CN106868045A - The construction method of Management of HBeAg negative mouse model - Google Patents
The construction method of Management of HBeAg negative mouse model Download PDFInfo
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Abstract
The invention discloses a kind of construction method of Management of HBeAg negative mouse model, including step：1) the patients serum DNA for being diagnosed as HBeAg negative infections is extracted, entering performing PCR using primer pair HBV F/HBV R expands；2) PCR primer sequencing, redesigns the specific PCR amplimer for the sequence to re-starting PCR amplifications；3) PCR primer is cloned into pEASY Blunt Zero carriers；4) plasmid is extracted, digestion is reclaimed fragment and purified, is cyclized and purifies, and obtains the negative cccDNA of HBV e；5) cccDNA is injected into mouse, that is, constructs Management of HBeAg negative mouse model.The present invention successfully constructs Management of HBeAg negative mouse model first, and the study mechanism and drug-treated effectiveness study to the infection of HBeAg negative HBs are significant.
Description
Technical field
The invention belongs to genetic engineering field, and in particular to a kind of structure side of Management of HBeAg negative mouse model
Method.
Background technology
Virus B hepatitis be one by hepatitis type B virus (hepatitis B virus, HBV) cause it is great
Global health problem.The chronic hepatitis B that HBV infection causes may develop into cirrhosis and hepatocellular carcinoma.Recently it has been reported that the whole world
About 2.48 hundred million people suffer from chronic hepatitis B (Chronic hepatitis B, CHB).The serological classification of chronic hepatitis B is very
Complexity, e antigen positives and e antigen negative chronic hepatitis B infections can be classified as according to serum e antigen levels.It is global in recent years
The number of the infected of HBV has declined, but wherein HBeAg negative infections quantity is more and more.But at present to e antigen negative patients couple
Antiviral therapy responsiveness is low, causes process time to increase, additionally, e negative chronic hepatitis B patients develop into the several of hepatocellular carcinoma
Rate is higher.Therefore, development history and pathogenesis to e feminine genders HBV needs deeper research.And at present to Chronic HBV sense
The research of dye and treatment is mainly based upon the expansion of e animals showing positives model, and up to the present, still none example is to e in world wide
The research of negative HBV infection animal model and report, the at present research to e negative HBV infections are only limitted at pathogenesis, medicine
Reason and drug screening.Therefore, an animal model that can be used in e feminine gender HBV persistent infections research is badly in need of at present.
The content of the invention
The purpose of the present invention provides a kind of construction method of Management of HBeAg negative mouse model regarding to the issue above,
Comprise the following steps：
1) the patients serum DNA for being diagnosed as HBeAg negative infections is extracted, performing PCR is entered using primer pair HBV-F/HBV-R
Expand, primer sequence is：
HBV-F：5 '-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3 ',
HBV-R：5′-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGTG-3′；
2) PCR primer is sequenced, the specific PCR amplimer for the sequence is redesigned according to sequencing result
It is right, re-start PCR using the patients serum DNA of primer pair HBeAg negative infections and expand；
3) by step 2) obtain PCR primer be cloned into pEASY-Blunt Zero carriers, select positive colony；
4) extraction step 3) positives clone plasmid, the plasmid that will be extracted BspQI endonuclease digestions carry out agar
Sugared gel electrophoresis, reclaims the fragment of size about 3.2kb and purifies, and is cyclized and is purified with T4 ligases, obtains HBV e cloudy
The cccDNA of property；
5) by step 4) cccDNA that obtains is injected into mouse, that is, construct Management of HBeAg negative mouse model.
In the above-mentioned technical solutions, step 2) described in primer pair be HBV-VF/HBV-VR, sequence is：
HBV-VF：CCGGATGCTCTTCTTTTTCACCTCTGCCTAATCATC,
HBV-VR：CCGGATGCTCTTCAAAAAGTTGCATGGTGCTGGTG.
Step 5) in cccDNA is injected into mouse using hydrodynamic(al) force method.
In step 5) in, the concentration that every mouse injects 2 μ g cccDNA, cccDNA is 1 μ g/mL, injection time is 5~
8s。
The beneficial effects of the invention are as follows：Management of HBeAg negative mouse model is successfully constructed first, solves mesh
Preceding only hepatitis B e animals showing positives model is without the problem of e antigen negative animal models.The hepatitis B e that the present invention builds resists
Former negative mice model can be used for e feminine gender HBV persistent infections research, the study mechanism hepatitis B infected to e antigen negatives and medicine
Thing treatment effectiveness study is significant.
Brief description of the drawings
Fig. 1 is expression figure of the HBV virus specific markers in mice serum, wherein, A is blood after water under high pressure powered inj ection
The expression of clear HBsAg, B is the percentage of different time points HBsAg in serum positive findings after injection, and C is serum HBV
The level of DNA, D is the expression of serum HBeAg after injection.
Fig. 2 is the qPCR testing result figures of HBVDNA expression in hepatic tissue.
Fig. 3 is the result that rolling circle amplification-PCR methods detect cccDNA, wherein, 1：100bp ladder, 2：pEASY-HBV/
HBeAg- feminine gender plasmid as template, 3：Linear HBV as template, 4：The DNA that the experimental group of 21 days is extracted after injection is used as mould
Plate, 5：After injection 21 days experimental group extract DNA as template, 6：CccDNA is used as template.
Fig. 4 is 21 days and 70 days IHC testing results of murine liver tissue HBcAg after injection, wherein, a, b, c are respectively notes
The hepatic tissue IHC results of 21 days after penetrating, a is physiological saline, and b is pAAV/HBV1.2, and c is the HBV DNA of cyclisation；D, e, f distinguish
It is the hepatic tissue IHC results of 70 days after injecting, d is physiological saline, and e is pAAV/HBV1.2, and f is the HBV DNA of cyclisation.
Fig. 5 is 21 days and 70 days IHC testing result figures (× 400) of murine liver tissue HBsAg after injection, wherein, a, b, c
It is respectively the hepatic tissue IHC results of 21 days after injecting, a is physiological saline, and b is pAAV/HBV1.2, and c is the HBV DNA of cyclisation；
D, e, f are respectively the hepatic tissue IHC results of 70 days after injection, and d is physiological saline, and e is pAAV/HBV1.2, and f is the HBV of cyclisation
DNA。
Fig. 6 is hepatic tissue changes in histopathology figure (× 400) of 21 days and 70 days after HE dyeing detection injections, wherein,
A, b, c are respectively the murine liver tissue pathology HE analysis results of 21 days after injection, and a is physiological saline, and b is pAAV/HBV1.2,
C is the HBVDNA of cyclisation；D, e, f are respectively the murine liver tissue pathology HE analysis results of 70 days after injection, and d is physiology salt
Water, e is pAAV/HBV1.2, and f is the HBV DNA of cyclisation.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Main agents and the producer：
PEASY-BluntZero carriers (Quan Shi King Companies, China)
PrimeSTAR GXLDNAPolymerase (Takara companies, Japan)
Plasmid extraction kit (QIAGEN companies, Germany)
BspQI restriction endonucleases (NEB companies, the U.S.)
Glue reclaim kit (Takara companies, Japan)
T4 ligases (Takara companies, Japan)
Radio-immunity identification kit (Beijing North Institute of Biological Technology, China)
Mouse ELISA kit (Wuhan bio tech ltd of gene U.S., China)
Plasmid-safeATP-dependentDNase PSAD (Epicentre companies, the U.S.)
Phi29DNA polymerases (NEB companies, the U.S.)
FastStartUniversal SYBR-Green Master mix (Roche companies, Germany)
Sequencing service (Invitrogen companies, Shanghai)
The experimental technique of unreceipted actual conditions in embodiment, generally according to normal condition, or is built according to manufacturer
The condition of view.
Embodiment one, the mouse model for building Management of HBeAg negative
Operate in accordance with the following steps：
First, the clone of HBV e negative virals strain full length DNA
1st, HBV DNA total lengths are expanded：Extraction is diagnosed as No. 11061008 patients serums of HBeAg negative infections (by weight
Infectious Disease of the second affiliated hospital of celebrating medical university provides) DNA, enter performing PCR using primer pair HBV-F/HBV-R and expand, primer sequence
It is classified as：
Sense primer HBV-F：
5 '-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3 ',
Anti-sense primer HBV-R：
5′-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGTG-3′。
2nd, the PCR primer that step 1 is obtained is sequenced, is obtained its sequence, primer is redesigned according to sequencing result, newly
The upstream and downstream primer of design respectively deletes 7 bases, and the degeneracy base of sense primer has been changed into spy according to sequencing result
Isobase, improves the specificity and amplification efficiency and subsequent purification efficiency of PCR.New primer is：
HBV-VF：CCGGATGCTCTTCTTTTTCACCTCTGCCTAATCATC, HBV-VR：
CCGGATGCTCTTCAAAAAGTTGCATGGTGCTGGTG.Using primer pair HBV-VF/HBV-VR, with what is above extracted
No. 11061008 patients serum DNA are template, enter performing PCR amplification：
PCR reaction conditions are：95℃3min；(95℃50S；58℃20S；72 DEG C of 3min30S) 30 circulations；72℃
5min。
3rd, the PCR primer that step 2 is obtained is cloned into pEASY-Blunt Zero carriers：Add in 1.5mL centrifuge tubes
Enter 3 μ L PCR primers and 1 μ L pEASY-Blunt Zero room temperatures connection 30min.Add 100 μ LDH5 α in connection product, put
In ice bath 20min on ice, 42 DEG C of water-bath heat shock 30S, 2min on ice is gone to, adds 800 μ L SOC cultures to be based in heat shock product,
It is placed in shaking table 200rpm/min, 37 DEG C of restoration ecosystem 30min, 10000rpm centrifugation 1min and abandons most of supernatant, it is resuspended heavy to blow and beat
Form sediment, resuspended cell is coated and is pre-coated with the LBA flat boards of IPTG and X-Gal；It is placed in 37 DEG C of culture carton upside down cultures
Night.10 hickie monoclonals of picking carry out LBA Liquid Cultures, and plasmid is extracted after 12h, carry out BspQI digestion verifications, electrophoresis result
Display has successfully obtained 3 positive colonies, is sent company to be sequenced, and sequencing result shows and is positive pEASY-HBV
Plasmid.
2nd, the preparation of e feminine gender HBV cccDNA
CccDNA is that the method being cyclized by plasmid enzyme restriction is prepared, and process is as follows：First by foregoing positive colony
Bacterial strain is enlarged culture, and the foregoing positive pEASY-HBV plasmids for obtaining, the matter that will be extracted are extracted with plasmid extraction kit
Then grain BspQI endonuclease digestions, digestion products are used the fragment of size about 3.2kb with 1% agarose gel electrophoresis
Glue reclaim kit is reclaimed and purified, and is finally cyclized and is purified with T4 ligases, that is, obtain the negative cccDNA of HBeAg.
3rd, the foundation of animal model
The C57BL/6J male mices for taking 36 6-8 week old are randomly divided into 3 groups, 15 hydrodynamic(al) force method injections of experimental group
HBeAg feminine genders cccDNA (2 μ g/2mL/, 5-8s)；15 injection pAAV/HBV1.2 of control group (5 μ g/2mL/ are only)；Blank group
6 injecting normal salines (2mL).Adopt within 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks and 10 weeks after injection respectively
Collection tail vein.Every group gathers hepatic tissue at 3 weeks and 10 weeks and is detected respectively.
The detection of embodiment two, mouse model
Experimental group, control group to the structure of embodiment one, naive mice model are detected：
First, the detection of mice serum HBV special sign things
Experiment mice serum is to HBV special sign things such as HBsAg, HBeAg, viral DNA and glutamic-pyruvic transaminase (alanine
Aminotransferase, ALT) etc. detected at different time points, the detection of HBsAg and HBeAg uses radio-immunity
Identification kit；ALT detections use mouse ELISA kit.
The detection real time fluorescence quantifying PCR method of HBVDNA, taking 50 μ L serum carries out the extraction of viral DNA, RNA, with 2
μ L STb genes carry out qPCR, and analyze HBV copy numbers.Design of primers is carried out in the conserved region of HBV gene and expand, forward primer：
HBV-Q-F：5′-CCTCTTCATCCTGCTGCT-3′；Reverse primer HBV-Q-R：5 '-AACTGAAAGCCAAACAGTG-3 ', mark
Directrix curve presses 5 × 10 using pEASY-HBV/HBeAg- feminine genders HBVDNA3,5×104,5×105,5×106,5×107,and 5
×108Copy/μ L gradients are drawn, and PCR response procedures are：95℃3min；94 DEG C of 20s, 50 DEG C of 30s, 40 circulations；72℃
32s。
Testing result is as shown in figure 1, Figure 1A is the expression of serum HBsAg after water under high pressure powered inj ection, Monitoring lower-cut
It is the HBsAg expressions rapid increasing in a week after injection of 15ng/mL, as can be seen from the figure experimental group and positive controls
Adduction reaches peak value, is then remarkably decreased, and blank control group is to continue negative findings.Figure 1B is different time points serum after injection
Percentage (control group and experimental group n=15, blank group n=6) the wherein experimental group of middle HBsAg positive findingses is after injection 70 days
Still there is 60% positive rate.Fig. 1 C are the level of serum HBV DNA, and experimental group and positive controls respectively may be about 2.70
×108Copy/mL and 2.69 × 108Copy/mL.Fig. 1 D are the expression of serum HBeAg after injection, and Monitoring lower-cut is 1ng/
ML, the expression without HBeAg in experimental group consistent with expected results.
In sum, can be seen that we have been successfully established the small of e antigen negative infection from conventional detection index
Mouse model.
2nd, in murine liver tissue virus titer and cccDNA detection
The quantitative of HBV DNA is carried out with foregoing qPCR methods in hepatic tissue, as a result as shown in Fig. 2 cccDNA's continues
In the presence of the mark for being HBV Long-term Infections, therefore we have carried out the detection of cccDNA for 21 days and 70 days after injection respectively, experiment
Result shows that the cccDNA of the mouse model is persistently present, and level is all higher than 108Copy/mL.
The detection of cccDNA adds the method for PCR to carry out with rolling circle amplification, and step is as follows：Extract first 3 weeks and 10 after injection
The viral DNA of the murine liver tissue in week, with the safe DNA enzymatic (plasmid-safeATP-dependent of the plasmid of dependency ATP
DNase PSAD) 37 DEG C for the treatment of 12h；Rolling circle amplification then is carried out, is mixed with eight primers of 1 μ L DNA and 2 μ L and is added 10
× phi29 buffer solutions to final volume is 10 μ L, and 95 DEG C of 3min are carried out to above DNA mixed liquors, is gradually cooling to 50 DEG C of 15s, 30
DEG C 15s, 20 DEG C of 10min, are subsequently placed on ice；Followed by amplified reaction, 3 μ L will be added in above-mentioned 10 μ LDNA mixtures
The Phi29DNA polymerases of dNTPs, 10 × Phi29 buffer solutions and 10U, carry out 30 DEG C of reactions of 16h, and reaction is placed in 65 after terminating
DEG C 10min, the product of last rolling circle amplification is detected by being reacted across the PCR of cccDNA breach.Eight described primer sequences
It is classified as：
RCA1:AATCCTCACAATACC,
RCA2:GATGGGATGGGAATA,
RAC3:CCTATGGGAGTGGGC,
RAC4:GCAACGGGGTAAAGG,
RAC5:ATGCAACTTTTTCAC,
RAC6:TCCAAATTCTTTATA,
RAC7:TAGAAGAAGAACTCC,
RAC8:AGAATATGGTGACCC。
Testing result is as shown in figure 3,1:100bp ladder；2:PEASY-HBV/HBeAg- feminine gender plasmids are used as template；
3:Linear HBV is used as template；4:The DNA that the experimental group of 21 days is extracted after injection is used as template；5:The experimental group of 21 days after injection
The DNA of extraction is used as template；6:CccDNA is used as template.It can be seen that being implicitly present in the mark cccDNA of HBV infection in this model.
3rd, liver histopathology and immunohistochemical analysis
The method dyeed with haematoxylin Yihong (hematoxylin and eosin, HE) carries out liver histopathology observation.
The positioning of viral antigen and expression are detected with immunohistochemical method (immunohistochemistry, IHC).Poly first
The histotomy of the FFPE of 4.5 μ m-thicks that benzene is fixed is dyeed with HE and IHC respectively, uses horse anti-HBsAg (1:
1000) with rabbit anti-HBcAg (1:1000) secondary antibody carries out HBsAg and HBV core antigen in observation hepatic tissue section
(HBcAg) expression, in negative control, secondary antibody is replaced with physiological saline.
After injection the IHC testing results of 21 days and 70 days murine liver tissues HBcAg and HBsAg respectively as shown in Figures 4 and 5,
As can be seen from the figure there is the expression of HBcAg and HBsAg in experimental mice liver cell, wherein HBVsAg results are further confirmed
Foregoing Placenta function result, it is seen that the model construction is successful.
The hepatic tissue changes in histopathology of 21 days and 70 days is as shown in Figure 6 after HE dyeing detection injections, it is seen that the length of HBV
Phase infects and does not cause acute liver damage.
Sequence table
<110>Chongqing Academy of Science ＆ Technology
<120>The construction method of Management of HBeAg negative mouse model
<160> 14
<210> 1
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<223> HBV-F
<400> 1
ccggaaagcttatgctcttctttttcacctctgcctartcatc 43
<210> 2
<211> 42
<212> DNA
<213>Artificial sequence
<223> HBV-R
<400> 2
ccggagagctcatgctcttcaaaaagttgcatggtgctggtg 42
<210> 3
<211> 36
<212> DNA
<213>Artificial sequence
<223> HBV-VF
<400> 3
ccggatgctcttctttttcacctctgcctaatcatc 36
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence
<223> HBV-VR
<400> 4
ccggatgctcttcaaaaagttgcatggtgctggtg 35
<210> 5
<211> 18
<212> DNA
<213>Artificial sequence
<223> HBV-Q-F
<400> 5
cctcttcatcctgctgct 18
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<223> HBV-Q-R
<400> 6
aactgaaagccaaacagtg 19
<210> 7
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA1
<400> 7
aatcctcacaatacc 15
<210> 8
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA2
<400> 8
gatgggatgggaata 15
<210> 9
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA3
<400> 9
cctatgggagtgggc 15
<210> 10
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA4
<400> 10
gcaacggggtaaagg 15
<210> 11
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA5
<400> 11
atgcaactttttcac 15
<210> 12
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA6
<400> 12
tccaaattctttata 15
<210> 13
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA7
<400> 13
tagaagaagaactcc 15
<210> 14
<211> 15
<212> DNA
<213>Artificial sequence
<223> RCA8
<400> 14
agaatatggtgaccc 15
Claims (4)
1. a kind of construction method of Management of HBeAg negative mouse model, it is characterised in that：Comprise the following steps：
1) the patients serum DNA for being diagnosed as HBeAg negative infections is extracted, is entered performing PCR using primer pair HBV-F/HBV-R and is expanded
Increase, primer sequence is：
HBV-F：5 '-CCGGAAAGCTTATGCTCTTCTTTTTCACCTCTGCCTARTCATC-3 ', HBV-R：
5′-CCGGAGAGCTCATGCTCTTCAAAAAGTTGCATGGTGCTGGTG-3′；
2) PCR primer is sequenced, the specific PCR amplimer pair for the sequence is redesigned according to sequencing result,
PCR is re-started using the patients serum DNA of primer pair HBeAg negative infections to expand；
3) by step 2) obtain PCR primer be cloned into pEASY-HBV carriers, select positive colony；
4) extraction step 3) positives clone plasmid, the plasmid that will be extracted BspQI endonuclease digestions, reclaim size about
The fragment of 3.2kb is simultaneously purified, and is cyclized and is purified with T4 ligases, obtains the negative cccDNA of HBeAg；
5) by step 4) cccDNA that obtains is injected into mouse, that is, construct Management of HBeAg negative mouse model.
2. the construction method of Management of HBeAg negative mouse model as claimed in claim 1, it is characterised in that：Step 2)
Described in primer pair be HBV-VF/HBV-VR, sequence is：
HBV-VF：CCGGATGCTCTTCTTTTTCACCTCTGCCTAATCATC,
HBV-VR：CCGGATGCTCTTCAAAAAGTTGCATGGTGCTGGTG.
3. the construction method of Management of HBeAg negative mouse model as claimed in claim 1, it is characterised in that：Step 5)
Be injected into cccDNA in mouse by middle use hydrodynamic(al) force method.
4. the construction method of Management of HBeAg negative mouse model as claimed in claim 3, it is characterised in that：In step
5) in, the concentration of every 2 μ g cccDNA, cccDNA of mouse injection is 1 μ g/mL, and injection time is 5~8s.
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| CN109652452A (en) * | 2018-12-13 | 2019-04-19 | 重庆市科学技术研究院 | The long-term experimental animal model construction method for carrying hepatitis type B virus |
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| CN1701124A (en) * | 2002-09-27 | 2005-11-23 | 国家健康医学研究所 | Method for assaying replication of HBV and testing susceptibility to drugs |
| WO2016120186A1 (en) * | 2015-01-27 | 2016-08-04 | F. Hoffmann-La Roche Ag | Recombinant hbv cccdna, the method to generate thereof and the use thereof |
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