hTAP bi-transgenic mice；HLA-I transgenic mice is mated with hTAP mouse, gained double transgenic progeny is the HLA-I transgene mouse model of HLA-II antigen enhancing；The hTAP transgenic mice is the mouse for being transferred to following gene: source of people TAP1 gene, source of people TAP2 gene, source of people PSMB8 gene and source of people PSMB9 gene.The HLA-I/hTAP bi-transgenic mice that the present invention is bred out enhances the ability that antigen is offered compared with HLA-I single transgenic mice.As it can be seen that hTAP transgenic mice facilitates the HLA-II antigen of HLA-I transgenic mice HLA-I and enhances ctl response.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of construction method of Optimization-type HLA-I class transgenic mice, especially
It is related to a kind of method for improving HLA-I transgenic mice HLA-I molecular antigen and offering ability.
Background technique
It includes three phases that the antigen of MHC I class molecule, which offers approach: the generation of antigen peptide fragment, peptide fragment are transported to endoplasm
The combination and displaying of net, peptide fragment and MHC I class molecule.Firstly, antigen protein is degraded into 3-22 by proteasome in cytoplasm
The small peptide of a residue.These small peptides pass through the further trimming of heat shock protein HSP90, offer related transport protein by antigen
(transporter associated with antigen presentation, TAP) is selectively transported to asperities endoplasm
Net (rough endoplasmic reticulum, RER) is intracavitary.In RER, molecular chaperones calnexin (calnexin,
CNX) promote the folding of free MHC I heavy chain, and raise another molecular chaperones ERp57 and promote MHC I class molecular structure domain
The formation of middle disulfide bond；Then, β2-microglobulin (β 2m) is in conjunction with MHC I heavy chain, CNX dissociation, MHC I and molecular chaperones calcium
Plectin (calreticulin, CRT) combines.As TAP GAP-associated protein GAP Tapasin (TAP-associated glyprotein)
When TAP is transported near MHC I class molecule, the MHC I class molecule heavy chain/common shape of β 2m/ERp57/CRT/Tapasin/TAP
Complex (peptide-loading complex, PLC) is loaded at polypeptide.After polypeptide transports RER by TAP, in PLC
MHC I class molecule captures polypeptide.MHC I class molecule correctly assemble and combine polypeptide rear stability increase, and by from
It is released in PLC, reaches cell surface into secretory pathway, receive the monitoring of CTL herein to trigger possible be immunized and answer
It answers.Therefore, MHC I class molecular antigen processing presentation pathway is the complex process that a polymolecular participates in, wherein the molecule participated in
It all will affect the immune response of body.
In view of the importance of MHC I class molecule, currently, (i.e. HLA-I turns a variety of source of people MHC I class molecule transgenic mices
DNA murine) be built and be used for the important virus CTL epitope of people screening, identification and immunization therapy evaluation, as influenza virus,
SARS virus etc. is had laid a good foundation for the development and application of vaccine.But in view of the processing of internal antigen and presentation pathway
Complexity and multifactor property, the application of independent HLA-I transgenic mice often will cause the leakage choosing and wrong choosing of epitope, influences table
The correctness of position screening.There is document to point out, it is more by vaccinia virus (VACV) epitope screened in HLA-A2 transgenic mice
Peptide only has 1/3 can be consistent (Kotturi MF, et al. (2009) Of mice and humans:how with human test results
Good are HLA transgenic mice as a model of human immune responses? Immunome
Res 5:3.)。
Additionally, it is known that HLA-A3 superfamily (including HLA-A11, HLA-A33, HLA-A68 etc.) is in Chinese population HLA parting
In account 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 to be distributed in A3 superfamily most extensively
, statistical analysis also indicates that higher (quick cutting edge of a knife or a sword of beam etc. (2007) South China of HLA-A11 gene frequency in Chinese hepatitis B patient
Preliminary investigation and its meaning the liver phase of volume 12 4 of chronic hepatitis B patient HLA-A allele distributions: 240-243), therefore
The relevant HLA-I transgenic animal model progress epitope screening of the family is established to be of great significance.However some researches show that should
Family member's preference combination C-terminal be basic amino acid polypeptide, this feature just in conjunction with mouse TAP polypeptide Preference
Be not consistent (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 HLA-I transgenic mice
In, matched polypeptide will be substantially reduced by the efficiency in mouse TAP molecular transport to endoplasmic reticulum, to influence epitope
Screening.
In summary content, we are badly in need of constructing a kind of Optimization-type HLA-I transgenic mice, with higher epitope
Screening efficiency, to be preferably applied for the evaluation of the screening of CTL epitope and vaccine 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 models of building HLA-II antigen enhancing
Method.
The method of the HLA-I transgene mouse model of building HLA-II antigen enhancing provided by the present invention, specifically may be used
Include the following steps: to construct hTAP transgenic mice, HLA-I transgenic mice and the hTAP transgenic mice are handed over
Match, the bi-transgenic mice in gained offspring is the HLA-I transgene mouse model of HLA-II antigen enhancing；
The hTAP transgenic mice is the mouse for being transferred to following gene: source of people TAP1 gene, source of people TAP2 gene, people
Source PSMB8 gene and source of people PSMB9 gene.
In the present invention, the hTAP transgenic mice be also transferred into two HLA-II class molecule HLA-DOB genes and
HLA-DMB gene.
Wherein, the bi-transgenic mice is the foreign gene being both transferred in the HLA-I transgenic mice, is also transferred to
The mouse of foreign gene in the hTAP transgenic mice.
Specifically, the nucleotides sequence of the source of people TAP1 gene is classified as the sequence 1 in sequence table；The source of people TAP2 gene
Nucleotides sequence be classified as the sequence 2 in sequence table；The nucleotides sequence of the source of people PSMB8 gene is classified as the sequence 3 in sequence table；
The nucleotides sequence of the source of people PSMB9 gene is classified as the sequence 4 in sequence table.The nucleotides sequence of the HLA-DOB gene is classified as
Sequence 5 in sequence table；The nucleotides sequence of the HLA-DMB gene is classified as the sequence 6 in sequence table.
Wherein, sequence 1 is made of 8058 nucleotide；Sequence 2 is made of 9328 nucleotide；Sequence 3 is by 3455 cores
Thuja acid composition；Sequence 4 is made of 5304 nucleotide；Sequence 5 is made of 3728 nucleotide；Sequence 6 is by 6403 nucleotide
Composition.
More specifically, the hTAP transgenic mice is prepared by a method comprising the following steps:
(1) RP11-10A19BAC plasmid is injected by the Folliculogenesis of the sperm and purpose mouse 2 of purpose mouse 1
In the protokaryon of fertilized eggs, zygote transplation enters develops in replace-conceive female rat, and F0 is obtained after birth for allophenic mice；
The F0 is that the positive hTAP head of PCR verifying identification builds mouse for allophenic mice；
(2) the F0 chimera of step (1) acquisition is carried out for mouse (positive head builds mouse) with the purpose mouse 2 miscellaneous
It hands over, is analyzed by PCR identification and Western blotting, obtained from F1 generation mouse and be transferred to the RP11-10A19BAC
The heterozygote mouse of plasmid, the heterozygote mouse are hTAP transgenic mice.
Certainly, the hTAP transgenic mice can also be to carry out the heterozygote mouse of male and the heterozygote mouse of female
One or many hybridization obtain the homozygote mouse for being transferred to the RP11-10A19BAC plasmid from filial generation.
In the present invention, the purpose mouse 1 is specially B6D2F1 mouse, and the purpose mouse 2 is specially C57BL/6 small
Mouse, the replace-conceive female rat are specially ICR mouse.
In the present invention, the GenBank ID of the above all of RP11-10A19BAC plasmid is 207834.
The method of building hTAP transgenic mice as described above belongs to the scope of protection of the present invention.
It is a further object to provide a kind of for constructing the HLA-I transgenic mice of HLA-II antigen enhancing
The kit of model.
The kit of the HLA-I transgene mouse model provided by the present invention for being used to construct HLA-II antigen enhancing,
It specifically may include the HLA-I transgenic mice, the hTAP transgenic mice and readable carrier；The readability carrier
In record building HLA-II antigen enhancing as described above HLA-I transgene mouse model method.
A further object of the present invention is to provide a kind of kit for constructing hTAP transgenic mice.
The kit provided by the present invention for being used to construct hTAP transgenic mice, specifically may include RP11-10A19BAC
Plasmid and readable carrier；The method of building hTAP transgenic mice as described above is recorded in the readability carrier.
Application of the hTAP transgenic mice in the HLA-II antigen of enhancing HLA-I transgenic mice also belongs to this
The protection scope of invention.
In the present invention, the HLA-I transgenic mice can be HLA-A2, HLA-A11, HLA-A1, HLA-A24, HLA-
B7, HLA-B27, HLA-B4 etc. currently reported HLA-I transgenic mice.
In one embodiment of the invention, the HLA-I transgenic mice be specially HLA-A2 transgenic mice or
HLA-A11 transgenic mice.The HLA-A2 transgenic mice is the C57BL/6J background mice for being transferred to HLA-A2 gene；It is described
HLA-A11 transgenic mice is the C57BL/6J × BALB/c background mice for being transferred to HLA-A11 gene.
The antigen offers to offer for the HLA-I restriction epi polypeptide to corresponding transgenic mice.Of the invention
In one embodiment, the HLA-I transgenic mice is specially HLA-A11 transgenic mice；The HLA-A11 transgenic mice
Corresponding antigen is offered to offer to HLA-A11 restriction epi polypeptide, and the HLA-A11 restriction epi polypeptide is specially
HBc141-151, amino acid sequence are STLPETTVVRR (sequence 7).
In the present invention, the HLA-II antigen of the 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, by hTAP transgenic mice and HLA-I transgenic mice
It mates, breeds out HLA-I/hTAP bi-transgenic mice, carry out functional experiment, discovery and HLA-I single transgenic mice phase
Than HLA-I/hTAP bi-transgenic mice enhances the ability that antigen is offered.As it can be seen that hTAP transgenic mice facilitates HLA-I
The antigen of HLA-I offers and enhances ctl response in transgenic mice.
Detailed description of the invention
Fig. 1 is that PCR identifies that people's MHC I class antigen offers approach relevant molecule in hTAP transgenic mice.M:DNA molecular weight
Standard, each band is descending to be followed successively by 500bp, 400bp, 300bp and 200bp.
Fig. 2 is the expression that Western blotting identifies 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 indicates HLA-A2/hTAP bi-transgenic mice；A2 indicates HLA-A2 single transgenic mice；WT table
Show wild type C57BL/6J mouse.B:HLA-A2 expression quantity statistical chart.
Fig. 4 is in HLA-A11/hTAP bi-transgenic mice, and the expression of HLA-A11 molecule is significantly raised.A: spleen
Cell surface HLA-A11 expresses streaming figure (left side) and its statistical chart (right side), P < 0.001 * * *.B: splenocyte surface H2-KbExpression
Measure streaming figure (left side) and its statistical chart (right side).A11-hTAP indicates HLA-A11/hTAP bi-transgenic mice；A11het is indicated
HLA-A11 single transgenic mice；TAP indicates hTAP transgenic mice；WT indicates wild type C57BL/6J mouse.
Fig. 5 is that HLA-A11/hTAP bi-transgenic mice is significantly stronger than HLA- to the ctl response of HLA-A11 restricted epitope
A11 single transgenic mice.Ctl response of the A:Elispot two kinds of mouse of detection to HBc141-151, A11-hTAP (n=5),
A11het (n=5), 1 μ g/ml of peptide concentration；B:Elispot points statistics histogram, P < 0.01 * P < 0.05, * *.
Fig. 6 is that ctl response significantly increases in HLA-A11/hTAP bi-transgenic mice.A:IFN- γ dyeing scatterplot intracellular
Figure；B:INF- γ+CD8+Cell percentages statistical chart,*P < 0.05,**P<0.01。
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
RP11-10A19BAC plasmid, GenBank ID:207834, the plasmid are U.S. CHOIR (Children ' s
Hospital Oakland Research Institute) product.People's MHC I class antigen that RP11-10A19BAC plasmid includes
Offering approach relevant molecule has TAP1, TAP2, PSMB8 and PSMB9, in addition also contains two people's MHC II class relevant molecule HLA-
DOB、HLA-DMB。
HLA-A2 transgenic mice: it is given by Meng Songdong seminar, Institute of Microorganism, Academia Sinica, genetic background
For C57BL/6J mouse.Be recorded in " appoint the B of (2010) New Type of HLA-A2 such as Yulin limitation, c-type HBV specific CTL epitope
In screening and identification Science Bulletin (55) 30:2915~2924 " text, the public can obtain from Institute of Microorganism, Academia Sinica
?.
HLA-A11 transgenic mice: Taconic company, male model mice number 9660-M, female model mice are come from
Number 9660-F.Its genetic background is C57BL/6J × BALB/c mouse.
C57BL/6J mouse: Beijing HFK Bio-Technology Co., Ltd., phone: 010-60753558 are come from.
ICR mouse: female comes from Beijing Vital River Experimental Animals Technology Co., Ltd..
B6D2F1 mouse: male comes from Beijing Vital River Experimental Animals Technology Co., Ltd..
PCDNA3.1-HBc (D) plasmid: it is given by Meng Songdong seminar, Institute of Microorganism, Academia Sinica.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
In Bao29 (12): a 1765-75 " text, the public can obtain from Institute of Microorganism, Academia Sinica.The plasmid carries hepatitis B
The gene order of virus core antigen, wherein the gene order of hepatitis B virus core antigen is sequence 8 in sequence table.
The building of embodiment 1, hTAP transgenic mice
One, acquisition of the F0 for allophenic mice
1, acquisition of the F0 for mouse
With BAC injection buffer (formula are as follows: 10mM Tris-HCl, 0.1mM EDTA, 100mM NaCl, 10 μM of sub- essences
Amine, 30 μM of spermine, pH7.2) by RP11-10A19BAC plasmid be configured to concentration be 1ng/ μ l solution.By prepared solution
Full of injection needle, injection needle is installed on microinjection microscope, micromanipulation is carried out under the microscope, that is, pushes away
Dynamic injection needle utilizes pressure through fertilized eggs (sperm source derives from C57BL/6 mouse in B6D2F1 mouse, ovum) hyalomitome band
DNA of the continuous (about 150hPa) of power pump into protokaryon injection injection needle, shows to inject successfully (injection if protokaryon expansion
The extremely difficult accurate control of volume occurs to be expanded to directive standard obviously with protokaryon volume).Fertilized eggs are placed in 37 DEG C of carbon dioxide
It is transplanted in replace-conceive female mice (ICR mouse) after incubator culture 24 hours to 2- cell stage, the final F0 that obtains is for mouse.
2, F0 is prepared for mouse genome
F0 adds 400 μ l lysates (by the limited public affairs of Shanghai south model organism for mouse 14 days clip rat-tail 0.3cm of birth
Department provides, catalog number: NMS014) and 40 μ l Proteinase Ks (concentration 10mg/ml), 56 DEG C of digestion are overnight.In centrifuging and taking
Clearly, two volumes dehydrated alcohol precipitates, and 75% ethanol washing is dissolved in 100 μ l pure water, 50 DEG C of baking oven hydrotropies 1 after slightly drying
Hour.
3, F0 is identified for murine genes type
Using step 2 obtain each F0 for mouse genome as template, respectively to source of people TAP1, TAP2, PSMB8,
PSMB9, HLA-DOB and HLA-DMB gene (sequence of 6 human source genes is followed successively by sequence table shown in sequence 1-6) carry out PCR
Amplification.It is as follows for the primer sequence of each gene:
For the primer pair of source of people TAP1 gene are as follows:
TAP1-F:5 '-GCCTCTTATCGTGTGCTTCT-3 '；
TAP1-R:5 '-CCATCTCCACCCAAGGTC-3 '.
TAP1-F/TAP1-R theoretical amplification primer size is 319bp.
For the primer pair of source of people TAP2 gene are as follows:
TAP2-F:5 '-CGCCCACAGTGTTAGGT-3 '；
TAP2-R:5 '-GAAGAGGCGTTTGGAATAG-3 '.
TAP2-F/TAP2-R theoretical amplification primer size is 234bp.
For the primer pair of source of people PSMB8 gene are as follows:
PSMB8-F:5 '-AGTACGTCAGGTATTTAGC-3 '；
PSMB8-R:5 '-GAGGGAGTAGGAGTATATG-3 '.
PSMB8-F/PSMB8-R theoretical amplification primer size is 438bp.
For the primer pair of source of people PSMB9 gene are as follows:
PSMB9-F:5 '-CACTTAATGTCTCAGTGGGA-3 '；
PSMB9-R:5 '-TATTTCTCTTCCTGTTCCTC-3 '.
PSMB9-F/PSMB9-R theoretical amplification primer size is 289bp.
For the primer pair of source of people HLA-DOB gene are as follows:
HLA-DOB-F:5 '-TAAACGTGTCTGGTATGTC-3 '；
HLA-DOB-R:5 '-AGTTAAAGATGAATCTGACC-3 '.
HLA-DOB-F/HLA-DOB-R theoretical amplification primer size is 347bp.
For the primer pair of source of people HLA-DMB gene are as follows:
HLA-DMB-F:5 '-CAAAGGAACATCCAGATGAAG-3 '；
HLA-DMB-R:5 '-TCCCCCATACTCCCTGAAG-3 '.
HLA-DMB-F/HLA-DMB-R theoretical amplification primer size is 228bp.
It tests while being arranged with H2Blank control of the O as template, using C57BL/6J mouse (wild type) genome as
The negative control of template, and using human genome as the positive control of template.
The results show that Recipient mice number is 4 by fertilized eggs microinjection and transplanting, pregnancy number is 4, pregnancy rate
100%；Transplanting ovum number is 30 pieces/, and mouse (F0 generation) number of being born in total is 24, birth rate 20%.It is 2 positive, positive rate
8.3%.6 human source genes expand obtain the F0 of corresponding purpose band for mouse be the injection of RP11-10A19BAC plasmid and
Successful F0 is transplanted for allophenic mice.
Two, the acquisition and genotype identification of F1 generation heterozygote mouse
1, the acquisition of F1 generation mouse
According to step 1 to F0 for the genotype identification of mouse as a result, choosing positive F0 for allophenic mice and wild type
The mating of C57BL/6J mouse, obtains F1 generation mouse (wild type and heterozygous).
2, prepared by F1 generation mouse genome
It is carried out referring to step 12.
3, F1 generation murine genes type is identified
It is carried out referring to step 13.
It is to identify positive to be transferred to RP11- that six human source genes, which expand and obtain the F1 generation mouse of corresponding purpose band,
The F1 generation chimeric mice of 10A19BAC plasmid.Part is transferred to the F1 generation heterozygote mouse (Founder2 of RP11-10A19BAC plasmid
And Founder14) PCR qualification result it is as shown in Figure 1.
4, F1 generation mouse expression identification
It takes F1 generation Mouse spleen cells as sample extraction albumen, carries out Western for source of people TAP2 albumen
Blotting detection.Wherein, primary antibody used is antibody (source of mouse, MBL Products, the catalogue of anti-human source TAP2 albumen
Number be K0137-3)；Secondary antibody be anti-mouse IgG antibody (goat source, Beijing Bioisystech Co., Ltd, Zhong Shan Golden Bridge product,
Catalog number is ZDR-5307).Experiment simultaneously using β-actin as internal reference, primary antibody for anti-β-actin antibody (source of mouse, three
Arrow Products, catalog number DKM9001)；(goat source, Beijing Zhong Shan Golden Bridge are raw for the antibody that secondary antibody is anti-mouse IgG
Object Technology Co., Ltd. product, catalog number ZDR-5307).
It tests while C57BL/6J mouse (wild type) is set as control.
The F1 generation mouse for having obvious source of people TAP2 Protein Detection signal is the expression positive for being transferred to RP11-10A19BAC plasmid
F1 chimeric mice.Part is transferred to the F1 generation heterozygote mouse (Founder2 and Founder14) of RP11-10A19BAC plasmid
Western blotting qualification result is as shown in Figure 2.The background of the mouse is C57BL/6J × B6D2F1, can be bred for trying
It tests.
In order to further confirm that the present inventor has carried out gene sequencing to it to F1 generation heterozygote mouse,
The results show that F1 generation heterozygote mouse (Founder2 and Founder14) is really containing 6 shown in sequence 1-6 in ordered list
Human source gene (TAP1, TAP2, PSMB8, PSMB9, HLA-DOB and HLA-DMB).
Three, acquisition and genotype identification of the F10 for C57BL/6J background hTAP heterozygote mouse
The F1 generation heterozygote mouse that selecting step two obtains mates with wild type C57BL/6J, passes through genotype identification
(step 1,3) and expression identification (step 2 4) and sequencing obtain positive F2 for heterozygote mouse.Again by positive F2 generation
Heterozygote mouse is bred with wild type C57BL/6J, obtains positive F3 for heterozygote mouse, and so on into breeding, finally
F10 is obtained for the positive hTAP heterozygote mouse of C57BL/6J background.
Four, the acquisition and genotype identification of C57BL/6J background hTAP homozygote mouse
The male F10 that selecting step three obtains carries out for heterozygote mouse and female F10 for heterozygote mouse primary or more
Secondary mating obtains mouse (wild type, heterozygote, homozygote) and passes through genotype identification (step 2 3) and expression identification (step 2
4) positive homozygote mouse is obtained, can be used for conservation experiment.
The judgement of Mice homozygous:
Meet following I simultaneously) and II) mouse of two conditions is judged to being transferred to RP11-10A19BAC plasmid and can express
It is transferred to the hTAP Mice homozygous of source of people albumen:
I it) identifies by above step 23 and step 24 and obtains positive findings；
II) identification of all offsprings hybridized with C57BL/6J wild-type mice Jing Guo above step 23 and step 24,
Positive findings equally can be obtained.
The optimization function research of embodiment 2, hTAP transgenic mice to HLA-I transgenic mice
One, HLA-A2 gets a promotion in the expression quantity of cell surface in HLA-A2/hTAP bi-transgenic mice
1, the acquisition of HLA-A2/hTAP bi-transgenic mice
The F10 that embodiment 1 is constructed is for hTAP transgenosis heterozygote mouse and HLA-A2 transgenic mice (C57BL/6 back
Scape heterozygote) it mates, filial generation is identified as follows:
(1) genotype identification
To the Molecular Identification of source of people TAP1, TAP2, PSMB8, PSMB9, HLA-DOB and HLA-DMB gene, referring to embodiment
1 step 13 carries out.The mouse of 6 gene PCR result positives is hTAP positive mice, otherwise is hTAP negative mice.
To the primer pair of the Molecular Identification of HLA-A2 gene are as follows:
HLA-A2F:5 '-GCCCCGAACCCTCGTC-3 '；
HLA-A2R:5 '-TGAGTGGGCCTTCACTTTC-3 '；
Theoretical amplification clip size is 270bp.The mouse of the PCR amplification positive is HLA-A2 positive mice, otherwise is HLA-
A2 negative mice.
(2) protein expression is identified
Western blotting detection is carried out to source of people TAP2 albumen, is carried out referring to 1 step 24 of embodiment.Source of people
TAP2 protein expression is hTAP positive mice, is not expressed as hTAP negative mice.
Detection to the expression of HLA-A2, the method expressed using detection mouse peripheral blood onthe surface of monocytes HLA-A2,
The step 2 that colouring method sees below.
According to the qualification result of (1) and (2) of each filial generation, determines that the HLA-A2/hTAP in filial generation is bis- and turn base
Because of mouse, HLA-A2 single transgenic mice, hTAP single transgenic mice and wild type WT mouse.
2, HLA-A2 is detected in the expression quantity of cell surface in HLA-A2/hTAP bi-transgenic mice
The splenocyte of HLA-A2/hTAP bi-transgenic mice and HLA-A2 single transgenic mice is taken to carry out fluidic cell respectively
Padding, concrete operations are as follows:
(1) mouse is put to death, mouse left abdomen is cut and takes out spleen, be placed in iron sieve, is containing about 10ml2% in advance
It is ground on the 6cm plate of FBS DMEM culture medium, gained cell suspension is transferred in 15ml centrifuge tube；
(2) be centrifuged: 4 DEG C of 1500 turns/min horizontal centrifugal 5min abandon supernatant, and gently concussion bounces bottom cell；
(3) it splitting erythrocyte: is placed on one side with 10 × PBS that 200 μ l pipettors draw 100 μ l for use, with 1ml pipettor
Draw 900 μ l sterilizing ddH2It walks in 15ml centrifuge tube in O addition, is shaken with whirlpool concussion instrument rapidly, be rapidly added side after about 5s
Ready 100 μ, the 10 × PBS of l in side continues concussion and mixes 5s, 2%DMEM to 10ml is added；
(4) be centrifuged: 4 DEG C of 1500 turns/min horizontal centrifugal 5min abandon supernatant, and gently concussion bounces bottom cell；
(5) it cleans: 2%FBS DMEM to 10ml, 4 DEG C of 1500 turns/min horizontal centrifugal 5min is added, abandon supernatant, gently shake
Swinging bounces bottom cell；
(6) it is resuspended and filters: 1640 culture medium of 5ml 10%FBS is added and is resuspended, is transferred to by 200 mesh nylon membranes new
It in 15ml centrifuge tube, mixes, is sucked out in 10 μ l to 1.5ml EP pipes for counting (cell counting board, microscope under count)；
(7) 1 × 10 is drawn according to count results6A splenocyte/only, carry out cell surface molecule dyeing, the molecule of dyeing
It is respectively anti-mouse CD4APC for CD4, CD8, B220 and transgene expression product HLA molecule, corresponding antibody
(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) detects CD4+T cell, CD8+T cell and B cell (B220+) surface
The expression of HLA-A2 molecule.
Every kind of mouse randomly selects 6-8 week old 5 and is only tested.
Brood hTAP mouse and wild type WT mouse are tested while are arranged as control.
The results show that the expression quantity of the HLA-A2 on HLA-A2/hTAP bi-transgenic mice splenocyte surface is higher than individually
The HLA-A2 expression quantity of HLA-A2 transgenic mice, and significant difference (Fig. 3).
Two, the expression quantity of HLA-A11 is significantly improved in HLA-A11/hTAP bi-transgenic mice
1, the acquisition of HLA-A11/hTAP bi-transgenic mice
The F10 that embodiment 1 is constructed for hTAP transgenosis heterozygote mouse and HLA-A11 transgenic mice (heterozygote) into
Row mating, by the identification of step 23 and step 24 in embodiment 1, respectively obtain HLA-A11/hTAP bi-transgenic mice,
HLA-A11 single transgenic mice, hTAP mouse and wild type WT mouse.
2, HLA-A11 is detected in the expression quantity of cell surface in HLA-A11/hTAP bi-transgenic mice
A. thin using Flow cytometry HLA-A11/hTAP bi-transgenic mice and HLA-A11 single transgenic mice spleen
The expression of cellular surface HLA-A11 molecule, concrete operations are as follows:
(1) splenocyte acquisition methods are the same as (1)-(6) in aforementioned step 12.
(2) 1 × 10 is drawn according to count results6A splenocyte/only, carry out cell surface molecule dyeing, the molecule of dyeing
It is respectively anti-mouse CD4APC for CD4, CD8, B220 and transgene expression product HLA molecule, corresponding antibody
(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) detects CD4+T cell, CD8+T cell and B cell (B220+) surface
The expression of HLA-A11 molecule.
Every kind of mouse randomly selects 6-8 week old 4-6 and is only tested.
Brood hTAP mouse and wild type WT mouse are tested while are arranged as control.
The results show that compared with independent HLA-A11 transgenic mice, the HLA- of HLA-A11/hTAP bi-transgenic mice
A11 expression has obtained significant up-regulation (A in Fig. 4).This up-regulation implies in bi-transgenic mice, due to obtaining people
The auxiliary of source TAP molecule etc., the probability that HLA-A11 is integrated to appropriate epitope greatly increase, thus antigen is stablized submission to thin
The ability of cellular surface can also be been significantly enhanced.
B. the present inventor further has detected up-regulation effect to be directed to HLA-A11/hTAP double transgenic small
HLA-A11 molecule or mouse itself MHC I class molecule H2-K in mouseb.Concrete operations are as follows:
(1) splenocyte acquisition methods are the same as (1)-(6) in aforementioned step 12.
(2) 1 × 10 is drawn according to count results6A splenocyte/only, carry out cell surface molecule dyeing, the molecule of dyeing
It is respectively anti-mouse CD4APC for CD4, CD8, B220 and transgene expression product HLA molecule, corresponding antibody
(Biolegend company, Catalog:100412), anti-mouse CD8 α FITC (Biolegend company, 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) detects CD4+T cell, CD8+T cell and B cell (B220+) surface
H2-KbThe expression of molecule.
Every kind of mouse randomly selects 4-6 and is only tested.
Brood hTAP mouse and wild type WT mouse are tested while are arranged as control.
The results show that up-regulation effect is mainly for HLA-A11 molecule in HLA-A11/hTAP bi-transgenic mice, mouse
Itself MHC I class molecule H2-KbThere is no significant expression up-regulation (B in Fig. 4) occurs.
Three, compared with HLA-A11 single transgenic mice, HLA-A11 is restricted in HLA-A11/hTAP bi-transgenic mice
The CTL activity of epitope significantly increases
In order to further verify hTAP transgenic mice to the optimization function of HLA transgenic mice, the present inventor
By the method for DNA immunization, compare HLA-A11/hTAP bi-transgenic mice and HLA-A11 single transgenic mice to same antigen
The ctl response of epitope.
HLA-A11/hTAP bi-transgenic mice and HLA-A11 transgenic mice respectively randomly select 6-8 week old 5-7 only, right
Mouse carries out intramuscular injection hepatitis B virus core antigen carrier for expression of eukaryon pCDNA3.1-HBc (D) plasmid (per injection twice
Amount be 100 μ g/ only, the interval of double injection 14 days), the 11st day two kinds of mouse of detection resist for HBc core after final injection
The ctl response of HLA-A11 restricted epitope HBc141-151 (STLPETTVVRR, sequence 7) in original.Concrete operations are as follows:
(1) splenocyte acquisition methods carry out following IFN-γ Elispot detection with (1)-(6) in aforementioned step 12 later
It is dyed with IFN-γ intracellular, detects ctl response.
(2) IFN-γ Elispot is detected: mouse IFN-γ Elispot detection kit comes from BD company, article No.
(551083), concrete operation step is operated by kit specification.Process is as follows: the first step, with (the examination of anti-mouse IFN-γ antibody
Agent box includes) coating 8~16 hours of 96 orifice plate of Elispot；Second step, on the day of obtaining splenocyte, accordingly to add in gaging hole
Enter to contain 1640 culture medium of 10%FBS, the 100 μ l of specific stimulant (polypeptide), is added and contains 1 × 106A sample to be tested cell
1640 culture medium of 10%FBS, 100 μ l, forms the cultivating system of 200 μ l, and cell is equably layered on flat plate bottom；Third step, will
Tissue culture plate added with stimulant is put into CO218~20 hours of incubator culture, in stimulating course, polypeptid specificity
CTL cell will stimulate continuous release IFN-γ by corresponding polypeptide, and the IFN-γ of secretion will be in cell position by Elispot
Preparatory coated IFN-γ antibody capture on plate；4th step, discards cellular liquid, and the anti-IFN- of Biotin label is added in board-washing
The cell position of gamma antibodies, secretion of gamma-IFN marks the anti-IFN-γ antibody marked by Biotin；5th step, chromogenic reaction,
The position of kit AEC Substrate Set (BD company, article No. 551951), secretion of gamma-IFN will form the red of a certain size
Color spot point, 1 spot represent a specific antigentic specificity CD8+T cell, spot number embody CTL immune response
Intensity.
(3) polypeptide stimulation is dyed with IFN-γ intracellular: the first step, 96 hole U-shaped bottom tissue culture plates (Costar, article No.:
Costar-3799) accordingly in gaging hole be added object containing particular stimulation (polypeptide) 1640 culture medium of 10%FBS, 100 μ l, add
Enter to contain 1 × 1061640 culture medium of 10%FBS, the 100 μ l of a sample to be tested cell forms the cultivating system of 200 μ l, stimulant
Monensin is added simultaneously when preparation, and (article No.: 420701) Biolegend, makes final cultivating system Monensin concentration become 1
× (stoste is 1000 ×)；Second step, 37 DEG C of CO2Incubator culture 4~6 hours；Third step, cell surface dyeing, using anti-
Body is anti-mouse CD4PerCP (three arrow companies, Catalog:M10043-32C), anti-mouse CD8 α PE
(Biolegend company, Catalog:100708)；4th step, IFN-γ dyeing intracellular, the antibody used 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, compared with negative control (polypeptide stimulation is not added), CD8+IFN-γ+Cell be anti-
The CTL cell of former specificity, CD8+IFN-γ+Cell percentage it is higher, illustrate that corresponding ctl response is stronger.
Elispot testing result is as shown in figure 5, the ctl response intensity in HLA-A11/hTAP bi-transgenic mice is obvious
Greater than HLA-A11 single transgenic mice, and significant difference.This shows that hTAP transgenic mice not only increases HLA-A11 and turns
The expression of HLA-A11 in DNA murine, while also enhancing HLA-A11 and restricted epitope is offered, promote intracorporal CTL
Reaction.
Polypeptide stimulation with IFN-γ coloration result intracellular as shown in fig. 6, compared with HLA-A11 single transgenic mice,
IFN-γ intracellular dyeing shows that HLA-A11/hTAP bi-transgenic mice expresses more IFN- after the stimulation of HBc141-151 polypeptide
γ activates out stronger ctl response, and significant difference.
Claims (6)
1. a kind of method of the HLA-I transgene mouse model of building HLA-II antigen enhancing, includes the following steps: to construct
HTAP transgenic mice；The hTAP transgenic mice is mated with HLA-I transgenic mice, double turns in gained offspring
DNA murine is the HLA-I transgene mouse model of HLA-II antigen enhancing；
The hTAP transgenic mice is the mouse for being transferred to following gene: source of people TAP1 gene, source of people TAP2 gene, source of people
PSMB8 gene, source of people PSMB9 gene；
The hTAP transgenic mice has also been transferred into source of people HLA-DOB gene and source of people HLA-DMB gene；
The nucleotides sequence of the source of people TAP1 gene is classified as the sequence 1 in sequence table；The nucleotides sequence of the source of people TAP2 gene
The sequence 2 being classified as in sequence table；The nucleotides sequence of the source of people PSMB8 gene is classified as the sequence 3 in sequence table；The source of people
The nucleotides sequence of PSMB9 gene is classified as the sequence 4 in sequence table；The nucleotides sequence of the HLA-DOB gene is classified as in sequence table
Sequence 5；The nucleotides sequence of the HLA-DMB gene is classified as the sequence 6 in sequence table；
The HLA-I transgenic mice is HLA-A2 transgenic mice or HLA-A11 transgenic mice.
2. according to the method described in claim 1, it is characterized by: the hTAP transgenic mice is according to including the following steps
Method be prepared:
(1) RP11-10A19BAC plasmid is injected into the fertilization of the Folliculogenesis by the sperm and purpose mouse 2 of purpose mouse 1
In the protokaryon of ovum, will treated that the zygote transplation enters develops in replace-conceive female rat, F0 is obtained after birth for allophenic mice；
(2) F0 that step (1) obtains is hybridized for allophenic mice with the purpose mouse 2, from F1 generation mouse
Obtain the heterozygote mouse for being transferred to the RP11-10A19BAC plasmid, the as described hTAP transgenic mice.
3. according to the method described in claim 2, it is characterized by: further including by the heterozygosis of male in the method
Body mouse with female the heterozygote mouse carry out it is one or many hybridize, obtained from filial generation be transferred to it is described
The step of homozygote mouse of RP11-10A19BAC plasmid, the homozygote mouse are also the hTAP transgenic mice.
4. according to the method in claim 2 or 3, it is characterised in that: the purpose mouse 1 is B6D2F1 mouse, the mesh
Mouse 2 be C57BL/6 mouse, the replace-conceive female rat be ICR mouse.
5. a kind of method for constructing hTAP transgenic mice is the hTAP transgenic mice described in claim 2-4 is any
Preparation method.
Application of the 6.hTAP transgenic mice in the HLA-II antigen of enhancing HLA-I transgenic mice；The hTAP turns base
Because mouse is to construct resulting mouse according to claim 5 the method；
The HLA-I transgenic mice is HLA-A2 transgenic mice or HLA-A11 transgenic mice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510136784.5A CN106148405B (en) | 2015-03-26 | 2015-03-26 | A kind of construction method of Optimization-type HLA-I class transgenic mice |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510136784.5A CN106148405B (en) | 2015-03-26 | 2015-03-26 | A kind of construction method of Optimization-type HLA-I class transgenic mice |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106148405A CN106148405A (en) | 2016-11-23 |
| CN106148405B true CN106148405B (en) | 2019-07-12 |
Family
ID=57340479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510136784.5A Active CN106148405B (en) | 2015-03-26 | 2015-03-26 | A kind of construction method of Optimization-type HLA-I class transgenic mice |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106148405B (en) |
Citations (3)
| 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 |
-
2015
- 2015-03-26 CN CN201510136784.5A patent/CN106148405B/en active Active
Patent Citations (3)
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| improved transgenic mouse model for studying hla class antigen presentation;manhuang et al.;《SCIENTIFIC REPORTS》;20160916;1-12 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106148405A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mackay et al. | T-box transcription factors combine with the cytokines TGF-β and IL-15 to control tissue-resident memory T cell fate | |
| CN104039132B (en) | The method for preparing MHC genetic modification mouse | |
| Kedl et al. | T cells compete for access to antigen-bearing antigen-presenting cells | |
| US8282935B2 (en) | Materials and methods relating to improved vaccination strategies | |
| CN105578876B (en) | The method for preparing the non-human animal of expression HLAI classes | |
| CN101146550B (en) | Use of immunogenicity composition in preparing kit for initiating and enhancing immune responses | |
| CN107636152A (en) | Novel immunogenic t cell epitope and the method for separating neoantigen specific t-cell receptor are detected by MHC cell libraries | |
| CN103570818B (en) | Tumor antigenic polypeptide and the purposes as tumor vaccine thereof | |
| Naruse et al. | Diversity of MHC class I genes in Burmese-origin rhesus macaques | |
| Wakenell et al. | Association between the Rfp-Y haplotype and the incidence of Marek's disease in chickens | |
| CN110055223A (en) | A kind of preparation method and applications of the immunodeficient animals of B2m genetic modification | |
| EP1878798A1 (en) | Method of producing a multichimeric mouse and applications to study the immunopathogenesis of human tissue-specific pathologies | |
| Ru et al. | Development of a humanized HLA-A2. 1/DP4 transgenic mouse model and the use of this model to map HLA-DP4-restricted epitopes of HBV envelope protein | |
| Gao et al. | Establishment of six homozygous MHC-B haplotype populations associated with susceptibility to Marek’s disease in Chinese specific pathogen-free BWEL chickens | |
| CN101595128A (en) | Immunne response enhanced transgenic animal and the method that is used to prepare it | |
| CN103209988B (en) | KIR allele and the typing based on molecular determinants of KIR-part | |
| CN109929875A (en) | A kind of construction method of LAG3 gene humanized animal's model and its application | |
| WO2013017545A1 (en) | Humanized hla-a2 / hla-dp4 transgenic mice and uses thereof as an experimental model for biomedical research and development | |
| CN106148405B (en) | A kind of construction method of Optimization-type HLA-I class transgenic mice | |
| JP2005530509A (en) | Methods for the development of animal models | |
| Shedlovsky et al. | Strong teratocarcinoma transplantation loci, Gt-1 and Gt-2, flank H-2 | |
| Imaeda et al. | Influence of swine leukocyte antigen haplotype on serum antibody titers against swine erysipelas vaccine and reproductive and meat production traits of SLA-defined selectively bred Duroc pigs | |
| CN110195057A (en) | The preparation method and application of the genetically modified non-human animal of Hr gene or its filial generation | |
| CN108251456A (en) | A kind of preparation method of the atherosclerosis mouse model of NOD genetic backgrounds | |
| Patel et al. | Alloantigen copy number as a critical factor in RBC alloimmunization |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |