CN101948827A - Method for cloning whole genome of low-load hepatitis B virus - Google Patents
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Abstract
The invention relates to a method for cloning the whole genome of low-load hepatitis B virus (HBV). The method comprises the following steps: extracting HBV DNA from the serum of a patient, performing amplification through the nested polymerase chain reaction to obtain the CP-S region and RT-X region of HBV, performing enzyme digestion with incision enzyme, then connecting, and using the obtained genome as the template to perform PCR amplification and cloning. The method of the invention is used to perform amplification, cloning and dsequence analysis of the whole genome of low-load HBV and can provide effective help for the basic and clinical researchs which further analyze the low-load HBV.
Description
Technical field:
The present invention relates to the method for the full genomic clone of a kind of low carrying capacity hepatitis B virus, particularly relate to and adopt two fragment connection methods to hang down the complete genomic clone of carrying capacity hepatitis B virus.
Background technology:
The full genomic clone of hepatitis B virus (the following HBV that also claims) has the important clinical meaning for research HBV virusology characteristics and biological characteristics.
Because the full genome of HBV is the relaxed configuration of a non-closure of two strands, two chains are not isometric, add that genome length is 3.2kb, increased HBV whole genome amplification and clone's difficulty, just successfully carry out the full gene amplification of HBV up to nineteen ninety-five American scholar Gunther etc.But this method requires the HBV virus load can not be lower than 10
5Copies/ml, and low carrying capacity is difficult to obtain PCR product [Gunther S, Li B, Miska S, et al.J Virol, 1995,69:5437-44.].
Present widespread use along with anti-HBV medicine, the interior HBV carrying capacity of patient's body often maintains lower level and duplicates, if want to study the biology characteristics of HBV under these medicine pressure, the method for the full genomic clone of low carrying capacity HBV of highly sensitive, high specificity must be arranged.There is the scholar to set up a kind of two fragments and clones the HBV sequence respectively and check order respectively and obtain the method (not obtaining full genomic clone) of complete sequence information, be lower than 10
5Copy/mL carrying capacity also can detect, but might not be the cloned sequence of same virus, does not have biologic activity.[Lu Jianxi, Guangdong medical science 2008 such as Li Li, 29 (7): 1095-7] also having the full gene amplification method of some HBV is the improved method [Li Rui of Gunther method, Yan Yongping grade in an imperial examination four army medical university's journals 2002,23 (6): 572-3], can not effectively improve the recall rate of the low full gene amplification of carrying capacity HBV.
Be limited to the research of a certain gene fragment mostly for the low full genome research of carrying capacity HBV, by some subgenomic fragment in the HBV gene group in the pcr amplification individuality, directly order-checking then, can under the lower condition of template amount, obtain the subgenomic fragment amplified production, but because there is regulatory gene HBV gene inside, overlapped again between gene fragment, form probably HBV different genes group fragment constitute one can be for the HBV gene fragment of analyzing, make the sudden change complicacy that appears on the individual gene molecule and cause a disease and the relation propagated can't be accurately and multianalysis.Discover that owing to have the Different Variation strain in the virosome, the method for this direct survey partial sequence can accurately not pointed out the relation of the virogene structure and the cause of disease and propagation.
Take two fragments of common PCR method amplification, unsatisfactory for the expanding effect of low carrying capacity HBV DNA, specificity is not strong, and it adopts two time cloning steps, has increased the complexity and the cost budgeting of experiment.
If can research and develop a kind of highly sensitive, high specificity, the method for the low full genomic clone of carrying capacity HBV simply and easily, amplification and clone HBV whole genome sequence from the low carrying capacity serum of patient, to help experiment in vitro to estimate hbv replication power, HBV transgenation feature and frequency, HBV Infection in Vitro power, significant for monitoring antiviral curative effect and deeply illustrating the HBV biologic activity.
Summary of the invention:
The purpose of this invention is to provide a kind of under low carrying capacity hepatitis B virus situation, to the effective ways of the full genomic clone of HBV.
Main means of the present invention and method are:
The method of the full genomic clone of a kind of low carrying capacity hepatitis B virus, adopt Auele Specific Primer that the HBVDNA that extracts in the serum is carried out the nest-type PRC reaction, amplify two fragments in CP-S district and RT-X district of HBV virus, carry out respectively after enzyme cuts, the two is connected into the full genome of complete HBV, again with connect product be template with the full genome of PCR method amplification HBV after, clone;
The sequence of described Auele Specific Primer is as follows:
SEQ ID NO:1～SEQ ID NO:2 is the outer primer sequence in CP-S district;
SEQ ID NO:3～SEQ ID NO:4 is the outer primer sequence in RT-X district;
SEQ ID NO:5～SEQ ID NO:6 is the inner primer sequence in CP-S district;
SEQ ID NO:7～SEQ ID NO:8 is the inner primer sequence of RT-X.
Count the 1st Nucleotide (nt 1) with the genomic EcoRI restriction enzyme site of ring-type HBV that 3215 Nucleotide are long, the initial termination to the S district is CP-S district (nt 1777-nt 274) from the core promoter district, and another is that initial the termination to the X district is RT-X district (nt 3194-nt 1797) from the reversed transcriptive enzyme district.
The concentration of the interior outer primer when adjusting nest-type PRC can improve the sensitivity of reaction, makes it can carry out tubular type nest-type PRC amplification.Best primer concentration is: the outer primer final concentration is 10nmol/L; The inner primer final concentration is 200nmol/L.
Described enzyme is cut, be two fragments of amplification after, cut using restriction endonuclease to carry out enzyme behind the product purification respectively; Restriction endonuclease preferably uses XbaI.
Described connection is to use ligase enzyme to carry out, and preferably uses the T4DNA ligase enzyme.
Present method also can adopt following one or more means to be optimized, and can make the invention better effects if or reach best:
Add BspQ I restriction enzyme site in primer SEQ ID NO:5 and SEQ ID NO:7 sequence, the amplification total length is convenient to not contain fully from the carrier acquisition HBV full-length gene group of exogenous array later on.
If in the PCR reaction system, mixed adding Pfu archaeal dna polymerase and general T aq archaeal dna polymerase with 1: 10, can improve the specificity of amplification, can significantly reduce cost again.Be convenient to carry out in the laboratory.
Described when amplifying two fragments with PCR method, adopt a tubular type nest-type PRC technology, can reduce pollution, improve output; And only need to have simplified operation steps through a time cloning.
Adopt method of the present invention to reach goal of the invention: to experiment showed, that the virus that amplifies has the power of duplicating (seeing embodiment 2).
The invention has the beneficial effects as follows:
1, highly sensitive: can increase obtains initial dna profiling concentration 10
3～10
4Copy/mL sample.The concentration of the interior outer primer when adjusting nest-type PRC and the reaction conditions of whole genome amplification can improve the sensitivity of reaction, reach 10
3Copy/mL (improving 2 orders of magnitude) than internationally recognized Gunther method.
2, high specificity: by the optimization to selection of primers and reaction system, specificity is significantly improved, the pseudomutation generation probability of PCR reaction is controlled at below 1,/10 ten thousand.
3, easy and simple to handle: as to use two fragments of tubular type nest-type PRC technology amplification, can reduce pollution, improve output; And, simplified operation steps only through a time cloning.
4, cost is low: main agents can adopt domestic reagent, and low price is easy to carry out in the laboratory.Cost as the Pfu/Taq enzyme mixture is about 1/10 of the anti-synthase of import high-fidelity heat-resistant dna, and reaction effect is identical.
Description of drawings:
Fig. 1 is the amplification 1772bp in HBV DNA CP-S district;
Fig. 2 is the amplification 1814bp in HBV DNA RT-X district;
Fig. 3 is that two fragments connect back whole genome amplification 3215bp as a result;
Fig. 4 is complete sequence clone transfection HepG 2 cell culture supernatant hepatitis B surface antigen result comparison after 72 hours that the amplification of two fragment methods obtains;
Fig. 5 is the complete sequence clone transfection HepG 2 cell that obtains of two fragment methods amplifications HBVDNA quantitative result in the cell pyrolysis liquid after 72 hours.
Embodiment:
Below used main experiment material source as follows:
Pfu and Taq archaeal dna polymerase and viral DNA extract test kit: be sky, Beijing bounties Gene Tech. Company Limited product.Restriction endonuclease XbaI, T4DNA ligase enzyme: be NEB company product.
The embodiment 1 low full genomic clone of carrying capacity hepatitis B virus
Amplimer design: from GenBank, choose 729 HBV full-length gene group sequences, wherein distinctive B of Chinese and C genotype sequence are 209, with NTI software compare HBV CP-S district and RT-X district conserved regions design primer, the design of total length primer reference, and the adding restriction enzyme site, help the analysis of next step the power of duplicating.
The primer sequence that table 1PCR reaction is used
One. CP-S district and the RT-X district of nest-type PRC amplification HBV
First round PCR reaction (total system 25 μ L)
2 * Mix Buffer (adding 0.3% orange G in advance), 12.5 μ L
Primer 2 and 4 (10 μ mol/L) the 0.25 μ L of dilution in 1: 10
Taq archaeal dna polymerase (5U/ μ L) 0.50 μ L
Pfu archaeal dna polymerase (5U/ μ L) 0.05 μ L
14.0μL
Template DNA 11.0 μ L
Illustrate:
1, in when reaction nest-type PRC first round, in the 25 μ L standard reaction systems, outer primer concentration is configured to the storage liquid of 10 μ mol/L, gets 0.25 μ L behind 10 times of the dilute with waters and adds in the 25 μ L reaction systems.
2, for improving atopic, the ratio according to 10: 1 when adding general T aq archaeal dna polymerase (final concentration reaches 0.1U/ μ L) in the system adds Pfu archaeal dna polymerase (final concentration reaches 0.01U/ μ L).
3, first round PCR reaction parameter:
94 ℃ of pre-sex change of 5min,
94 ℃ of 45s, 59 ℃ of 50s, 2 circulations of 72 ℃ of 100s,
94 ℃ of 45s, 57 ℃ of 50s, 2 circulations of 72 ℃ of 100s,
94 ℃ of 45s, 55 ℃ of 50s, 2 circulations of 72 ℃ of 100s,
94 ℃ of 45s, 53 ℃ of 50s, 2 circulations of 72 ℃ of 100s,
94 ℃ of 45s, 51 ℃ of 50s, 2 circulations of 72 ℃ of 100s,
94 ℃ of 45s, 56 ℃ of 50s, 30 circulations of 72 ℃ of 100s,
72 ℃ of 5min, 68 ℃ of 5min drop to 4 ℃ at last.
Second takes turns PCR reaction (total system 50 μ L comprise that 25 μ L newly join damping fluid and 25 μ L templates) primer 5 and 7 amplification CP-S district sheet segment length 1668bp, primer 6 and 8 amplification RT-X district sheet segment length 1846bp
H
2O 8.5μL
2 * Mix Buffer (adding 0.3% orange G in advance), 15 μ L
Primer 5 and 7 (10 μ mol/L) 0.5 μ L
Taq archaeal dna polymerase (5U/ μ L) 0.45 μ L
Pfu archaeal dna polymerase (5U/ μ L) 0.05 μ L
25.0μL
Template (the 1st takes turns the PCR product) 25.0 μ L
Illustrate:
1, the nest-type PRC first round 25 μ L product is all as second template of taking turns PCR, second takes turns the storage liquid that the PCR primer concentration is configured to 10 μ mol/L, getting 0.5 μ L adds in the 50 μ L systems, two take turns reaction solution and directly be added in first pipe, the 25 μ L reaction solutions what prepare, finish single tube nest-type PRC reaction.
2, second take turns the PCR reaction parameter: 94 ℃ of pre-sex change of 3min, 94 ℃ of 40s, 55 ℃ of 45s, 35 circulations of 72 ℃ of 90s, 72 ℃ of 5min, 68 ℃ of 5min drop to 4 ℃ at last.
Get 3 μ L PCR end products, carry out electrophoresis with 1% agarose.All the other 47 μ L PCR end product purifying reclaim (carrying out according to Qiagen PCR product purification test kit specification sheets).
Two, cut purifying with the enzyme in RT-X district and be connected in the CP-S district of HBV
1. to cut system as follows for enzyme:
Xba?I 1.5μL
PCR purified product 38.5 μ L
10×M?Buffer 5μL
0.1％BSA 5μL
50μL
Illustrate: the enzyme tangent condition: 37 ℃ are spent the night.In order to improve the activity of enzyme, increase the efficient that enzyme is cut, before cutting, enzyme wants purifying nest-type PRC product.
2. linked system is as follows:
10×Buffer 4μL
T4DNA ligase enzyme 1.5 μ L
Sterilization ddH
2O 14 μ L
CP-S district enzyme is cut after product 10 μ L
RT-X district enzyme is cut after product 10 μ L
40μL
Illustrate: condition of contact: 37 ℃ are spent the night.All only there are single XbaI enzyme cutting site in the CP-S district of HBV virus and the nest-type PRC product in RT-X district, so the two can directed be connected to the full genome of complete HBV.
Three, HBV whole genome amplification
Reaction system:
10×LA?PCR?Buffer 2.5μL
2.5mmol/L?dNTP?Mixture 2μL
51 μ L of dilution in 1: 5
71 μ L of dilution in 1: 5
LATaq(5U/μL) 0.5μL
H
2O 17μL
Explanation
1, when whole genome amplification, in the 25 μ L standard reaction systems, primer concentration is configured to the storage liquid of 10 μ mol/L, gets 1 μ L behind 5 times of the dilute with waters and adds in the 25 μ L reaction systems.
2. reaction parameter:
94 ℃ of pre-sex change of 5min;
94 ℃ of 40s, 60 ℃ of 1min40s, 10 circulations of 72 ℃ of 5min;
94 ℃ of 40s, 60 ℃ of 1min40s, 10 circulations of 72 ℃ of 6min;
94 ℃ of 40s, 60 ℃ of 1min40s, 15 circulations of 72 ℃ of 7min;
72 ℃ of 10min drop to 4 ℃ at last
Four, PCR product end adds A
Reaction system:
PCR product 50 μ L
Taq archaeal dna polymerase 1 μ L
dATP 6μL
10×LA?buffer 6μT
63μL
Illustrate: reaction conditions: 72 ℃ of 20min.Because LA Taq enzyme itself has 5 prime excision enzyme activity, so, be necessary the PCR product is carried out purifying, to remove its influence to connecting for the T-A clone's that improves efficient.
Five, connect
Reaction system
2 * connection buffer, 5 μ L
PGEM T-easy carrier 1 μ L
DNA 3 μ L behind the purifying
10μL
Illustrate: condition of contact: 4 ℃ are spent the night.Through optimum experimental, DNA: the pGEMT-easy carrier is that 3: 1 o'clock joint efficiencies are higher.
Six, transform
Get competent cell and place 5min on ice, melt standby, 5 μ L connect and add 25 μ L competent cells (operation on ice), ice bath 20min, 42 ℃ of thermal shock 1min in the product, behind the ice bath 2min, the SOC substratum that adds 950 μ L preheatings, 37 ℃ of 130rpm/min cultivated 200 μ L coated plates behind the centrifugal 10min of 1000g 1 hour, 37 ℃ of overnight incubation are selected white bacterial plaque sequencing analysis.
Seven, the result obtains 1 HBV full-length gene group cloned sequence, submits GenBank to, Accession Number:GQ372968
Embodiment 2 hangs down the complete genomic clones of carrying capacity hepatitis B virus and duplicates the power analysis
Low carrying capacity HBV DNA chronic hepatitis B patient serum sample to PLA's the 302nd hospital admission is cloned, and the power of duplicating is analyzed as follows:
Case: 3 routine patients are chronic hepatitis B patient, and Case definition meets Xi'an viral hepatitis Case definition in 2000, and [Chinese Medical Association infects sick and parasitosis association; China hepatopathy association: viral hepatitis diagnosis and treatment standard. Chinese hepatopathy magazine 2000; 6:324-329.].Serum HBV DNA carrying capacity is 9.77 * 10 during patient's 6380 samplings
3Copy/mL, serum HBV DNA carrying capacity is 6.85 * 10 during patient Z3 sampling
3Copy/mL.Serum HBV DNA carrying capacity is 2.48 * 10 during patient Z31 sampling
3Copy/mL.
Experimental procedure
One, two-stage method difference pcr amplification CP-S district and RT-X district: concrete steps are with embodiment 1.
Two, HBV full-length gene group clone: concrete steps are with embodiment 1.
Three, 1.0 times of HBV genome cell transfectings:
1. enzyme is cut: BspQ I and Sca I (purchasing the company in NEB) enzyme is cut the T-easy carrier that contains HBV full-length gene group and is spent the night, and glue reclaims the HBV sequence of 3.2kb, obtains not contain the complete HBV full-length gene group of exogenous array, and ultraviolet spectrophotometer is measured its concentration.
2. transfection: the HepG2 cell is pressed 1 * 10
5/ hole is divided foster in 12 orifice plates, and 37 ℃, 5%CO
2It is fully adherent to cultivate 24 hours reliefs, 1 * PBS washes 2-3 time, the nutrient solution that adds no FBS (foetal calf serum), every hole adds plasmid and the 2 μ L Fugene HD (purchasing the company in Roche) that 1 μ g glue reclaims, cultivate that 1 * PBS washes 2-3 time after 4-5 hour, add the complete culture solution that contains FBS, 37 ℃, 5%CO
2Cultivate harvested cell after 72 hours.
Four, ELISA (enzyme linked immunological) method detects culture supernatant secretion HBsAg: carry out according to hepatitis B surface antigen(HBsAg) (HBsAg) ELISA detection kit (purchasing the hygienic articles technology company limited in Beijing section) specification sheets.
Five, quantitative fluorescent PCR is measured HBV genome duplication power: the HepG2 cell of transfection after 72 hours used 0.5%NP-40 cracking 20 minutes, the centrifugal precipitation of abandoning of lysate, add 20U DNase and 37 ℃ of water-bath 2-3 of 10U RNase hours, add 37 ℃ of water-bath digestion of protease K digesting liquid 2-3 hour, add isopyknic phenol and chloroform extracting HBV DNA, Virahol and ethanol sedimentation, be dissolved at last in the 30 μ L distilled water, get 3 μ L with HBV fluorescent quantitation (SYBGreen I) detection kit (purchasing) detection by quantitative in Hangzhou BIOER Technology Co., Ltd.
The complete genomic surface antigen analysis of different HBV DNA carrying capacity hepatitis B viruses as a result: use 6 total length HBV genomic clones difference transfection HepG 2 cells that two fragment methods obtain from the low virus load patients serum of 3 examples, collect the supernatant euzymelinked immunosorbent assay (ELISA) and detect the surface antigen (see figure 4), the OD value is between 0.13～0.58.Prompting: by the full genome of HBV of two fragment methods splicings, can finish voluntarily whole life cycle after being transfected into liver cell, and successfully secrete surface antigen, prove that this method has feasibility, the HBV genome of amplification has biologic activity.
The complete genomic quantitative fluorescent PCR analysis of the HBV of two different HBV DNA carrying capacity as a result: the cracking transfectional cell, extract HBV DNA, carry out the quantitative fluorescence analysis (see figure 5).Prompting: by the full genome of HBV that the splicing of two fragment methods obtains, HBV dna replication dna power is between 6.75 * 10
6～5.8 * 10
7Copy/mL.Explanation not only can obtain total length HBV DNA by this method, also has the stronger power of duplicating, for research HBV virusology characteristics provide instrument.
In a word, the method of the full genomic clone of low carrying capacity hepatitis B virus of the present invention is highly sensitive, has good prospect in actual applications, the relation of can be accurately and comprehensively analyzing the low sudden change complicacy of virus load sample on the individual gene molecule and causing a disease and propagate, significant for clinician's early intervention.
Claims (9)
1. the method for the one kind low full genomic clone of carrying capacity hepatitis B virus adopts Auele Specific Primer that the hepatitis B virus DNA that extracts in the serum is carried out the nest-type PRC reaction; Two fragments in CP-S district and RT-X district that amplify hepatitis B virus are carried out respectively the two being connected into the full genome of complete virus after enzyme cuts, again with connect product be template with this full genome of PCR method amplification after, clone; Described CP-S district is nt 1777～nt 274, and the RT-X district is nt 3194～nt 1797; The sequence of described Auele Specific Primer is as follows:
SEQ ID NO:1～SEQ ID NO:2 is the outer primer sequence in CP-S district;
SEQ ID NO:3～SEQ ID NO:4 is the outer primer sequence in RT-X district;
SEQ ID NO:5～SEQ ID NO:6 is the inner primer sequence in CP-S district;
SEQ ID NO:7～SEQ ID NO:8 is the inner primer sequence of RT-X.
2. claim 1 or 2 described methods, when carrying out the nest-type PRC reaction, the concentration of outer primer extremely in adjusting: the outer primer final concentration is 10nmol/L, and the inner primer final concentration is 200nmol/L.
3. claim 1 or 2 described methods, described enzyme is cut, be two fragments of amplification after, cut using restriction endonuclease to carry out enzyme behind the product purification respectively.
4. the described method of claim 3, described restriction endonuclease is XbaI.
5. claim 1 or 2 described methods, described connection is to use ligase enzyme to carry out.
6. the described method of claim 5, described ligase enzyme is T4DNA.
7. claim 1 or 2 described methods add BspQ I restriction enzyme site in primer SEQ ID NO:5 and SEQ ID NO:7 sequence.
8. claim 1 or 2 described methods are mixed with 1: 10 volume ratio in the PCR reaction system and are added Pfu archaeal dna polymerase and general T aq archaeal dna polymerase.
9. claim 1 or 2 described methods when amplifying two fragments with PCR method, adopt a tubular type nested PCR method.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559657A (en) * | 2011-11-09 | 2012-07-11 | 中国人民解放军第三〇二医院 | Method for performing HBV (Hepatitis B Virus) whole genome cloning with two-fragment connecting method |
| CN103282497A (en) * | 2010-08-17 | 2013-09-04 | 默沙东公司 | Rna interference mediated inhibition of hepatitis b virus (hbv) gene expression using short interfering nucleic acid (sina) |
| US10513703B2 (en) | 2014-11-10 | 2019-12-24 | Alnylam Pharmaceuticals, Inc. | Hepatitis B virus (HBV) iRNA compositions and methods of use thereof |
| US11324820B2 (en) | 2017-04-18 | 2022-05-10 | Alnylam Pharmaceuticals, Inc. | Methods for the treatment of subjects having a hepatitis b virus (HBV) infection |
| US11492623B2 (en) | 2018-08-13 | 2022-11-08 | Alnylam Pharmaceuticals, Inc. | Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof |
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2010
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103282497A (en) * | 2010-08-17 | 2013-09-04 | 默沙东公司 | Rna interference mediated inhibition of hepatitis b virus (hbv) gene expression using short interfering nucleic acid (sina) |
| CN103282497B (en) * | 2010-08-17 | 2018-07-10 | 瑟纳治疗公司 | The inhibition led using the mediated rnai of hepatitis type B virus (HBV) gene expression of short interfering nucleic acid (siNA) |
| CN102559657A (en) * | 2011-11-09 | 2012-07-11 | 中国人民解放军第三〇二医院 | Method for performing HBV (Hepatitis B Virus) whole genome cloning with two-fragment connecting method |
| US10513703B2 (en) | 2014-11-10 | 2019-12-24 | Alnylam Pharmaceuticals, Inc. | Hepatitis B virus (HBV) iRNA compositions and methods of use thereof |
| US11060091B2 (en) | 2014-11-10 | 2021-07-13 | Alnylam Pharmaceuticals, Inc. | Hepatitis B virus (HBV) iRNA compositions and methods of use thereof |
| US11324820B2 (en) | 2017-04-18 | 2022-05-10 | Alnylam Pharmaceuticals, Inc. | Methods for the treatment of subjects having a hepatitis b virus (HBV) infection |
| US11492623B2 (en) | 2018-08-13 | 2022-11-08 | Alnylam Pharmaceuticals, Inc. | Hepatitis B virus (HBV) dsRNA agent compositions and methods of use thereof |
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