CN1726287A - Method of distinguishing drug-resistance of hepatitis b virus - Google Patents
Method of distinguishing drug-resistance of hepatitis b virus Download PDFInfo
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- CN1726287A CN1726287A CNA2003801059163A CN200380105916A CN1726287A CN 1726287 A CN1726287 A CN 1726287A CN A2003801059163 A CNA2003801059163 A CN A2003801059163A CN 200380105916 A CN200380105916 A CN 200380105916A CN 1726287 A CN1726287 A CN 1726287A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/706—Specific hybridization probes for hepatitis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
Abstract
It is intended to provide a method of conveniently distinguishing the drug-resistance of HBV. A method of distinguishing the drug-resistance of HBV which comprises confirming a gene mutation in the nucleic acid sequence of the core promoter region of hepatitis B virus (HBV).
Description
Technical field
The present invention relates to discern the chemical sproof method of HBV to lamivudine chronic hepatitis B curatives such as (ラ ミ Block ジ Application) by the genotype of confirming hepatitis B virus (HBV) core promoter zone.
Background technology
The infected of hepatitis B virus (HBV), the whole world is estimated to have reached about 300,000,000 people.The infection of HBV causes acute and chronic hepatitis (hepatitis B), and causes liver cirrhosis, liver cancer.
The diagnosis of hepatitis B except that the hepatic tissue picture, is also used serum markers such as ALT, HBsAg, HBsAb, HBcAb, HBeAg, HBeAb, HBV polysaccharase, HBV-DNA amount.Wherein, the efficacy determination of antiviral drug is paid attention to HBV-DNA amount in the blood.Be that HBV-DNA amount obviously descends or reaches and measures following and this state of long term maintenance of sensitivity, when other indexs are also good, can be judged as and show the height curative effect.
The HBV gene incomplete double-stranded DNA of ring-type that about 3200 base pairs are formed of serving as reasons.There is the reverse transcription process in duplicating of this HBV gene.Reproduction process roughly can be divided into following 4 stages.
1. form the cyclic DNA of complete two strands by endonuclear endogenous dna polysaccharase, and closed loop and form covalently closed circle double-stranded DNA (covalently closed circular DNA, stage cccDNA).This cccDNA has superhelix.
2. do template is transcribed into mRNA by the rna plymerase ii of cell stage with cccDNA.Wherein the RNA of the longest 3.5Kb becomes the template of reverse transcription as pregenome RNA.There is " ε " signal (encapsidation signal, encapsidation signal) in 5 ' and the 3 ' end of this RNA, and wherein the signal of 5 ' side is packaged into pregenome RNA and varial polymerases in the particle of core and plays an important role.
3. the archaeal dna polymerase of the dependenc RNA in core particle synthesizes the oligonucleotide as primer, and former genomic dna carries out reverse transcription as template, the stage of synthetic (-) chain DNA.
4. make primer with 5 ' the terminal oligonucleotide that remains in pregenome RNA, do the stage of synthetic (+) chain DNA of template with (-) chain DNA.
It is generally acknowledged by suppressing the some of these processes, the gene replication of HBV is ended, just can suppress the propagation of virus.Particularly there is the reverse transcription process in duplicating of HBV, and human immunodeficiency virus's (HIV) propagation also has the reverse transcription process, therefore begins to screen the archaeal dna polymerase inhibitor of dependenc RNA as curative.
Lamivudine is the reverse transcription in HIV inhibiting (HIV) reproduction process specifically also, developed as the HIV curative originally, but finding also has inhibited proliferation to HBV, develops as the curative of chronic hepatitis B from beginning in 1992.
Lamivudine is not understood the mechanism of action of HBV is clear, but it is believed that following mechanism.Lamivudine is nucleosides (cytidine) derivatives class antiviral drug, forms the triphosphoric acid derivative through phosphorylation in cell, becomes active form.This medicine is to suppress the reverse transcription of pregenome RNA by the archaeal dna polymerase (reversed transcriptive enzyme) that suppresses dependenc RNA to one of HBV inhibition of proliferation mechanism.
Another mechanism thinks to be impregnated in the viral DNA chain, ends its elongation.Also report in addition, as to immune remote effect, if cause that by taking lamivudine virus reduces, then the viral protein in the blood flow reduces, the T cell just recovers low reactivity (Boni C etc., Journal of ClinicalInvestigation, 102 to HBV antigen, 968-975,1998).
According to such mechanism of action, lamivudine is widely used as the curative that HBV infects disease, but also has the case that virus quantity is difficult to reduce in the blood, occurs problem also a lot of of persister and hepatitis recurrence thereafter.In order to select the effective case of lamivudine, changing research with the relation of curative effect as virogene is discussed, mainly is much about as the B of the archaeal dna polymerase of the target of lamivudine and research (Ono-Nita SK etc., the Hepatology in C-structure territory, 29,939-945,1999) (Allen MI etc., Hepatology, 27,1670-1677,1998), the report of inquiring into other zones seldom.
And as the genotypic research of HBV, reported the relatively sequence in HBs zone, determine genotype A-D genotypic example (Journal of Medical Virology, 2002,68,522-528).This report discloses, because this genotypic difference, Interferon, rabbit is to the curative effect difference of HBr.In addition, reported that also HBV-DNA 1896 bit base G are mutated into A in the gene in core promoter zone, 1899 bit base G are mutated into A etc.Yet the variation of these genes, be considered to not be the genotype of virus, but metainfective sudden change, and also unclear (the Tang Zeda letter waits, Japanese clinically increase number of the edition, the basic clinical prevention last volume of molecule hepatitis virus first, second, third, fourth, hepatitis E virus, nineteen ninety-five, 52-57) with the relation of HBV treatment.Therefore, though reported the transgenation in core promoter zone, the sudden change of not reporting this zone is the genotypic notion of HBV.
Non-patent literature 1
Hepatology such as Ono-Nita, 29,939-945,1999
Non-patent literature 2
Hepatology such as Allen MI, 27,1670-1677,1998
Non-patent literature 3
Journal of Virology such as Yuh, 66,4073-4084,1992
Non-patent literature 4
Honigwachs etc., Journal of Virology, 63,919-924,1989
Non-patent literature 5
Proceedings of the national Academy of Sciences of theUnited States of America such as Lopez-Cabrera, 87,5069-5073,1990
Non-patent literature 6
Tang Zeda letter etc., clinical number of the edition, the basic clinical prevention last volume of molecule hepatitis virus first, second, third, fourth, hepatitis E virus, nineteen ninety-five, the 52-57 of increasing of Japan.
The announcement of invention
In the pharmacological agent of HBV hepatitis, understanding the effect of this medicine and the relation of HBV objectively, is useful information for the selection for the treatment of policy and medicine.The invention provides from the genetically engineered angle and obtain method for the useful information of the treatment policy of HBV hepatitis and medicament selection.
The present inventor supposes that the effect when HBV hepatitis is carried out pharmacological agent is subjected to the influence of the factor of viral aspect.The genotypic difference, the particularly diversity of the base sequence in HBV core promoter zone of new discovery participation virus multiplication process are relevant to the difference on effect of HBV with medicine, thereby have finished the present invention.
That is, the present invention is the chemical sproof method of identification HBV, comprises the transgenation in the nucleotide sequence of confirming hepatitis B virus (HBV) core promoter field.
And, the invention still further relates to by the HBV-DNA in the separation and purification blood, make the amplification of HBV core promoter zone, determine base sequence, thereby identify the method for the core promoter transgenation that influences curative effect of medication through PCR.
Variation (sudden change) aid forecasting that the invention still further relates to evaluation (detecting) HBV core promoter nucleotide sequence is the method for the effect that infects of the inhibiting pharmacological agent hepatitis B virus of the archaeal dna polymerase that dependenc RNA is arranged (HBV) of representative with the lamivudine.Be tested and appraised at least one sudden change of (detecting) HBV core promoter nucleotide sequence especially, the decline of HBV amount in the blood after the aid forecasting administration.
The invention still further relates to the method for at least one sudden change of the sequence (for example sequence of sequence numbering 2, sequence numbering 3, sequence numbering 4, sequence numbering 5, sequence numbering 6, sequence numbering 7 records) of the specific part of evaluation (detecting) HBV core promoter nucleotide sequence, better is the method for the variation of any or more than one nucleic acid among 7 nucleic acid C identifying 1 record of (detecting) sequence numbering, 28 nucleic acid T, 33 nucleic acid A, 57 nucleic acid A, 73 nucleic acid T, 106 nucleic acid A, 107 nucleic acid T, 200 nucleic acid A, 250 nucleic acid G or 253 the nucleic acid G.
Being more preferably 7 nucleic acid C that the present invention relates to identify 1 record of (detecting) sequence numbering becomes T or 28 nucleic acid T and becomes C or 33 nucleic acid A and become G or 57 nucleic acid A and become G or C or 73 nucleic acid T and become G or 106 nucleic acid A and become T or C or G or 107 nucleic acid T and become C or G, 200 nucleic acid A and become T or C, 250 nucleic acid G and become A, 253 nucleic acid G and become any or the method for more than one sudden change among the A.Difference that it is generally acknowledged the resistance of HBV or susceptibility results from the difference of the genotypic kind of HBV.Therefore, there is the genotype of the HBV of above-mentioned arbitrary sudden change in the core promoter that the invention provides HBV, provides utilization this genotypic resistance Forecasting Methodology.The present invention also provides the genotypic method of HBV of identifying.
The method of evaluation of the present invention (detecting) transgenation comprises the method for definite kernel acid sequence.Also comprising with nucleic acid makes nucleic acid form the method for two strands (hybridization) mutually as probe or primer.
The present invention also is provided for detecting diagnostic reagent, the diagnostic kit of the transgenation in above-mentioned HBV core promoter zone.
The invention still further relates to the novel HBV curative of the nucleic acid that utilizes sequence numbering 1 with HBV core promoter or its a part of gene order.This nucleic acid is as decoy nucleic acid, makes the wild strain gene order of HBV core promoter be incorporated into protein as target specifically, and hindering original target is core promoter and this protein bound of HBV self, thereby has antagonistic action.Therefore,, can suppress transcribing of HBV core promoter, thereby suppress the propagation of HBV by giving body with decoy nucleic acid of the present invention.
About the core promoter zone of HBV, each reports more or less difference, will transcribe basically the beginning position the about 200bp in upstream as the core promoter zone identify (Yuh CH, etc., Journal ofVirology, 66,4073-4084,1992; Honigwachs J etc., Journal of Virology, 63.919-924,1989; Lopez-Cabrer M etc., Proceedings of the national Academyof Sciences of the United States of America, 87,5069-5073,1990).
The present invention represents first T of the limiting enzyme EcoRI position GAATTC of the double-stranded DNA of HBV the numbering of base sequence for convenience of explanation as nucleic acid numbering 1.When using this easy numbering, the zoning of the said HBV core promoter of the present invention is the nucleic acid numbering 1900 from nucleic acid numbering 1631 to the core protein initial amino acid.
This zone exists breeding very important enhanser II zone (Enh II) and pregenome RNA being wrapped into necessary encapsidation signal (ε) zone in the core particle, become the archaeal dna polymerase of HBV and the bound fraction of liver cell specificity nuclear intrinsic factor, in case therefore think and undergo mutation, can make viral propagation, duplicate the generation important change.This ε zone for the initiation of the archaeal dna polymerase of HBV and reverse transcription activity be essential (Urban M. waits Journal of General Virology (1998), 79,1121-2231).In addition, exist the regulatory element of the footprint II that identifies with footprinting (footprinting) or methylation interference analysis (methylation interference) or Box α, footprint I, Box β in enhanser II zone (Enh II).
The present invention is conceived to the zone in conjunction with polysaccharase etc., and to the enhanser zone that the adjusting that virogene duplicates plays an important role, and identifies the transgenation to the curative of hepatitis B, the core promoter that especially curative effect of lamivudine exerted an influence.
Detect diagnosing the transgenation of useful HBV core promoter before chronic hepatitis B patient's the treatment according to the present invention, can make contributions to persister and the few テ イ ラ one メ one De medical treatment of hepatitis recurrence, and can develop new diagnostic reagent.In addition, owing to can identify the virus that is difficult to obtain curative effect with reverse transcription inhibitor such as lamivudines, therefore by using such mutated viruses sequence can be developed also higher curative effect to this viroid novel anti HBV medicine.
The present invention is specially and comprises the nucleotide sequence of confirming the core promoter zone and the method for identifying the transgenation of HBV.This zone is important position to virus multiplication, because the difference of gene order, for the susceptibility difference of the medicine that is used for the HBV treatment, present inventor's research inference has proved this relation.Find the zone by confirming 6 weak points in the core promoter zone in this research process and be the variation of wherein specific nucleotide sequence, can discern the chemical sproof difference of HBV easily.
Below, be example explanation the present invention with representative HBV curative lamivudine, but the present invention is not limited in the treatment of lamivudine.
The representational curative lamivudine therapy of apparatus HBV, the mark of the HBV-DNA of virus quantity amount and reflection liver cell inner virus amount is the case that the height of HBe antigen amount is tired and existed a lot of virus quantities to be difficult to reduce in the case in showing blood, is much occurring the problem of persister and hepatitis recurrence thereafter.
Yet, have also in the height of HBV-DNA amount and HBe antigen amount is tired case that lamivudine proves effective, a HBV-DNA amount case of minimizing, staticization of hepatitis rapidly in the blood.
The present inventor is the serum after 6 months with preceding serum of the chronic hepatitis B patient's who accepts lamivudine therapy treatment and treatment beginning, the HBV-DNA that measures in the blood with the TMA method measures, in treatment when HBV-DNA amount drops to (below the 5000 copy/ml) below the detection limit after 6 months, be judged to be the negative group of virus, when detection limit is above,, seek prognostic factor by significance of difference calibrating as the virus-positive group.
Then, method (Journal of Medical Virology by six POLOs etc., 62,471-478,2000), extract HBV-DNA with commercially available nucleic acid extraction kit serum sample before the treatment of gathering, make the zone amplification of HBV core promoter with the PCR method, in 3% sepharose, carry out electrophoresis after, from gel, cut out positive band, refining nucleic acid is determined base sequence with direct sequencing.The base sequence in the core promoter zone before will treating again and wild strain are relatively, select the position of producer sudden change, the virus-positive group for the treatment of after 6 months is carried out the significance of difference calibrating of index with negative group, analyze the transgenation position of measurable lamivudine curative effect.
Consequently, the HBe antigen positive of report, HBV-DNA amount can be used as significant prognostic markers before finding, but age, sex, medical history, sick type, thrombocyte value, albumin value, ALT value, bilirubin value were not seen significant difference in the past.Analyze the transgenation in core promoter zone, detect the transgenation that significant correlation is arranged with the lamivudine therapy effect on 3 positions.
And, when the transgenation in this HBV core promoter zone has one at least, find that also there were significant differences (P＜0.0001) between virus-positive group after 6 months and the negative group, prove by confirming that this sudden change can predict the curative effect of lamivudine.Can think that it is the HBV of different genotype that the HBV of this transgenation and the HBV of this sudden change are not arranged.This genotypic difference may with the biology functional correlations such as multiplication capacity of HBV.
Like this, the sudden change of having illustrated HBV core promoter of the present invention zone is relevant with the inhibition effect of breeding, duplicating with the archaeal dna polymerase of the HBV HBV relevant with liver cell specificity nuclear intrinsic factor, be tested and appraised this variation, can impel the efficient raising of lamivudine therapy.Thereby the transgenation in the core promoter zone of HBV can be used as the predictive marker of lamivudine therapy effect.
The working of an invention mode
The evaluation of transgenation of the present invention (detecting) method, the nucleotide sequence in core promoter zone that for example can be by determining HBV carries out.The method of definite kernel acid sequence is used the improved method of Maxam and Gilbert method or dideoxy method basically.Its detailed explanation is seen " Molecular Cloning:A Laboratory Manual, ThirdEdition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) " or " neonatology experiment lecture 2 nucleic acid II structure and character, p.59-100 ".In addition, all ingredients box is sold by many manufacturers, also can be used.
The DNA that the definite kernel acid sequence is used can make core promoter of the present invention zone amplification with PCR, directly uses direct sequencing definite kernel acid sequence.Determine sequence after also PCR product one time cloning can being advanced carrier.Perhaps can use the sequencing of finding point mutation.
The method for detecting of the sudden change of nucleotide sequence of the present invention except that determining gene order, also can use to detect a different any method to several bases.For example, can utilize the method that detects of transgenations such as SSCP, DGGE, CFLP, RFLP.In addition, also can utilize the invader method that detects of the SNP that is used for a base mutation.In addition, the sudden change that utilizes complementary nucleic acid to form double-stranded hybridization principle detects method and also can be used.The method that detects of these genes, nucleic acid mutation has the method for using probe or primer or the method that both are made up.These probes or primer are not limited to nucleic acid, comprise passing through hybridization and bonded probe or primer with target nucleic acid.
Following embodiment is an illustration of the present invention, but scope of the present invention is not limited to these.
Embodiment
＜embodiment 1〉with the turn out cloudy searching of relevant prognostic factor of lamivudine therapy and virus
Give the oral day after day lamivudine of chronic hepatitis B infected patient 53 people (male sex's 40 examples, women's 13 examples, 49.8 ± 9.4 years old mean age, chronic hepatitis 38 examples, liver cirrhosis 15 examples), every day, 100mg analyzed preceding data of administration and the data after the administration.Take lamivudine in the time of 6 months, when the HBV-DNA that measures with the TMA method is brought down below sensitivity (3.7LGE/ml is following), is judged to be virus and turns out cloudy.
For virus cloudy change and still positive patient's age, sex, HBeAg positive rate, treatment before HBV-DNA amount, medical history, sick type, thrombocyte value, albumin, ALT, bilirubin value etc. in the past, with the direct method of Fisher, the significance of Mann-Mhitney U checking method calibrating difference, inquire into the predictor whether it can become curative effect.
Table 1
| The group of turning out cloudy (n=40) | Positive group (n=13) | P | |
| Age | 49.1±9.3 | 52.0±9.9 | 0.3007 |
| The sex male sex (%) | 31(77.5) | 9(69.2) | 0.7119 |
| The HBeAg positive (%) | 14(35.0) | 12(92.3) | 0.0003 |
| HBV DNA(LGE/mL) | 5.9±1.3 | 7.8±0.8 | ＜0.0001 |
HBe antigen positive, the HBV-DNA amount of report can be used as significant prognostic markers before finding out according to table 1, and age, sex, medical history, sick type, thrombocyte value, albumin value, ALT value, bilirubin value were not seen significant difference in the past.
＜embodiment 2〉determine the base sequence in HBV core promoter zone
Carry out in 53 examples of lamivudine therapy at embodiment 1, before the treatment of 43 examples, extract virus N DA with DNA extraction test kit (ス マ イ テ ス ト, (strain) ゲ ノ system サ イ エ Application ス institute) by operation instruction the patients serum 50 μ l.
Reaction solution (200 μ M dNTP, 50mM KCl, 10mM TrisHCl[pH 8.3], 2.0mM MgCl
20.001% gelatin) the AmpliTaq Gold (PE appliedBiosystems) of DNA 2 μ l, the 0.5U that adding is extracted among the 25 μ l, each 0.25mM increases with following primer, that is is 2:5 '-GAG ACC ACC GTG ACCGCC CA (1611-1630) and CB4:5 '-AAA AGA GAG TAA CTC CAC AG (1954-1935).
The PCR scheme is to carry out 94 ℃ of 30 seconds, 55 ℃ circulations of 1 minute after preheating, repeats 43 circulations.Thermal cycler use DNA Engine (MJ Research Corp.Watertown, MA).The PCR negative sample is after carrying out the PCR first time with outer primer set, and PCR repeats 30 circulations for the second time.The sample of this PCR feminine gender is after using es2:5 '-ACG TCG CAT GGA GAC CAC CG (1601-1620), CB2:5 '-GGAAAG AAG TCA GAA GGC AA (1974-1955) to carry out the PCR first time as outer primer set, repeat 30 circulations with is2 and these two primers of CB4, carry out the PCR second time.
This PCR product cuts out band from gel after carrying out electrophoresis with 3% sepharose, and is refining with GFX PCR DNA andGel Band Purification Kit (Amersham Pharmacia Biotec Inc).Dideoxy method is then adopted in order-checking, makes it reaction with Taq Dye Deoxy Terminator cycle sequencing kit, analyzes with fluorescent DNA sequencer (Applied Biosystems).43 routine patients' of sequence core promoter regional sequence such as Fig. 1-1～Fig. 1-3 expression have been determined.
H5 to 262 totally 31 samples be through lamivudine therapy after 6 months in the blood DNA amount reduce to the sequence of the patient below the detection limit.A17 to A5 is totally 11 sequences that sample is the patient that DNA does not reduce to below the detection limit, the curative effect of lamivudine is little.
The sequence as the sequence (wild-type) on basis is shown in each figure tabulation topmost.As seen respectively scheme top 31 samples and following 12 its sequences of sample there are differences.And as seen its difference concentrates on certain zone.Lamivudine is effectively organized in (effectively group), and the zone (sequence numbering 2) that the base numbering is 1671～1682 has than multiple sample and has the sequence different sequence basic with conduct.The A that the nucleic acid T of particularly 1674 (sequence numbering 1 28) becomes C, 1679 (sequence numbering 1 33) becomes G, and lamivudine invalid group (invalid group) is not seen such variation.
In addition, lamivudine is effectively in the group, have in the zone (sequence numbering 3) of 1701～1721 of base numberings than multiple sample have with as the basic different sequence of sequence.The T that the nucleic acid A of particularly 1703 (sequence numbering 1 57) becomes C or G, 1719 (sequence numbering 1 73) becomes G, does not manifest invalid group of result of treatment at lamivudine and does not see such variation.
Equally, in effective group base be numbered 1736～1761 zone (sequence numbering 4) have than multiple sample have with as the basic different sequence of sequence.The nucleic acid A of particularly 1752 (sequence numbering 1 106) becomes T or C or G, and the T of 1753 (sequence numbering 1 107) becomes G or C, does not see such variation at invalid group.
In addition, effectively the zone (sequence numbering 5) of base numbering 1799～1817 has than multiple sample and has the sequence different sequence basic with conduct in the group, occur to change at 1802,1803,1809,1810,1811,1812,1814, and invalid group do not seen such variation.And effectively group has in 1843～1860 zone (sequence numbering 6) than multiple sample and has the sequence different sequence basic with conduct.The nucleic acid A of particularly 1846 (sequence numbering 1 200) becomes T or C, does not see such variation at invalid group.Effectively group has in 1893～1902 zone (sequence numbering 7) than multiple sample and has the sequence different sequence basic with conduct.The G that the nucleic acid G of particularly 1896 (sequence numbering 1 250) becomes A, 1899 (numbering 1 253) becomes A, does not see such variation at invalid group.
＜embodiment 3〉analyze according to the transgenation in core promoter zone
(A) carry out statistical study according to the transgenation position
Press the method for embodiment 1 again, effective patient 9 people and invalid patient's 2 sample is determined the sequence of core promoter region, the curative effect of considering the lamivudine of 53 samples altogether and nucleotide sequence change the relation of (sudden change), and the direct method of usefulness Fisher is examined and determine the significance of difference.Table 2 expression sudden change that there were significant differences and positive group are not seen and the sudden change that occurs in the group of turning out cloudy.
Table 2
| The nucleic acid variation portion | The group of turning out cloudy (n=40) | Positive group (n=13) | P |
| C1653T | 16 | 3 | 0.3340 |
| T1674C | 11 | 0 | 0.0474 |
| A1703G/C | 7 | 0 | 0.1737 |
| T1719G | 8 | 0 | 0.1764 |
| A1752T/C/G | 5 | 0 | 0.3174 |
| T1753C/G | 9 | 0 | 0.0924 |
| A1846T/C | 18 | 0 | 0.0021 |
| G1896A | 18 | 0 | 0.0021 |
| G1899A | 8 | 0 | 0.1764 |
The group (n=40) of turning out cloudy of table 2 is that lamivudine therapy is effectively organized, and positive group (n=13) is invalid group an of lamivudine therapy.Therefore show that significant difference appears in several points.
In addition, for core promoter sudden change (A1762T, G1764A), the group of turning out cloudy, positive component do not have 82.5%, 84.6%, do not see significant difference.
(B) statistical study of the sudden change in whole core promoter zone
Whether can predict the curative effect of lamivudine for sample that the nucleic acid shown in the table 2 changes and the sample of not seeing variation fully occurring, examine and determine the significance of difference with the direct method of Fisher.
Table 3
| A variation does not all have | A variation is arranged | Add up to | |
| Virus-positive after 6 months | 10 | 3 | 13 |
| Negative | 5 | 35 | 40 |
| Add up to | 15 | 38 | 53 |
P＜0.0001
The patient who a certain variation occurs, 53 philtrums have 38 people, and wherein 35 people's lamivudine therapy has effect.And the patient that variation is not seen yet has 15 people, and wherein 10 people do not see curative effect.This variation that shows the nucleotide sequence in this core promoter zone has remarkable related with the lamivudine curative effect.Lamivudine has the effect of the archaeal dna polymerase that suppresses dependenc RNA, can think that its propagation that participates in HBV suppresses.
According to the present invention, by detecting the virogene sudden change before treatment or in the treatment, measurable is the effect of the HRV treatment of representative with the lamivudine, the effect that improves the treatment achievement or can expect control persister and hepatitis recurrence etc.
The simple declaration of accompanying drawing
The comparison of the nucleotide sequence in the patient's of Fig. 1-1 expression carrying out lamivudine therapy HBV core promoter zone.The most above-listed expression is as the sequence on basis, above 31 samples represent the medicable sample of lamivudine, below 12 samples represent not have the sample of result of treatment.
Fig. 1-2 represents to carry out the patient's of lamivudine therapy the comparison of nucleotide sequence in HBV core promoter zone.The most above-listed expression is as the sequence on basis, above 31 samples represent the medicable sample of lamivudine, below 12 samples represent not have the sample of result of treatment.
The comparison of the nucleotide sequence in the patient's of Fig. 1-3 expression carrying out lamivudine therapy HBV core promoter zone.The most above-listed expression is as the sequence on basis, above 31 samples represent the medicable sample of lamivudine, below 12 samples represent not have the sample of result of treatment.
Sequence table
＜110〉Advanced Life Sciences Research Institute Co., Ltd (Advanced Life Science Institute, Inc.)
＜120〉the chemical sproof recognition methods of hepatitis B virus
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gtcttacata agaggactct tggactctca gcaatgtcaa cgaccgacct tgaggcatac 60
ttcaaagact gtttgtttaa agactgggag gagttggggg aggagattag gttaaaggtc 120
tttgtactag gaggctgtag gcataaattg gtctgttcac cagcaccatg caactttttc 180
acctctgcct aatcatctca tgttcatgtc ctactgttca agcctccaag ctgtgccttg 240
ggtggctttg gggcatggac attgacccgt ataaagaatt tggagcttct gtggagtta 299
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ggagttgggg gaggagatta ggttaaa 27
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ctgttcacca gcaccatgc 19
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gctttggggc at 12
Claims (12)
1. the chemical sproof method of identification HBV is characterized in that, the transgenation in the nucleotide sequence in affirmation hepatitis B virus (HBV) core promoter zone.
2. the method for claim 1, its feature is that also the resistance of hepatitis B virus (HBV) is the resistance to the inhibiting medicine of archaeal dna polymerase with dependenc RNA.
3. method as claimed in claim 2, its feature are that also the inhibiting medicine of archaeal dna polymerase with dependenc RNA is a lamivudine.
4. as each described method in the claim 1～3, its feature is that also the nucleotide sequence in core promoter zone is the sequence of sequence numbering 1 record.
5. as each described method in the claim 1～4, its feature also is, the transgenation at least a portion of the partial sequence that is equivalent to the nucleotide sequence shown in sequence numbering 2, sequence numbering 3, sequence numbering 4, sequence numbering 5, numbering 6 or the sequence numbering 7 in the nucleotide sequence in affirmation HBV core promoter zone.
6. as each described method in the claim 1～5, its feature also is, confirms that the transgenation that changes has taken place any or the more than one nucleic acid among 7 nucleic acid C of sequence numbering 1,28 nucleic acid T, 33 nucleic acid A, 57 nucleic acid A, 73 nucleic acid T, 106 nucleic acid A, 107 nucleic acid T, 200 nucleic acid A, 250 nucleic acid G or 253 the nucleic acid G.
7. method as claimed in claim 6, its feature also is, 7 nucleic acid C that confirm sequence numbering 1 record become T, 28 nucleic acid T and become C, 33 nucleic acid A and become G, 57 nucleic acid A and become G or C, 73 nucleic acid T and become G, 106 nucleic acid A and become T or C or G, 107 nucleic acid T and become C or G, 200 nucleic acid A and become T or C, 250 nucleic acid G and become A or 253 nucleic acid G and become any or more than one transgenation among the A.
8. identify the genotypic method of HBV, it is characterized in that the HBV genotype has any or the transgenation of more than one nucleic acid among 7 nucleic acid C of sequence numbering 1 record, 28 nucleic acid T, 33 nucleic acid A, 57 nucleic acid A, 73 nucleic acid T, 106 nucleic acid A, 107 nucleic acid T, 200 nucleic acid A, 250 nucleic acid G or 253 the nucleic acid G.
9. identify the genotypic method of HBV, it is characterized in that 7 nucleic acid C that the HBV genotype has sequence numbering 1 record become T, 28 nucleic acid T and become C, 33 nucleic acid A and become G, 57 nucleic acid A and become G or C, 73 nucleic acid T and become G, 106 nucleic acid A and become T or C or G, 107 nucleic acid T and become C or G, 200 nucleic acid A and become T or C, 250 nucleic acid G and become A or 253 nucleic acid G and become any or more than one transgenation among the A.
10. as each described method in the claim 1～9, its feature also is, carries out the affirmation or the genotypic evaluation of transgenation by the base sequence of determining HBV.
11. as each described method in the claim 1～9, its feature also is, carries out the affirmation or the genotypic evaluation of transgenation by the method for having used probe or primer.
12.HBV curative is characterized in that, has used the nucleic acid of being made up of each described nucleotide sequence in the sequence numbering 1～7 or its part.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP359372/2002 | 2002-12-11 | ||
| JP2002359372 | 2002-12-11 |
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| CN1726287A true CN1726287A (en) | 2006-01-25 |
| CN100422343C CN100422343C (en) | 2008-10-01 |
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| CNB2003801059163A Expired - Fee Related CN100422343C (en) | 2002-12-11 | 2003-12-10 | Method of distinguishing drug-resistance of hepatitis b virus |
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| Country | Link |
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| JP (1) | JP4398379B2 (en) |
| KR (1) | KR20050095584A (en) |
| CN (1) | CN100422343C (en) |
| AU (1) | AU2003289016A1 (en) |
| WO (1) | WO2004053162A1 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880731A (en) * | 2009-05-06 | 2010-11-10 | 香港中文大学 | The multiple detection of hepatitis virus variations |
| CN101177715B (en) * | 2006-11-09 | 2010-12-29 | 中山大学达安基因股份有限公司 | Method and reagent case for detecting hepatitis b virus anterior C/BCP region gene mutation |
| CN101948834A (en) * | 2010-09-16 | 2011-01-19 | 山东大学 | siRNA for treating HBV |
| CN101812537B (en) * | 2009-10-27 | 2012-08-08 | 重庆医科大学 | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus |
| CN113645998A (en) * | 2019-03-26 | 2021-11-12 | 富士胶片株式会社 | Pharmaceutical composition for inhibiting hepatitis B virus protein production and screening method |
-
2003
- 2003-12-10 AU AU2003289016A patent/AU2003289016A1/en not_active Abandoned
- 2003-12-10 WO PCT/JP2003/015810 patent/WO2004053162A1/en active Application Filing
- 2003-12-10 KR KR1020057009350A patent/KR20050095584A/en not_active Application Discontinuation
- 2003-12-10 JP JP2004558464A patent/JP4398379B2/en not_active Expired - Fee Related
- 2003-12-10 CN CNB2003801059163A patent/CN100422343C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177715B (en) * | 2006-11-09 | 2010-12-29 | 中山大学达安基因股份有限公司 | Method and reagent case for detecting hepatitis b virus anterior C/BCP region gene mutation |
| CN101880731A (en) * | 2009-05-06 | 2010-11-10 | 香港中文大学 | The multiple detection of hepatitis virus variations |
| CN101880731B (en) * | 2009-05-06 | 2013-08-07 | 香港中文大学 | Multiplex detection of hepatitis virus variations |
| CN101812537B (en) * | 2009-10-27 | 2012-08-08 | 重庆医科大学 | Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus |
| CN101948834A (en) * | 2010-09-16 | 2011-01-19 | 山东大学 | siRNA for treating HBV |
| CN113645998A (en) * | 2019-03-26 | 2021-11-12 | 富士胶片株式会社 | Pharmaceutical composition for inhibiting hepatitis B virus protein production and screening method |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20050095584A (en) | 2005-09-29 |
| CN100422343C (en) | 2008-10-01 |
| AU2003289016A1 (en) | 2004-06-30 |
| WO2004053162A1 (en) | 2004-06-24 |
| JPWO2004053162A1 (en) | 2006-04-13 |
| JP4398379B2 (en) | 2010-01-13 |
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