CN1332804A

CN1332804A - Means and methods for monitoring nucleoside reverse transcriptase inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of HIV/AIDS

Info

Publication number: CN1332804A
Application number: CN 99813479
Authority: CN
Inventors: 赫里斯托斯·J·彼得罗普洛斯; 珍尼特·惠特科姆
Original assignee: Virologic Inc
Current assignee: Monogram Biosciences Inc
Priority date: 1998-06-24
Filing date: 1999-06-24
Publication date: 2002-01-23
Anticipated expiration: 2019-06-24
Also published as: BR9912209A; WO1999067427A1; EP1015642A4; MXPA00012843A; JP2003503004A; WO1999067427A9; AU769927B2; AU4961199A; EP1015642A1; CA2341679A1; NZ508981A; CN1154747C

Abstract

This invention relates to antiviral drug susceptibility and resistance tests to be used in identifying effective drug regimens for the treatment of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome (AIDS) and further relates to the means and methods of monitoring the clinical progression of HIV infection and its response to antiretroviral therapy, particularly nucleoside reverse transcriptase inhibitor therapy using phenotypic susceptibility assays or genotypic assays.

Description

Monitoring nucleoside reverse transcriptase inhibitor antiretroviral therapy and the approach and the method that determine in the treatment of treatment HIV/AIDS middle finger training

The application is the part continuation application of the U. S. application submitted on June 24th, 1998 number 09/104,295, and its content is incorporated herein by reference.

All kinds of in this application reference mark in bracket, and these documents are incorporated herein by reference with its complete form, more fully to describe the technology contents that the present invention relates to.

The present invention relates to antiretroviral drugs susceptibility and resistant proof, test is used for identifying that treatment human immunodeficiency virus (HIV) infects and the active drug preparation of acquired immune deficiency syndrome (AIDS) (AIDS).The invention further relates to phenotype and genotype sensitivity analysis method and monitor clinical HIV progression of infection and the reaction of antiretroviral therapy.The invention still further relates to the novel carriers, host cell and the component that are used to implement the phenotype sensitivity test.The invention further relates to and use range gene type method to differentiate the method that specific antiretroviral drugs preparation is produced the patient of resistance because of infection.The invention still further relates to the antiretroviral medicine of ability screening candidate that suppresses virus, the virus sequence of selecting and/or viral protein according to it.Present invention is specifically related to and use phenotype sensitivity test and/or genotype test to determine efabirenz.

The HIV infection character is to upgrade in the virus of pathogenic process high frequency, finally causes the extinction of cd4 cell and PD to worsen.Wei X, Ghosh SK, Taylor ME waits (1995) Nature343,117-122, and Ho DD, Naumann AU, Perelson AS waits (1995) Nature373,123-126.The purpose of antiretroviral therapy is to obtain substantive and suppress virus replication constantly, and the control virus that obtains to continue comprises the treatment of using series, and each treatment comprises the combination of 3 kinds or more antiretroviral drugs generally.Therefore select beginning and follow-up treatment should be based upon on the basis of the rational faculty that antagonism and crossed resistance pattern fully understand, this is most important for instructing treatment to determine.The ultimate principle of combined therapy relates to Synergist S-421 95 or additive activity to suppress virus replication to a greater extent.Yet the tolerance of pharmaceutical preparation is with most important, because treatment needs to continue many years.

Produce about 10 every day in the patient body who does not receive treatment ¹⁰New virion adds that hiv reverse transcriptase (RT) does not have the exonuclease correct functioning, can not proofread and correct transcription error, and this high-caliber virus is upgraded and caused having 10 every day in genomic each position of HIV ⁴To 10 ⁵Inferior sudden change, the result forms genomic extensive sudden change rapidly.In view of some template position or base pair replace (the Mansky LM that is more prone to make a mistake, Temin HM (1995) J Virol 69,5087-5094) (Schinazi RF, Lloyd RM, Ramanathan CS waits (1994) Antimicrob AgentsChemother 38,268-274), mathematical model is presented in the individuality of infection each possible single site, and many sudden changes to 10,000 times may take place every day.

The resistance of reverse transcription disease cytotoxic drug if create antagonism, the target enzyme must change under the condition that keeps its function when inhibitor existed.The point mutation that causes single amino acids to replace may cause the change of enzyme shape, size, the perhaps change of reactive site, substrate binding site or neighboring area.Detecting the sudden change that the antiretroviral preparation is had resistance on low-level before the initial therapy.(Mohri H, Singh MK, Ching WTW, Deng (1993) Proc Natl Acad Sci USA90,25-29) (Najera I, Richman DD, Olivares I, Deng (1994) AIDS Res HumRetroviruses 10,1479-1488) (Najera I, Holguin A, Quinones-Mateu E, Deng (1995) J Virol 69,23-31).Yet these mutant strains only account for the sub-fraction of whole virus quantity, and compare to be in wild-type virus and duplicate and compete inferior position.(Coffin?JM(1995)Science?267，483-489)。The selective pressure that antiretroviral therapy produces provides competitive edge for these anti-medicine mutant strains, they become advantage quasispecies (Frost SDW.McLeanAR (1994) AIDS 8 gradually like this, 323-332) (Kellam P, Boucher CAB, Tinagal JMGH (1994) J Gen Virol 75,341-351), finally cause anti-medicine and antiviral failure in the patient body.

Efabirenz

Hiv reverse transcriptase inhibitor zidovudine (Ruide furan pyridine) (ZVD:Retrovir, Glaxo Wellcome, Uxbridge that 7 kinds of nucleoside analogues are arranged at present, UK Britain), zalcitabine (Sai Xitai also) (ddC:HIVID, Hoffman-LaRoche, Basle, Switzerland Switzerland), didanosine (Da Nuosen) (ddI:Videx, Bristol-Myers Squibb, Syracuse, NY, the USA U.S.), stavudine (this too furan pyridine) (d4T:Zerit, Bristol-Myers Squibb, Syracuse, NY, the USA U.S.), and lamivudine (drawing the pyridine of miaow furan) (3TC, and abacavir (Ai Bokai furan) (ABC, Ziagen Epivir),, Glaxo Wellcome), (ADV, Preveon's adefovir (Ai Defo furan) GileadSciences) get the Green Light at US and European.In addition, 3 kinds of non-nucleoside reverse transcriptase inhibitors (NNRTIs) Viramunes (nevirapine) (viramune, Boehringer Ingelheim, Ingelheim am Rhein, Germany Germany), ground draws furan pyridine (delavirdine) (Rescriptor, Pharmacia ﹠amp; Upjohn, Kalamazoo, MI, the USA U.S.) and Chinese mugwort send out a furan auspicious now (efavirenz) (EPV) and get the Green Light in the U.S..These all preparations have all shown antiviral activity at least in a short time, therefore, naturally they can apply selective pressure to HIV, has produced anti-medicine mutant strain in therapeutic process.Though these medicines generally are used for combination preparation, many resistances that obtain are studied data from I/II phase single therapy.Viewed sudden change perhaps can not accurately reflect the resistance that sudden change causes in the single therapy process, because sudden change is to produce in the presence of the selective pressure of several medicaments, they act on identical site and same gene.

Novel mutation

Though set up in the research work in vitro and in vivo and cause that reverse transcriptase inhibitors (RTIs) is had the relevant genotype catastrophic model of phenotype resistance, other resistant mutation that produces often in clinical study seldom has report.Identify 62,75,77,116 and 151 individualities that the aminoacid replacement unique pattern takes place of codon in the patient who accepts to add with ZDV the combined therapy that ddI or ddC prolong: these individualities all have resistance to 2 kinds of medicines, (this too furan pyridine) has crossed resistance to stavudine, and 3TC is had the part crossed resistance.There do not have consistent genotype to change to be relevant with phenotype d4T resistance, or really relevant with the forfeiture of this medicament viral therapy effect.

The relevant sudden change of reversed transcriptive enzyme (RT) inhibitor with nucleoside analogues

Zidovudine (Ruide furan pyridine)

The HIV variant that ZDV susceptibility is reduced reported first in 1989; The concentration that the some of them variant will suppress 50% of virus replication and need ZDV increase more than 100 times (Larder BA.Darby G, Richman DD (1989) Science 243,1731-1734).The ZDV resistant phenotype show in vivo quite stable, treatment stops can also to detect resistance virus sometimes after 1 year, (BoucherCA, O ' Sullivan E, (1992) J Infect Disease 165 such as Mulder JW, 105-110), and with whether use didanosine (Da Nuosen) treatment irrelevant (Smith MS, Koerber KL, Pagano JS, (1994) J Infect Disease 169,184-188).

The hiv reverse transcriptase nucleotide sequencing has been found the sudden change of many energy influence viruses to ZDV susceptibility, it can be used as genotype mark (Kellam P.Boucher CAB, (1994) J Gen Virol 75 such as Tinagal JMGH, 341-351) (Boucher CAB when the ZDV resistance exists, Tersmette M, Lange JMA waits (1990) Lancet 336,585-590) (Lopez-GalindezC, Rojas JM, Najera R waits (1991) PNAS 88,4280-4284).Have a series of sudden changes that resistance level increases sequentially to have occurred, these sudden changes that occur in proper order reduce relevant with virus to ZDV susceptibility incremental.(the same) (Larder BA, Kellam P.Kemp SD, (1991) AIDS 5,137-144).The replacement of codon 70 (arginine 70-Methionin) may moment dominance, failure plays a crucial role that (Stilianakis NI waits (1996) PNAS 93,9501-9506) for DeJong MD, Weenstra J for treatment virus in the ZDV single therapy.Continuation is selected further sudden change with the ZDV treatment at 215 codons, this is a comparatively stable sudden change, the replacement of Threonine 215-Methionin and Threonine 215-phenylalanine takes place in existing report sudden change, and may there be (Mayers DL simultaneously, McCutchan FE, Sanders-Buell EE waits (1992) J Acq Imm DefSynd 5,749-759).The virus that additional mutations then may occur, modal is that 41 bit codons replace (methionine(Met) 41-leucine), follow in 67 bit codons (aspartic acid 67-l-asparagine) and 219 bit codons (Methionin 219-glutamine) additional mutations, or reappear 70 bit codon mutations (the same).

Rite-directed mutagenesis has been used to estimate the interaction (the same) that causes different sudden changes.Experiment shows ZDV high level resistance (IC ₅₀＞1uM) typically relevant with the existence of multisite mutation.Though sudden change often has synergy, but also has antagonistic action, for example, observed 74 bit codon mutations (leucine 74-Xie Ansuan) have been reported in external ZDV215 is suddenlyd change in ddI and ddC treatment antagonistic action, resistance level (St Clair MH, Martin JI, Tudor-Williams F have been reduced to ZDV, Deng (1991) Science 253,1557-1559).181 password sudden changes that also have report to select by non-nucleoside reverse transcriptase inhibitor, and lamivudine (drawing the pyridine of miaow furan) has antagonistic action (Larder BA external to 215 password sudden changes with 184 bit codon mutations that uncommon ddC and ddI cause, (1992) Antimicrob Agents Chemother 36,2664-2669) (Boucher CAB, Cammack N, Schipper P, et al (1993) Antimicrob AgentsChemother 37,2231-2234) (Tisdale M, Kemp SD, Parry NR, Deng (1993) PNAS 90,5653-5656) (Larder BA, Kemp SD, Harrigan PR (1995) Science269,696-699) (Zhang D, Cliendo AM, Eron JJ, Deng (1994) AntimicrobAgents Chemother 38,282-287).Yet, in the combination therapy process, can observe novel mutation pattern or extra " compensatory " sudden change in vivo, this has just impelled dual or multiple drug resistance (as follows).

There is the virus strain of resistance to show to ZDV to including 3 '-azido group as 3 '-nitrine-2 ', crossed resistance (the Rooke R of other nucleoside analog of the auspicious pyridines of 3 ' two deoxidations (AZU), ParniakAM, Tremblay M, Deng (1991) Antimicrob Agents Chermother 35,988-991).Research group reported a HIV laboratory strains and clinical separation strain to lack 3 '-the thymus pyrimidine analogue stavudine (this too furan pyridine) of nitrine part has crossed resistance (the same).Most researchers finds to select in the ZDV single therapy sudden change can not influence susceptibility (the Rooke R of ddI, ddC and 3TC, Tremblay M, Soudeyns H waits (1989) AIDS 3,411-415) (id) (Larder BA, Chesebro B, Richman DD (1990) Antimicrob Agents Chemother34,436-441) (id) (Dimitrov DH, Hollinger FB, Baker CJ waits (1993) J InfectDisease 167,818-823).Yet, almost be not reported in the resistance (id) (Japour AJ, the Chatis PA that continue to produce after ZDV treats to ddI, Eigenrauch HA, Deng (1991) PNAS 88,3092-3096), there is one to report in the clinical separation strain to 10 times of the every reductions of the susceptibility of ZDV, to 2.2 times of the corresponding minimizings of susceptibility of ddI, susceptibility to ddC reduces by 2 times of (Mayers DL, Japour AJ, Arduino JM, Deng (1994) Antimicrob Agent Chemother 38,307-314).And, the possibility that obtains RNA reaction than those patients at the baseline wild-type virus baseline has the patient of resistance to add ddC and ddI to ADV after obviously reduces (Holodniy M, Katzenstein D, Mole L, Deng (1996) J Infect Disease 174,854-857).

Zalcitabine (Sai Xitai also) and Didanosine (Da Nuosen)

The ddI resistance suddenlyd change by leucine 74-Xie Ansuan regulate, sudden change has produced the reduction of 6 times to 26 times of susceptibility, but has partly recovered susceptibility to ZDV by external antagonism 215 bit codon mutations.This sudden change also causes the susceptibility of ddC is reduced about 10 times.Existing be reported in one group in totally 64 people's the investigation, the mutation frequency of 74 bit codons is increased to 56% of 24 whens week from 0 of begin treatment, these patient's cd4 cell average quantity baselines are 129/mm, accept the ZDV treatment in the past and then accepted ddI treatment (Kozal MJ, Kroodsma K, Winterrs MA waits (1994) Annals Intern Med 121,263-268).Equally, in the research of ACTG143, accepting the ddI single therapy, the cd4 cell number being received treatment first and accepting in patient's the population mixture of ZDV treatment 200-500/mm's, there are 17 people to undergo mutation in 26 individualities in the time of 1 year at 74 bit codons, in this research, accept among the patient of ZDV/ddI combination therapy 55 people and have only 2 people undergo mutation at 74 bit codons (Shafer RW, Iversen AKN, Winters MA, Deng (1995) JInfect Disease 172,70-78).

From the several patients that accept ddI and ddC treatment for a long time, isolated virus, the IC of this and ddI at 65 bit codon mutations (Methionin 65-arginine) ₅₀3 times to 5 times increases, the minimizing that ddC susceptibility is 5 times to 10 times and to the rare passes of 3TC susceptibility 20 demultiplications (id) (Fang H waits (1994) Antimicrob Agents Chemother 38,275-281) for Gu Z, Gao Q.69 bit codon mutations (Threonine 69-aspartic acid) are that external ddC selects modal sudden change, it causes 5 times of ddC susceptibility reductions but does not cause other nucleoside analog crossed resistance (id) (Fitzgibbon JE, Howell RM, Haberzettl CA, Deng (1992) Antimicrob Agents Chemother 36,153-157).Summarize the formation of ddC resistance is existing recently (Craig C, Moyle G (1997) AIDS11,271-279).

Combination therapy-zidovudine (Ruide furan pyridine)+zalcitabine (Sai Xitai also) or didanosine (Da Nuosen)

May influence the probability that resistance occurs with ZDV/ddC or ZDV/ddI combination therapy, and may be suppressed at more observed sudden changes in the single therapy, but can cause the appearance (therefore may be more harmful) of novel mutation pattern.

The novel mutation pattern may appear in combination therapy.Identified the virus strain of aminoacid replacement unique pattern in accept continuing to add with ZDV the patient of ddI or ZDV/ddC combination therapy once in a while at 62,75,77,116 and 151 bit codons: these viruses all have resistance (id) (Shafer RW to two kinds of medicines, Kozal MJ, Winter MA, Deng (1994) J Infect Disease 169,722-729) (Shirasaka T, Kavlick MF, Ueno T, Deng (1995) PNAS 92,2398-2402), and make that (this too furan pyridine) has crossed resistance and 3TC is had the part crossed resistance to stavudine.The probability of ddU changes from 0 to＞10% (ibid) in treating greater than the patient in 1 year with ZDV.The sudden change of being selected by 3TC (184 Xie Ansuan) and Viramune (nevirapine) (181 halfcystine) may be added to (Shafer RW in this background external, Winters MA, IversenAKN, Deng (1996) Antimicrob Agents Chemother 40,2887-2890), external existing report 184 Xie Ansuans and 103 aspartic acids sudden change (loviride resistance) (Schmit JC, CogniauxJ, Hermans P waits (1996) J Infect Disease 174,962-968).These virus mutants duplicate advantage when medicine exists, detecting at single therapy perhaps can not be relevant with those advantage mutant strain competitions with them less than the possible cause of these novel mutations.

Lamivudine (drawing the pyridine of miaow furan)

In single therapy and combination therapy observed in the body 184 bit codons replace (id) (Kuritzkes DR, Quinn JB, Benoit SL waits (1996) AIDS 10,975-981) (BartlettJA, Benoit SL, Johnson VA waits (1996) Annal Intern Med 125,161-172) (Eron JJ, Benoit SL, Jemsek J waits (1995) NEJM 333,1662-1669) (Katlama C, Ingrand K, Loveday C waits (1996) JAMA 276,118-125) (Staszewski S, Loveday C, Picazo JJ waits (1996) JAMA 276,111-117), resistance (modal is methionine(Met) 184-Xie Ansuan) to 3TC will appear at once, the appearance of this sudden change at least in part with viral therapy failure relevant (id) (Moyle GJ (1996) Drugs 52,168-185) (GouldenMG, Cammack N, Hopewell PL waits (1996) AIDS 10,101-102).This sudden change causes the high-caliber resistance (IC to 3TC ₅₀Increase by 500 times to 1000 times), and to ddI and some crossed resistance of ddC (susceptibility reduces 4 times to 8 times) (id) (LiX waits (1992) J Virol 66,7128-7135) for Gu Z, Gao Q.At the external possibility antagonism ZDV (id) (id) (id) that should suddenly change, although have report that ZDV/3TC is had two resistances at external and clinical separation strain.In addition, may be by way of compensation, for the two resistances of ZDV/3TC, be necessary such as 135 and 333 bit codon mutations, this problem is (id) just under study for action.In NUCA3002 research, when the patient who used ZDV is treated with 3TC, there is 82% people to produce the phenotype that 3TC is had resistance among back 33 patients of 12 weeks.In 10 patients that 3TC had a resistance, (pass through IC at the baseline place of ZDV resistance ₅₀＞0.2nM determines) the existence virus strain responsive more to ZDV in 4 human bodies arranged, and 6 people have two resistances to ZDV/3TC, this explanation body inner virus is not general to the sensitivity once more of ZDV.

Stavudine (this too furan pyridine)

The external HIV that determines by rite-directed mutagenesis selects to identify a sudden change at 75 bit codons to the d4T resistance, and this sudden change makes to the IC of ddI and ddC ₅₀Increase by 7 times, and to its susceptibility reduce (Lacey SF, Larder BA (1994) Antimicrob Agent Chemother 38,1428-1432).The d4T susceptibility that causes 50 bit codon mutations reduces 30 times, but it to external other nucleoside analog of having found do not have crossed resistance (Fank H waits (1994) Leukemia 8 for Gu Z, Gao Q, Suppl.1,166-169).The a series of amino acid changes of existing report comprise 75 bit codon mutations but are not that 50 bit codons replace yet in vivo.Do not accept to have reduced 12 times in the back susceptibility maximum of treatment in 18 to 22 months among the ZDV treatment patient at 13 to d4T, yet, there are 5 people that the susceptibility of ZDV has been reduced 9 times to 176 times, there are 3 people that the susceptibility of ddI has been reduced by 7 times to 29 times (Lin PF, Samanta H, Rose RE waits (1994) JInfect Disease 170,1157-1164), this explanation uses d4T may limit later treatment selection in some patient.Therefore, also do not set up the catastrophic model of the unanimity of d4T resistance.

Abacavir (Ai Bokai furan)

Confirm that by rite-directed mutagenesis the HIV strain shows that in external resistance selection to Abacavir (Ai Bokai furan) individual plant mutant only causes the low-level resistance to Abacavir (Ai Bokai furan), the resistance that produces 10 times needs multisite mutation (at least 3).M184V is external at Abacavir (Ai Bokai furan) modal selection resistant mutation when existing, and causes susceptibility 2-5 reduction doubly.Sudden change in L74V and F115Y position also cause to Abacavir (Ai Bokai furan) susceptibility forfeiture (Tisdale M, Alnadaf T, Cousens D (1997) Antimicrob Agent Chemother 41,1094-1098).Observe ddC and ddI crossed resistance, but d4T or ZDV are not had crossed resistance.From the HIV resistance of patient's virus population owing to the previous sudden change relevant with the efabirenz resistance, the two unites ZDV resistant mutation (M41L, L210W T215Y) and 3TC resistant mutation (M184V) and shows Abacavir (Ai Bokai furan) susceptibility is reduced by 8 times.Multinuclear glycosides resistance mixture (A62V, V75I, F77L, Y116F and Q151M) and susceptibility reduce by 17 times, and relevant (Daluge S waits (1998) 2 for Lanier R, Danehower S ^NdInternational Workshop onHIV Drug Resistance and Treatment Strategies).

Adefovir (Ai Defo furan)

In adefovir (Ai Defo furan) appearance that external selection causes K65R or K70E to suddenly change when existing, this makes adefovir (Ai Defo furan) susceptibility 16-or 9-is doubly reduced.To having reported the K70E sudden change in patient's the research, but do not report the K65R sudden change.The virus of many AZT resistances, 3TC resistance and multi-drug resistance to adefovir (Ai Defo furan) responsive (Mulato AS, Lamy PD, (1998) Antimicrob Agent Chemother 42 such as Miller MD, 1620-1628).

The clinical meaning of resistance

Infect to select beginning and the treatment action subsequently should be firm for HIV, but also should be planned and on the rational basis of understanding resistance and crossed resistance model, carry out, so that there is broad treatment to select in the future.

An object of the present invention is to provide and to illustrate that whether patient body's inner virus population has the drug susceptibility and the resistant proof of resistance to given prescription drug.Another object of the present invention is that patient provides the test that can substitute one or more medicines in the treatment prescription for the doctor when in therapeutic process given one or more medicines being produced resistances.Another purpose of the present invention provides the test that can select the active drug prescription in treatment HIV infection and/or AIDS.Another purpose of the present invention has provided one and has identified that patient has produced the medicine of resistance, particularly identifies the resistance to efabirenz.Another purpose of the present invention provides test and the method for a kind of role of evaluation in the drug candidate preparation biological effectiveness of specific virus, virogene and/or viral protein, particularly relevant with efabirenz viral drug-fast test and method.A further object of the present invention provides method and the composition of estimating HIV antiretroviral drugs resistance and susceptibility, obviously this purpose of the present invention and other purpose technical requirements be do as a whole.

The present invention relates to use phenotype and genotype method monitor para-immunity defective virus progression of infection and it to resist the method for viral therapeutic response.The present invention also is based in part on and has found to make reversed transcriptive enzyme (RT) heritable variation of HIV antagonism viral therapy generation resistance can use phenotype or genotype method directly to determine rapidly by the HIV RNA of patient's blood plasma, these methods are based on using the amplification oligonucleotide analysis, polymerase chain reaction (PCR) for example refers to the analysis of PCR-based here based on the analysis of amplification oligonucleotide.Perhaps, do not use the method for the viral nucleic acid of amplification step in-service evaluation viral protein can make the present invention monitor and/or improve antiretroviral therapy yet.The present invention also is based in part on and has found that hiv reverse transcriptase 69 bit codons suddenly change separately/insert or unite sudden change with 41 and 215 bit codons in the patient of efabirenz (NRTI) treatment, the existence of these sudden changes is with relevant to the reduction of d4T susceptibility, and to AZT, ddC, ddI, the susceptibility of 3TC and abacavir (Ai Bokai furan) reduces relevant, and these sudden changes were found in blood plasma HIV RNA after treatment beginning for some time.We find that hiv reverse transcriptase can be used as the indication that forms resistance and final immunocompromised at additional 41 and 215 bit codon mutations of 69 bit codon mutation/insertions.Particularly reversed transcriptive enzyme is at 69 bit codon mutations/insertion (T69SSA, T69SSG, T69SSS) may with and AZT (M41L for example, L210W, T215Y) relevant with 3TC (M184V) or ddI/ddC (L74V) resistance sudden change is relevant, these sudden changes reduce relevant with efabirenz (NRTI) susceptibility, comprise that d4T susceptibility reduces and to AZT, ddC, ddI, the susceptibility of 3TC and abacavir (Ai Bokai furan) reduces, find that also reversed transcriptive enzyme is at 69 bit codon mutations/insertion (T69SSA, T69SSG, T69SSS) may observe in 69 bit codon mutation/insertions (T69SSG) and 75 bit codon mutations (V75M) relevant with the substantive reduction of AZT susceptibility (30 times) for the first time with relevant to the relevant sudden change of multiple efabirenz resistance with 62 bit codons (for example A62V) and/or 75 bit codon novel mutations (for example V75M) with d4T susceptibility reduction (3 times).The present invention also is based in part on and has found hiv reverse transcriptase 62,75,77,116 and 151 bit codon mutations relevant with multiple efabirenz resistance in the patient of efabirenz (NRTI) treatment, the existence of this sudden change and d4T, ddC, ddI reduces relevant with AZT susceptibility.Find that also sudden change is specifically relevant with resistance: 41,67,210,215 and 219 password sudden changes (M41L for example, D67N, L210W, T215Y is K219Q) with the AZT resistance; 184 password sudden changes (M184V) and 3TC resistance; 69 bit codons (T69D) and ddC resistance; Perhaps 215 bit codon novel mutations (T215V) follow relevant with multiple efabirenz resistance at 62,75,77,116 or 151 bit codon mutations, they and d4T, ddC, ddI reduces relevant with AZT susceptibility.The present invention also is based in part on and has found hiv reverse transcriptase 4 or the more sudden change relevant with the AZT resistance in the patient of ucleotides reverse transcriptase inhibitors treatment, sudden change is from by 41,67,70,210,215 and/or 219 codons (M41L for example, D67N, K70R, L210W, T215Y/F, K219Q) filter out in the cohort of Zu Chenging, their separately sudden change or with 74 (relevant with the ddI resistance-V74I), 69 (relevant with the ddC resistance-T69D), 75 (V75M, V75S) and/or 219 (K219N) codon mutations exist simultaneously, the existence of these sudden changes and d4T susceptibility reduce relevant.These sudden changes were found in blood plasma HIV RNA after treatment beginning for some time.Make up the resistance that is included in 75 bit codon unit points sudden changes (V75I) by rite-directed mutagenesis and detect the susceptibility that carrier finds not change d4T, but increased the susceptibility of AZT, used rite-directed mutagenesis and find that also 151 bit codon unit points sudden changes (Q151M) have reduced d4T and AZT susceptibility.Another discovery of the present invention is to have reduced d4T and AZT susceptibility in 75 and 151 bit codon dibit point mutation (V75I+Q151M), use rite-directed mutagenesis and also find to include 5 (M41L, D67N, K70R, T215Y, K219Q) or 6 (M41L, D67N, K70R, L210W, T215Y, K219Q) resistance of the sudden change relevant with AZT resistance detection carrier shows d4T and AZT susceptibility is reduced, and also finds to include 62, the sudden change of 69 and 75 bit codon unit points (A62V, T69SSA, V75I, resistance V75T) detects carrier does not have the susceptibility of attenuating to d4T.Yet finding slightly increases AZT susceptibility in 75 bit codon unit points sudden changes (V75I) or (V75T), T69SSA unit point sudden change slightly minimizing AZT susceptibility and the A62V sudden change to the not influence of AZT susceptibility.Using rite-directed mutagenesis, to discover that further the resistance that includes in 62 and 69 bit codon dibit point mutation (for example A62V+T69SSA) detects the minimizing amplitude of carrier d4T susceptibility big unlike the T69SSA single mutation, but can further reduce because of the single mutation of the T69SSA susceptibility to AZT.Use the further research of rite-directed mutagenesis and find that also the resistance detection carrier that includes in 62 and 75 bit codon dibit point mutation (for example A62V+V75I) does not influence d4T susceptibility, finds that also the A62V sudden change does not change the reduction to AZT susceptibility that is caused by the V75I sudden change.Use rite-directed mutagenesis and further discover and include the resistance of uniting sudden change (for example A62V+T69SSA+V75I) in 3 sites of 62,69 and 75 bit codons to detect the minimizing amplitude of carrier d4T susceptibility big unlike the T69SSA single mutation, also find to unite in the sudden change (for example A62V+T69SSA+V75I) V75I and suddenly change to have offset fully and unite the reduction that suddenlys change and cause AZT susceptibility by A62V and T69SSA in 3 sites of 62,69 and 75 bit codons.Use rite-directed mutagenesis further research also find to include resistance at 3 site mutations of 41,69 and 215 bit codons (for example M41L+T69SSA+T215Y) and detect carrier and show d4T and AZT susceptibility are significantly reduced.Use rite-directed mutagenesis and discover that further unite sudden change (for example M41L+A62V+T69SSA+T215Y) in 4 sites of 41,62,69 and 215 bit codons bigger than the minimizing amplitudes of T69SSA single mutation or the two sudden changes of T69SSA+A62V to the minimizing amplitude of d4T susceptibility, also find to unite all 4 sudden changes in the sudden change (for example M41L+A62V+T69SSA+T215Y) and unite the attenuating amplitude of AZT susceptibility bigger than the suddenly change reduction amplitude of uniting of M41L and T215Y in 4 sites of 41,62,69 and 215 bit codons.The application rite-directed mutagenesis is further discovered in 5 sites of 41,62,69,184 and 215 bit codons and is united in the sudden change (for example M41L+A62V+T69SSA+M184V+T215Y), the M184V sudden change has been offset by M41L, A62V, the reduction to AZT susceptibility that causes is united in T69SSA and T215Y sudden change.Use rite-directed mutagenesis and find that in another research (T69SSA-SSA69T) replied in the T69SSA sudden change among the clone in patient's virus, the revertant of T69SSA sudden change has reduced the resistance (promptly increasing susceptibility) of d4T, and has reduced the resistance (promptly increasing susceptibility) of AZT.

In the further research of using rite-directed mutagenesis, find that introducing the L210W sudden change in 41,69 and 215 sudden changes (for example M41L+T69SSA+T215Y) causes reducing AZT susceptibility is substantive, and if there are not 210 sudden changes to find that AZT susceptibility only reduces by 140 times.Use the further research of rite-directed mutagenesis and also find to show substantive reduce (greater than 1000 times) of AZT susceptibility, to the only slightly reduction of susceptibility of other efabirenz at 4 site mutations of 41,62,69 and 215 bit codons (for example M41L+A62V+T69SSA+T215Y).In the further research of using rite-directed mutagenesis, discovery is introduced the susceptibility almost not influence of L210W sudden change to medicine in 41,62,69 and 215 sudden changes (for example M41L+A62V+T69SSA+T215Y), and the resistance spectrum that shows resulting resistance spectrum when only having these 4 kinds of sudden changes to exist is similar.In the further research of using rite-directed mutagenesis, discovery is introduced the T215Y sudden change in 62 and 69 sudden changes (for example A62V+T69SSA) and is caused substantive reduce (greater than 1000 times) of AZT susceptibility, and, find that also introducing the L74V sudden change in 62 and 69 sudden changes (for example A62V+T69SSA) causes the susceptibility of AZT is returned to wild-type if there are not 215 sudden changes to find that AZT susceptibility only reduces by 7 times.In the further research of using rite-directed mutagenesis, discovery is introduced the susceptibility almost not influence of V75M sudden change to medicine in 41,69,210 and 215 sudden changes (for example M41L+T69SSA+L210W+T215Y), and the resistance spectrum that shows resulting resistance spectrum when only having these 4 kinds of sudden changes to exist is similar.

In yet another embodiment of the present invention, can use the pcr analysis that comprises phenotype and gene type assay to detect 69 password sudden changes, and other codon mutation that comprises 41 and/or 215 on the hiv reverse transcriptase, these sudden changes reduce relevant with the particular model and the immunizing power subsequently that antiretroviral therapy are produced resistance.More clearly, in yet another embodiment of the present invention, can use the pcr analysis that comprises phenotype and gene type assay to detect 69 password sudden change (T69SSA, T69SSG, T69SSS), and comprise 41 (M41L) on the hiv reverse transcriptase, 210 (L210W), 215 (T215Y), 184 (M184V) and/or 74 (L74V) position are at other interior codon mutation, as described herein-in, these sudden changes reduce relevant with the particular model and the immunizing power subsequently that antiretroviral therapy are produced resistance.In yet another embodiment of the present invention, can use the pcr analysis that comprises phenotype and gene type assay to detect on the hiv reverse transcriptase 62,75,77,116 or 115 bit codons suddenly change separately, or simultaneously with it comprise 41,67,210,215,219,184,69 and/or 215 bit codons are at other interior codon mutation, as described herein-in, these sudden changes are with relevant with the immunizing power reduction to antiretroviral therapy generation resistance, and the codon of the above sudden change comprises, but be not limited only to (A62V, V75I, F77L, F116Y, Q151M) and (M41L, D67N, L210W, T215Y, K219Q, M184V/I, T69D, T215Y).In yet another embodiment of the present invention, can use the pcr analysis that comprises phenotype and gene type assay to detect 4 or more sudden change on the reversed transcriptive enzyme, these sudden change groups comprise 41,67,70,210,215 and/or 219 bit codons (M41L for example, D67N, K70R, L210W, T215Y/F, K219Q) sudden change separately, or simultaneous with it 71 (V74I) that comprise, 69 (T69D), 75 (V75M, V75S) and/or 219 (K219N) bit codon mutation.As described herein-in, these sudden changes are with relevant with the immunizing power reduction to antiretroviral therapy generation resistance.69 passwords suddenly change separately on the hiv reverse transcriptase in case detect in the patient body who accepts the NRTI antiretroviral therapy, or also have 41 and 215 bit codon mutations simultaneously, just must consider to change the medicine prescription.Equally, in case in the patient body who accepts specific NRTI antiretroviral therapy, detect 69 and/or 41,210,215,184 and/or 74 password sudden changes, just must consider to change the medicine prescription.Equally, in case detecting 62,75,77,116 and/or 151 passwords in the patient body who accepts the NRTI antiretroviral therapy suddenlys change separately, or also have simultaneously and AZT, 3TC, sudden change or T215V sudden change that the ddC69 resistance is relevant, just must consider change medicine prescription.Equally, in case in the patient body who accepts specific NRTI antiretroviral therapy, detect 4 or more sudden change from the group that forms by 41,67,70,210,215 and/or 219 passwords, filtering out, or also have 74 (V74I), 69 (T69D), 75 (V75M simultaneously, V75S) and/or 219 (K219N) bit codon mutation, just must consider to change the medicine prescription.Can use the pcr analysis that comprises phenotype and gene type assay to detect 69 bit codon mutations on the hiv reverse transcriptase, Guan Lian other codon mutation that comprises 41 and/or 215 bit codons with it, these sudden changes are relevant with the reduction of immunizing power subsequently with the particular model that antiretroviral therapy is produced resistance.Equally, can use the pcr analysis that comprises phenotype and gene type assay to detect 69 bit codon mutations on the hiv reverse transcriptase, Guan Lian other codon mutation that comprises 41,210,215,184 and/or 74 bit codons with it, these sudden changes are relevant with the reduction of immunizing power subsequently with the particular model that antiretroviral therapy is produced resistance.Can use the pcr analysis that comprises phenotype and gene type assay to detect 69 bit codon mutations on the hiv reverse transcriptase, Guan Lian other codon mutation that comprises 62 and/or 75 bit codons with it, these sudden changes are relevant with the reduction of immunizing power subsequently with the particular model that antiretroviral therapy is produced resistance.Can use the pcr analysis that comprises phenotype and gene type assay to detect 62,75,77,116 and 151 bit codon mutations on the hiv reverse transcriptase, perhaps with related with it other codon mutation that comprises 41,67,210,215,219,184,69 bit codon mutations and/or T215V, these sudden changes reduce relevant with the particular model and the immunizing power subsequently that antiretroviral therapy are produced resistance.After using pcr analysis to estimate antiretroviral therapy, should formulate the time of revising the medicine prescription, it depends on charge capacity, cd4 cell quantity and former historical this Several Factors of treatment that comprises patient's virus.

Another one of the present invention aspect has provided the method for estimating nucleoside reverse transcriptase antiretroviral drugs validity, comprising: the resistance that (a) will include the fragment that comes from patient and indicator detects carrier and imports host cell; (b) cultivation is from the host cell of step (a); (c) detect the expression of indicator in the target host cell, wherein the expression of indicator depends on the fragment that comes from patient; (d) will the two compare from the expression of detected indicator under the expression of step (c) indicator and the situation that does not add the NRTI inverase when implementation step (a)-(c), wherein the experimental concentration of NRTI, inverase is at step (a)-(c); At step (b)-(c); Or in step (c), exist.

The present invention also provides and has estimated the method for patient being carried out the validity of non-nucleoside reverse transcriptase antiretroviral therapy, comprising: (a) draw the drug susceptibility typical curve for the NRTI inverase; (b) use aforesaid sensitivity test to determine the susceptibility of patient NRTI inverase; And (c) typical curve of determining in the susceptibility of NRTI inverase in the step (b) and the step (a) is compared, wherein the anti-HIV susceptibility reduction of NRTI shows the resistance that has formed inverase in the patient body.

The present invention also provides the method for the biological effectiveness of estimating HIV antiretroviral drug candidate medical compounds, comprising: the resistance that (a) will include the fragment that comes from patient and indicator detects carrier and imports host cell; (b) cultivation is from the host cell of (a); (c) detect the expression of indicator in the target host cell, wherein the expression of indicator depends on the fragment that comes from patient; (d) will the two compare from the expression of the detected indicator when not adding antiviral candidate medication medication compound when implementation step (a)-(c) of indication expression of gene in the step (c), the experimental concentration of wherein antiviral candidate medication medication compound is at step (a)-(c): at step (b)-(c); Or in step (c), exist.

The expression that detects indicator on the carrier in resistance in the target cell finally depends on the behavior that comes from patient's fragment sequence, and the function of indicator is not essential.

Another aspect of the present invention is for instructing antiretroviral drugs susceptibility and the resistant proof at HIV/AIDS.Be used for the antiretroviral drugs susceptibility of HIV/AIDS and own evaluation of specific resistance detection carrier of resistant proof among the present invention.

Another aspect of the present invention has provided identifies and estimates method for the biological effectiveness of treatment HIV and/or the potential therapeutic anti retrovirus of AIDS medical compounds.On the other hand, the present invention is directed to a novel resistance and detect carrier, carrier includes a fragment that comes from patient, further includes sudden change and an indicator on the one or more pol genes.

Accompanying drawing is concisely described

Fig. 1

Resistance detects carrier.Include one with the figure signal and come from patient's the fragment and the resistance detection carrier of an indicator.

Fig. 2

Two cell analysis.Schematically show analytical procedure.Detect carrier by making up resistance in fragment cloning to the indicator virus vector that will come from patient, then this resistance is detected carrier and an expression vector cotransfection of expressing amphophilic murine leukemia virus (MLV) coat protein or other virus that can infect or cell protein, the pseudotyped viral particle of Chan Shenging includes proteolytic enzyme (PR) and reversed transcriptive enzyme (RT) gene product that comes from patient's sequence like this, and then collecting granules is used to infect new fresh cell.Use the PR and the RT sequence of defective to show that the activity of luciferase depends on the PR and the RT of function.After the transfection proteinase inhibitor is joined in the cell, in the particle ripening process, just have inhibitor like this.On the other hand, when virion infects or before in cell, add reverse transcriptase inhibitors, the a plurality of processing of medicine that do not add medicine and add different concns in the wide region are established in this analysis, measure the content of luciferase, calculate under the different pharmaceutical test concentrations and suppress per-cent (%).

Fig. 3

The phenotypic drug sensitivity legend.By drawing uciferase activity log10 concentration (um) is suppressed the percentage analysis data.This figure is used to calculate the 50% (IC that suppresses virus replication ₅₀) and 95% (IC ₉₅) required drug level, suppress curve and can be interpreted as drug-fast evidence to higher drug level displacement.Demonstrate a kind of efabirenz (AZT), a kind of non-nucleoside reverse transcriptase inhibitor (draw furan pyridine delavirdine) and these 3 kinds of typical curves of a kind of proteinase inhibitor (ritonavir) among the figure, compare with baseline (before the treatment) sample or control drug susceptible virus, PNL4-3 or the HXB-2 that selects for use when baseline sample be difficult for to obtain for example, the medicaments insensitive linearity curve has reflected that to higher drug level (to the right) displacement drug susceptibility (resistance) reduces.

Fig. 4

D4T resistance patient strain isolated: multiple efabirenz resistant mutation.These 4 kinds of viruses show d4T susceptibility are reduced (4-12 doubly), and comprise and multiple efabirenz resistance (A62V, V75I, F77L, F116Y, Q151M) relevant reversed transcriptive enzyme sudden change.Some viruses also include and AZT (M41L, D67N, L210W, T215Y, K219Q), 3TC (M184V/I) or the relevant specific sudden change of ddC (T69D) resistance, not have the sudden change (T215V) described before perhaps, examined viral sudden change be listed in examined viral figure below.Sudden change in the bracket shows that viral colony is made up of wild-type and mutant strain mixing.Genotype as described herein is a part wherein, and embodiment 3 is seen in the genotypic complete description of patient.

Fig. 5

D4T resistance patient strain isolated: T69SSX sudden change/insertion.These 4 kinds of viruses show d4T susceptibility are reduced (2-10 is doubly), and including on the reversed transcriptive enzyme before, do not have description sudden change/insertion (T69SSA, T69SSG, T69SSS).These viruses also include and AZT (M41L, L210W, T215Y) with 3TC (M184V/I) or the relevant sudden change of ddI/ddC (L74V) resistance, some viruses also include the sudden change (A62V) relevant with multiple efabirenz resistance, and/or not have in the past the sudden change (V75M) described, examined viral sudden change be listed in examined viral figure below.Sudden change in the bracket shows that viral colony is made up of wild-type and mutant strain mixing.Genotype as described herein is a part wherein, and embodiment 4 is seen in the genotypic complete description of patient.

Fig. 6

D4T resistance patient strain isolated: the sudden change relevant with the AZT resistance.These 4 kinds of viruses show d4T susceptibility are reduced (3-6 doubly), and include 4 or more sudden change (M41L, D67N, the K70R relevant with the AZT resistance, L210W, T215Y/F, K219Q), some viruses also include to ddI (V74I), the relevant sudden change of ddC (T69D) resistance, or not have in the past description sudden change (V75M, V75S, L219N).Examined virus sudden change be listed in examined viral figure below.Sudden change in the bracket shows that viral colony is made up of wild-type and mutant strain mixing.Genotype as described herein is a part wherein, and embodiment 4 is seen in the genotypic complete description of patient.

Fig. 7

Rite-directed mutagenesis: with the relevant sudden change of multiple efabirenz resistance.Make up the resistance that comprises unit point (V75I), (Q151M) and dibit point (V75I+Q151M) sudden change by rite-directed mutagenesis and detect carrier, demonstrate the resistance that includes these rite-directed mutagenesises among the figure and detect the phenotype susceptibility of carrier d4T and AZT.Left side hurdle: Q151M sudden change has approximately reduced 3 times to the susceptibility of d4T, and the V75I sudden change does not change the susceptibility to d4T.Right hurdle: Q151M sudden change has approximately reduced 5 times to the susceptibility of AZT, and the V75I sudden change has improved about 2 times to the susceptibility of AZT.

Fig. 8

Rite-directed mutagenesis: the sudden change relevant with the AZT resistance.Comprise 5 (M41L by the rite-directed mutagenesis structure, D67N, K70R, T215Y, K219Q) or 6 (M41L, D67N, K70R, L210W, T215Y, K219Q) resistance of the sudden change relevant with the AZT resistance detects carrier, demonstrates the resistance that comprises these rite-directed mutagenesises among the figure and detects the phenotype susceptibility of carrier to d4T and AZT.Hurdle, a left side: the resistance that comprises 5 or 6 relevant sudden changes with the AZT resistance detects carrier and compares according to resistance detection carrier little about 2 times to the susceptibility of d4T.Right hurdle: the resistance that comprises 5 or 6 relevant sudden changes with the AZT resistance detects carrier the susceptibility comparison of AZT is shone the little about 75-180 of resistance detection carrier doubly.

Fig. 9

Rite-directed mutagenesis: in the single mutation of reversed transcriptive enzyme 62,69 and 75 amino acids.Comprise unit point (A62V, T69SSA, V75I, V75T) resistance of sudden change detection carrier by the rite-directed mutagenesis structure.Demonstrate the resistance that comprises these rite-directed mutagenesises among the figure and detect the phenotype susceptibility of carrier d4T and AZT.Left side hurdle: T69SSA and V75T sudden change do not have big minimizing (less than 2 times) to the susceptibility of d4T, and A62V and V75I sudden change are to the not influence of susceptibility of d4T.Right hurdle: V75I and V75T sudden change have increased the susceptibility (about 2 times) to AZT a little, the susceptibility (about 2 times) of T69SSA sudden change the minimizing a little AZT, and the A62V sudden change is to the not influence of susceptibility of AZT.

Figure 10

Rite-directed mutagenesis: in the multisite mutation of reversed transcriptive enzyme 62,69 and 75 amino acids.Make up the resistance that comprises dibit point (A62V+T69SSA), (A62V+V75I) and three sites (A62V+T69SSA+V75I) sudden change by rite-directed mutagenesis and detect carrier.Demonstrate the resistance that comprises these rite-directed mutagenesises among the figure and detect the phenotype susceptibility of carrier d4T and AZT.Left side hurdle: A62V and T69SSA sudden change are united the reduction degree of d4T susceptibility big unlike independent T69SSA sudden change, yet these 2 sudden changes have reduced about 6 times to the susceptibility of AZT.Middle column: A62V and V75I sudden change are united the not influence of d4T susceptibility, and A62V can not change the reduction level of the AZT susceptibility that is caused by the V75I sudden change.Right hurdle: A62V, T69SSA and V75I sudden change are united the reduction degree of d4T susceptibility big unlike independent T69SSA sudden change, and 6 times that the AZT resistance that caused by A62V and T69SSA sudden change increases have been offset in the V75I sudden change fully.

Figure 11

Patient 285 clone: the T69SSA revertant of fixing a point.The T69SSA that uses rite-directed mutagenesis to reply from the resistance detection carrier molecule clone that patient samples 285 makes suddenlys change.Demonstrate the phenotype susceptibility of parent clone (T69SSA) and revertant clone (SSA69T) among the figure to d4T and AZT.The reverse mutation of left side hurdle: T69SSA sudden change reduces about 3 times to the resistance of d4T.The reverse mutation of right hurdle: T69SSA sudden change reduces about 30 times to the resistance of AZT.

Figure 12

Patient 770 clone :+/-to detect the carrier storehouse be inhomogenous for resistance that T69SSG+V75M. obtains from patient's sample 770, by mutant or do not have T69SSG and the T75M sudden change is formed.Demonstrate the resistance that contains or do not contain these sudden changes among the figure and detect the phenotype susceptibility of carrier d4T and AZT.Hurdle, a left side: the resistance detection carrier cloning that contains T69SSG and T75M sudden change is bigger more than 3 times to the resistance of d4T than the clone who does not have these sudden changes.Right hurdle: the resistance that contains T69SSG and T75M sudden change detects carrier cloning than the clone who does not have these sudden changes to the resistance of AZT about 30 times.

Figure 13

Rite-directed mutagenesis: in the multisite mutation of reversed transcriptive enzyme 41,62,69,184 and 215 amino acids.Make up the resistance that comprises three sites (M41L+T69SSA+T215Y) and four sites (M41L+A62V+T69SSA+T215Y) or five sites (M41L+A62V+T69SSA+M184V+T215Y) sudden change by rite-directed mutagenesis and detect carrier.Demonstrate the resistance that comprises these rite-directed mutagenesises among the figure and detect carrier one group of phenotype susceptibility of totally 6 kinds of efabirenzs (AZT, ddC, DDI, 3TC, d4T and Abaeavir (Ai Bokai furan)).M41L+T69SSA+T215Y significantly lowers all susceptibility for the examination efabirenz (2-150 doubly), adding A62V sudden change causes AZT, d4T and ddI susceptibility are further reduced, but does not influence the susceptibility to 3TC, ddC and Abacavir (Ai Bokai furan).The resistance that contains M41L+A62V+T69SSA+M184V+T215Y detects carrier and detects carrier to AZT than the resistance that contains 4 sudden change M41L+A62V+T69SSA+T215Y, d4T and ddI are responsive more, adding M184V causes 3TC susceptibility is reduced, but does not influence the susceptibility to ddC and Abacavir (Ai Bokai furan).

The present invention relates to monitor the patient of antiretroviral therapy, particularly accept ucleosides The Clinical course that HIV infects in the patient body of RTI ART.

In one embodiment, the invention provides a kind of evaluation patient ART The method of validity comprises: (i) gather biological sample from the patient that HIV infects; (ii) true Decide the nucleotides whether biological sample includes the hiv reverse transcriptase of encoding, have one at reverse transcriptase Individual or a plurality of mutational sites, these sudden changes really change with the sensitiveness/resistance of phenotype are relevant. In the specific embodiment, the invention provides a kind of evaluation patient NRTI anti-reverse transcription disease The method of poison treatment validity comprises: (i) gather biological sample from the patient that HIV infects; (ii) determine whether biological sample includes the HIV reverse transcription that is coded in 69 bit codon mutations The nucleotides of enzyme uses phenotype sensitivity analysis the present invention to confirm that hiv reverse transcriptase is at 69 Password separately sudden change or with 41 and 215 passwords unite suddenly change relevant with the d4T Reduced susceptibility. In another specific embodiment, the invention provides degeneration-resistant the turning to of a kind of evaluation patient NRTI The method of record viral therapy validity comprises: (i) gather biological sample from the patient that HIV infects Product; (ii) determine biological sample whether include coding contain from one group comprise 62,75,77, The nucleosides of the hiv reverse transcriptase of the one or more sudden changes in 116 and/or 151 bit codon mutations Acid, use phenotype sensitivity analysis the present invention confirmed hiv reverse transcriptase from 62,75,77, The password subgroup of 116 and/or 151 compositions is suddenlyd change separately, or with comprise that from one group HIV reverses The record enzyme on 41,67,210,215,219,184,69 and/or the T215V bit codon mutation in One or more sudden change simultaneous mutations, these the sudden change with d4T Reduced susceptibilities (resistance increase) Relevant. In these cases, the phenotype sensitiveness of the HIV virus of infected patient/resistance figure and gene Type figure has revised in order to reflect ARV preparation changes of reactivity. Anti-for NRTI Retrovirus treatment, the HIV virus of infected patient may to as described herein one or more NRTIs has resistance, but other NRTIs is not had resistance, therefore after detecting sudden change, need or The person increases the dosage of ARV preparation, perhaps changes other ARV preparation, Perhaps in the medical compounds of Case treatment, add one or more extra ARV systems Agent. For example, if patient accept Stavudine (this too furan pyridine) (d4T) when treating 62,75, 77,116 and/or 151 bit codons suddenly change separately, or with comprise hiv reverse transcriptase from one group Upper 41,67,210,215,219,184,69 and/or the T215V bit codon mutation in one Individual or more multimutation simultaneous mutation,, the Case treatment medical compounds may need to make following modification, (i) Perhaps change different NRTI ARV preparations and stop the d4T treatment; Perhaps (ii) increases The dosage of d4T; Perhaps (iii) adds other ARV system in the Case treatment medical compounds Agent. Can by detect the viral load amount for example HIV RNA copy number estimate modified treatment Validity, HIV RNA copy number reduces the validity positive correlation with the medicine compound.

Refer to that with the term " positive correlation " here specific result draws specific most possible conclusion, Rather than other conclusion.

Preferred, the non-limiting particular of another one of the present invention is as follows: a kind of evaluation is sick The method of people NRTI ART validity comprises: the patient who (i) infects from HIV The middle biological sample of gathering; (ii) RNA of amplification HIV coding from biological sample is about to RNA Reverse transcription is cDNA, and with HIV primer amplification HIV sequence, the PCR that amplifies like this produces Thing includes pol gene; (iii) carry out pcr amplification with primer, the PCR that amplifies like this Product includes wild type or 69 and 41 and 215 password sudden changes; And (iv) produce by PCR Thing determines whether to exist 69 or 41 or 215 password sudden changes or 3 sudden changes all to exist. The present invention Another preferred, non-limiting particular is as follows: a kind of evaluation patient NRTI is degeneration-resistant Transcribe the method for viral therapy validity, comprising: (i) from the patient that HIV infects, gather blood plasma Sample; (ii) RNA of amplification HIV coding from plasma sample, soon RNA reverse transcription is CDNA, and with HIV primer amplification HIV sequence, the PCR product that amplifies like this includes contrary The transcriptase gene; (iii) carry out pcr amplification with primer, the PCR product that amplifies like this includes Wild type or in the codon that comprises 62,75,77,116 and 151 compositions from a group Suddenly change, and/or comprise 41,67,210,215,219,184,69 bit codons from one group One or more sudden changes in the sudden change; And (iv) by the PCR product determine whether to exist 62,75, 77,116 and 151 bit codon mutations, and/or from one group comprise 41,67,210,215, 219, the one or more sudden changes in 184,69 bit codon mutations. Another is preferred in the present invention, Non-limiting particular is as follows: a kind of evaluation patient NRTI ART has The method of effect property comprises: (i) gather plasma sample from the patient that HIV infects; (ii) from blood plasma The RNA of amplification HIV coding in the sample, being about to the RNA reverse transcription is cDNA, and draws with HIV Thing amplification HIV sequence, the PCR product that amplifies like this includes pol gene; (iii) use Primer carries out pcr amplification, the PCR product that amplifies like this include wild type or 69 and 41, 210,215,184 or 74 bit codon mutations; And (iv) determine whether to deposit by the PCR product 69 (T69SSA, T69SSG, T69SSS) and 41 (M41L), 210 (L210W), 215 (T215Y), 184 (M184V) or 74 (L74V) codon mutations. This It is as follows to invent another preferred, non-limiting particular: a kind of evaluation patient NRTI The method of ART validity comprises: (i) gather from the patient that HIV infects Plasma sample; (ii) RNA of amplification HIV coding from plasma sample is about to the RNA reverse transcription Be cDNA, and with HIV primer amplification HIV sequence, the PCR product that amplifies like this comprises Pol gene is arranged; (iii) carry out pcr amplification with primer, the PCR product bag that amplifies like this Contain wild type or at 41,67,70,210,215 and 219 bit codon mutations; And (iv) By the PCR product determine whether to exist 41 (m41L), 67 (D67N), 70 (K70R), 210 (L210W), 215 (T215Y/F) and 219 (K219Q) codon mutations.

There are 69,41 and 215 bit codon mutations show existing or will in hiv reverse transcriptase The curative effect of the efabirenz treatment that comes may need to change, because such as institute of the present invention What show is such, and 69 bit codon mutations have reduced the sensitiveness of d4T. Here will provide and use this The method of invention changes the efabirenz therapeutic scheme. Equally, use is of the present invention Measure confirms that hiv reverse transcriptase exists 62,75,77,116 and/or 151 passwords prominent Becoming expression the curative effect existing or treatment of efabirenz in the future may need to change, Because as shown in the present invention, prominent at 62,75,77,116 and/or 151 bit codons Become the sensitiveness that has reduced d4T. Equally, use measure of the present invention to confirm that HIV reverses The record enzyme exist 69 (T69SSA, T69SSG, T69SSS) and 41 (M41L), 210 (L210W), 215 (T215Y), 184 (M184V) or 74 (L74V) position is close Code sudden change expression the curative effect existing or treatment of efabirenz in the future may need to change Become because as shown in the present invention, in the sudden change of 69 bit codons/insertion, or join same 41, 210,215,184 or 74 bit codon mutations have reduced the sensitiveness of d4T.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluation is to HIV The patient who infects carries out the method for ART validity, comprises that (a) infects from HIV Patient in gather biological sample; (b) determine whether biological sample includes coding HIV and reverse The nucleotides of record enzyme, reverse transcriptase contains from one group and comprises 41,67,70,210,215 and 219 In the bit codon 4 or multimutation more, or connection with 74 (V74I), 69 (T69D), 75 (V75M, V75S) or the simultaneous mutation of 219 (K219N) codons. The utilization phenotype is quick Perceptual analysis is found these 4 or more multimutation is clearly relevant with the d4T resistance. In another enforcement side In the case, hiv reverse transcriptase is encoded at 41,67,70,210,215 and 219 bit codon mutations 41L, 67N, 70R, 210W, 215Y/F and 219Q. In a further embodiment, contrary Transcriptase is at V74I, T69D, V75M, V25S, K219N bit codon mutation, or combination HIV 4 or more dashing forward on the reverse transcriptase in 41,67,70,210,215 and 219 bit codons Become.

Another preferred, non-limiting particular of the present invention is as follows: a kind of evaluation is to HIV The patient who infects carries out the method for ART validity, comprises that (a) infects from HIV Patient in gather biological sample; (b) determine whether biological sample includes coding HIV and reverse The nucleotides of record enzyme, reverse transcriptase contains from one group and comprises 67,75,77,116 and 151 In the codon 1 or multimutation more, or connection with (E6D, K20R, A33I, T39A, E44D, S68G, Y115F, I167V, E138A, G196A, I202V, T215V, D218E, and T240K) codon 1 or more sudden change in the cipher code set that consists of. Use the phenotype sensitivity analysis to find that HIV is contrary Transcriptase is at 62,75,77,116 and 151 bit codon mutations, or unite by 62,75,77, In 116 and 151 bit codons 1 or more sudden change cause the reduction to d4T sensitiveness.

The invention provides patient's ART validity that a kind of HIV of evaluation infects Method comprises that (a) gathers biological sample from the patient that HIV infects; (b) determine biological sample Whether include the nucleotides of the hiv reverse transcriptase of encoding, reverse transcriptase contains at 69 bit codons (T69SSA, T69SSG, T69SSS) sudden change/insert, or connection is same by V75M, A158S, K20R, V21I, K102M, V179I, V241L, L283I, E297R, E6D, Q174R, D177E, R284K, A288S, 1 or more sudden change in the cipher code set that the E291D codon consists of. The utilization phenotype is quick Perceptual analysis is found at the existence of 69 bit codon mutation/insertions and the reduction positive of d4T sensitiveness Close.

The invention provides patient's ART validity that a kind of HIV of evaluation infects Method comprises that (a) gathers biological sample from the patient that HIV infects; (b) determine biological sample The nucleotides that whether includes the hiv reverse transcriptase of encoding, reverse transcriptase contains by 41,67,70, 210,4 or more sudden change in the cipher code set that consists of of 215 and 219 codons, or connection with by P1L, P9R, K20R, T39D, K43E, E44D, K64Y, V75M/S, G99R, L109V, V118I, K173E/T, I202T, R211H/T, D218E, K219N, H221Y, L228H, L283I, R284K 1 or more sudden change in the cipher code set that consists of with the A288T codon. Use phenotype sensitiveness Analyze 4 in the cipher code set of finding to be consisted of by 41,67,70,210,215 and 219 codons Or the more existence of multimutation and the reduction positive correlation of d4T sensitiveness.

The present invention also provides and has used resistance to detect the measure of carrier, and carrier contains the HIV base Cause further contains the required efabirenz sudden change of drug screening. Especially Be, the invention describes the resistance that includes hiv reverse transcriptase and detect carrier that reverse transcriptase has 69,41 and 215 password sudden changes that drug screening is required. The present invention has also described the HIV reverse The record enzyme include by 4 in 41,67,70,210,215 and 219 cipher code set that form or The resistance of more codon mutation detects carrier. The invention further relates to for separating of and identify Novel carriers, host cell and the change of nucleoside HIV-1 reverse transcriptase inhibiting resistance mutant strain The compound composition, and use these carriers, host cell and component to implement Antiviral screening. This The bright ability screening drug candidate that suppresses described mutant strain according to it that also relates to.

The invention provides evaluation can influence the method for the compound of HIV-1 reversed transcriptive enzyme function, comprise compound is contacted with HIV-1 reversed transcriptive enzyme complete peptide chain or partial peptide chain, wherein 69 bit codons change over coding SSS, the insertion of SSG or SSA amino-acid residue, replace Threonine, if wherein the two can influence the function of above-mentioned reversed transcriptive enzyme in conjunction with this compound of explanation.

The invention provides evaluation can influence the method for the compound of HIV-1 reversed transcriptive enzyme function, comprise compound is contacted with HIV-1 reversed transcriptive enzyme complete peptide chain or partial peptide chain, wherein will be by one or more codon mutations in 62,75,77,116 and 151 cipher code set of forming, the amino acids coding residue just is different from original L-Ala, Xie Ansuan, phenylalanine, hydrocinnamamide and L-glutamic acid respectively like this, if wherein the two can influence the function of above-mentioned reversed transcriptive enzyme in conjunction with this compound of explanation.

The present invention also provides the method for identifying the compound that can influence HIV-1 reversed transcriptive enzyme function, comprise compound is contacted with HIV-1 reversed transcriptive enzyme complete peptide chain or partial peptide chain, wherein will be by 4 in 41,67,70,210,215 and 219 cipher code set of forming or more a plurality of codon mutation, the amino acids coding residue just is different from original methionine(Met), aspartic acid, Methionin, leucine, Threonine or Methionin respectively like this, if wherein the two can influence the function of above-mentioned reversed transcriptive enzyme in conjunction with this compound of explanation.

Comprise the fragment that comes from human and various animal kinds with here " coming from patient's fragment ", these animal kinds include but are not limited to orangutan, horse, ox, cat and dog.

The fragment that comes from patient can use any resistance that participates in several different clone technologies to detect in the carrier, and this number at the PCT international application: have a detailed description among the PCT/US97/01609, this patent and on January 29th, 1997 file, reference in the lump here.For example, the clone can be by introducing class II restriction enzyme site the plasmid main chain and come from patient's the fragment, or by ura DNA glycosyl enzyme primer clone.

The fragment that comes from patient can obtain by any method of molecular cloning or gene amplification, perhaps the fragment that comes from patient two ends that will introduce resistance detection carrier is modified by adding following acceptor site.For example, in method, can join two ends with the primer of PCR reaction with the corresponding restriction enzyme site of patient's sequence acceptor site such as the genetic modification of PCR.Equally, in molecular cloning method such as the cDNA clone, described restriction enzyme site can mix and be used for cDNA first or second chain synthetic primer end, perhaps in method such as the primer repair of DNA, whether no matter is the DNA that clones or do not clone, described restriction site can mix the primer that is used to repair reaction.The acceptor site of design patient sequence and primer are so that improve the segmental expressivity that comes from patient, and design has the serial resistance detection carrier of patient's acceptor site to make that the low fragment that comes from patient of expressivity is expressed again in an independent resistance detection carrier.

" resistance detection carrier " refers to comprise in the lump the DNA that is made up of the fragment that comes from patient and indicator or one or more carriers of RNA.Resistance detects the PCT international application no PCT/US97/01609 institute described method preparation of carrier according to filing on January 29th, 1997, here reference in the lump, it is the fragment that amplification or clone come from patient, and the sequence that will increase or clone is inserted in the indicator virus vector at patient's sequence acceptor site.Perhaps, resistance detects carrier (referring to that also resistance detects carrier system) by patient's sequence acceptor site is introduced package carrier, amplification or clone come from patient's fragment, and the sequence that will increase or clone is inserted in the package carrier this package carrier of usefulness indicator virus vector cotransfection at patient's sequence acceptor site.

" indication or indicator " is as the PCT international application no PCT/US97/01609 of filing on January 29th, 1997 is described, refer to that one of coding can be directly or provide one can measure or as seen albumen, DNA or the RNA structure of phenomenon by reaction, for example this phenomenon is the light that color maybe can be surveyed wavelength, is the change or the generation of specific DNA or RNA structure for DNA that is used to indicate or RNA.The preferred example of indicator is the intestinal bacteria lacZ gene of coding beta-galactosidase, coding is from for example luc gene of the luciferase of Photonis pyralis (Lampyridea) or Renilla reniformis (extra large violet), the intestinal bacteria phoA gene of coding alkaline phosphatase, the bacterium CAT gene of green fluorescent protein and coding E.C. 2.3.1.28.Indicator and indicator as the PCT international application no PCT/US97/01609 of on January 29th, 1997 filing is described, can be function arranged or do not have a function.

Phenotypic drug sensitivity of the present invention and resistance experiment can be implemented reference in the lump here in a kind of or a plurality of host cell as the PCT international application no PCT/US97/01609 that on January 29th, 1997 submitted to is described.Virus drugs susceptibility is determined (for example, the IC50 of anti-virus formulation is the concentration that suppresses indicator expression amount 50%) by the concentration of the required anti-virus formulation of the given percentage that suppresses the indicator expression amount.The drug susceptibility typical curve of given antiviral can use the described method of above-mentioned patent application to draw for viral section, it can be a standard laboratory virus section, perhaps from the patient who does not have medication history (promptly not accepting the patient of antiviral treatment).Correspondingly, for a particular patient, the virus drugs resistance is the reduction of virus drugs susceptibility, this can perhaps measure the increase of one or many indicator expression inhibiting and determine (promptly reducing the expression of indicator) in succession by drug susceptibility and given standard are relatively come to determine in for some time.

The selected interpolation time-dependent of antiviral that joins detection system is in the target of antiviral.For example, for the hiv protease inhibitor, comprise saquinavir, ritonavir, indinavir and nelfinavir, they are detecting carrier in proper concentration during transfection or join in the packaging host cell at once afterwards with resistance, for hiv reverse transcriptase inhibitor, comprise AZT, ddI, ddC, d4T, 3TC, Viramune (nevirapine) and draw furan pyridine (delavirdine), they detect with resistance the vector virus particle in proper concentration during transfection or before join in the packaging host cell.Perhaps, antiviral all exists in whole analysis.In wide range of concentrations, select detectable level, generally greatly about 0.1nM between the 100uM, and specific concentrations is arranged for following every kind of medicine: AZT, from about 6nM to about 400uM; DdI from about 15nM to about 1,000uM; 3TC, from about 9nM to about 600uM; D4T, from about 6nM to about 400uM; DdC, from about 15nM to about 1,000uM; Viramune (nevirapine), from about 0.7nM to about 50uM; Draw furan pyridine (delavirdine), from about 0.07nM to about 5uM; Saquinavir, from about 0.02nM to about 1.5uM; Indinavir, from about 0.02nM to about 1.5uM; Nelfinavir, from about 0.02nM to about 1.5uM; And ritonavir, from about 0.05nM to about 3uM.

In another embodiment of the invention, in phenotypic drug sensitivity and resistant proof, use the resistance of the reversed transcriptive enzyme that is included in 69,41 and 215 bit codon mutations to detect carrier sense candidate efabirenz antiviral drug compounds.In yet another embodiment of the present invention, in phenotypic drug sensitivity and resistant proof, use the resistance detection carrier sense candidate efabirenz antiviral drug compounds that is included in by the reversed transcriptive enzyme of one or more codon mutations of selecting in 62,75,77,116 and/or 151 codon groups of forming.In yet another embodiment of the present invention, in phenotypic drug sensitivity and resistant proof, use is included in by M41L, D67N, K70R, L210W, the resistance of the reversed transcriptive enzyme of 4 of selecting in the codon group that T215Y/F and/or K219Q position are formed or more a plurality of codon mutations detects carrier sense candidate efabirenz antiviral drug compounds.In yet another embodiment of the present invention, in phenotypic drug sensitivity and resistant proof, use comprises 69 bit codon (T69SSA, T69SSG, one among the T69SSS) and M41L and T215Y sudden change, and by M184V/I, L74V, A62V, the resistance of the reversed transcriptive enzyme of one or more codon mutations of selecting in the codon group that V75M forms detects carrier sense candidate efabirenz antiviral drug compounds.The candidate antiviral joins in the detection system in the proper concentration scope with in the transfection step.Perhaps, can detect more than one candidate antiviral, perhaps the candidate antiviral can join with verified antiviral and detects in the lump, verified antiviral is AZT for example, ddI, ddC, d4T, 3TC, ground draws furan pyridine (delavirdine), Viramune (nevirapine), saquinavir, ritonavir, indinavir, nelfinavir or just at the medicine of clinical trial, Abacavir (Ai Bokai furan) for example, or amprenavir or efavirenz.The validity of candidate antiviral will be estimated by expression or the inhibition of measuring indicator.In another aspect of the present invention, drug susceptibility and resistant proof can be used for screening virus mutation, behind the resistant mutation that identifies known antiretroviral drugs or candidate antiretroviral drugs, separation resistance sudden change and analyzing DNA, so just can set up virus resistance sudden change storehouse, this just can screen candidate efabirenz antiretroviral drugs independent or coupling.This just makes those skilled in the art also can identify effective efabirenz antiretroviral drugs, and prescription is effectively treated in design.

General material and method

The most of technology that are used for carrier construction, transfection and cells infected are widely used the technician, and most professionals are familiar with the material in these standard of having described specified conditions and method sources.Yet for convenience's sake, following paragraph can be used as guide.

" plasmid " and " carrier " uses small letter lattice p back word adding mother and/or numeral.Initial plasmid here or market can have been bought, and perhaps publish without restriction, perhaps can make up from available plasmid according to the method for having delivered.In addition, known in this field with the plasmid that those descriptions are equal to, also know for the technician who generally has the knack of.

Carrier construction uses among the present invention connection and restriction enzyme digestion technology (are seen Ausubel etc. in this field for everybody is known, (1987) Current Protocol in Molecular Biology, WileyInterscience or Maniatis etc., (1992) in Molecular Cloning:A laboratoryManual, Cold Spring Harbor Laboratory, N.Y).Isolating plasmid, dna sequence dna or synthetic oligonucleotide sequence are cut, tailing and connect into desirable form again, all DNA construct of having mixed the artificial-synthetic DNA by dna sequence analysis determine (Sanger etc. (1977) Proc.Natl.Acad.Sci.74,5463-5467).

" digestion " of DNA refers to only act on DNA particular sequence, restriction site with Restriction Enzyme catalyze cleavage DNA.Various Restriction Enzymes used herein city field energy has been bought, and their reaction conditions, cofactor and other requirement are known by the general technician who is familiar with.Be analysis purposes, the plasmid of general 1ug or the enzyme that dna fragmentation uses 2 units perhaps, use excessive Restriction Enzyme to guarantee the complete digestion of DNA substrate in the damping fluid of 20ul.Feasible usually in the time that 37 ℃ of incubations are about 1 to 2 hour, although mutant has resistance.Behind each incubation, remove albumen, and can use a kind of extracting wherein again, reclaim nucleic acid from aqueous phase with ethanol sedimentation by phenol/chloroform extracting.Can use standard method by polyacrylamide gel or agarose gel electrophoresis endonuclease bamhi to be carried out size exclusion and separate, the isolating general description of size exclusion is seen Enzymology method (Methods Enzymology) 65:499-560 (1980).

Restriction fragment can be handled flush endization with the big fragment of e. coli dna polymerase I, and reaction adds 4 kinds of deoxy-ribonucleoside triphosphates (dNTPs), and 20 ℃ are used 15 to 25 minutes incubation time, 50mM Tris (pH7.6) 50mM NaCl, 6mM MgCl ₂, 6mM DDT and 5-10dNTPs.Even have 4 kinds of dNTPs, the Klenow fragment can be mended flat 5 ' sticking end, and excises 3 ' outstanding strand.If desired, can carry out the selectivity reparation according to the sticky end characteristic by the dNTPs of a kind of or selection among the dNTPs only is provided.After the Klenow processing, mixture is separated any strand part with S1 nuclease or Bal-31 treating water with phenol/chloroform extracting and alcohol precipitation under appropriate condition.

Be connected under following standard conditions and the temperature and in the system of 15-50ul, carry out: 20mMTirs-HCl pH7.5, l0mM MgCl ₂10mM DTT, 33mg/ml BSA, 10mM-50mM NaCl and or 40uM ATP, the T4 dna ligase of 0.01-0.02 (Weiss) unit, 0 ℃ (being used for " cohesive end " connects), perhaps 1mM ATP, the T4 dna ligase of 0.3-0.6 (Weiss) unit, 14 ℃ (being used for " flush end " connects).Intermolecular cohesive end connection is carried out under the total DNA concentration of 33-100ug/ml (the total ultimate density of 5-100mM) usually, and intermolecular flush end connects (using the doubly excessive joint of mole of 10-30 usually) to carry out under the total terminal concentration of 1uM.

" transient expression " refers to that non-amplification is expressed in 1 day to 2 weeks of about transfection.The Best Times of a specific purpose heterologous protein transient expression may depend on Several Factors and different, and this for example comprises, the trans-acting factor that may use, translational control mechanism and host cell.When the certain plasmid of transfection works when promptly transcribing and translating, just begun transient expression.The plasmid DNA that enter cell this moment is transferred in the nuclear, and DNA is non-integrated state, freely has the transcribing this section period that occurs in of the plasmid that is absorbed by cell in nuclear.Plasmid DNA may be degraded or diluted by cell fission after the transfection, and the random integration in the cyto-chromatin also can take place.

In general, carrier comprises promotor and control sequence, and they derive from the kind compatible with the host cell of particular host cell coupling.Be applicable to the illustrative β-Nei Xiananmei and the lactose promoter systems of comprising of promotor of prokaryotic hosts, alkaline phosphatase, tryptophane (trp) promoter systems and hybrid promoters be the tac promotor for example.Yet other functional bacterium promotor is also available.Except that prokaryotic organism, also can use for example yeast culture of eukaryotic microorganisms, yeast saccharomyces cerevisiae or normal Bei Shi yeast are the most frequently used eukaryotic microorganisms, although many other kinds are also commonly used.Can obtain the promotor that the control of mammalian host cell carriers is transcribed by several sources, for example, viral genome is for example: polyomavirus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus and act cell virus, or from the mammiferous promotor of allos, for example beta-actin promotor.Can obtain early stage and late promoter easily and comprise the simian virus 40 restriction fragment of simian virus 40 replication initiation as simian virus 40 (SV40), the early promoter that can obtain human adenovirus easily is as the HindIII restriction fragment.Certainly, also be available from host cell or relevant promotor of planting here.

Carrier used herein can comprise the selection gene, is defined as alternative mark again.The albumen of selecting genes encoding for transfection the survival and the growth of host cell of plasmid be essential.Example for the suitable selected marker of mammalian cell comprises dihydrofolate reductase gene (DHFR), ornithine deamination formylase gene, multiple drug resistance gene (mdr), adenosine deaminase gene and glutamine synthetase gene.When these selected markers successfully imported mammalian cell, mammalian cells transfected just can be survived under selective pressure.The completely different widely used selection prescription of two classes is arranged, and the first kind is the metabolism according to cell, uses the cell strain of sudden change, and they lack energy for growth, depend on the interpolation substratum.Second class is meant the dominance selection, and the selection scheme of indication is used for any cell type, does not need to use mutant clone.These schemes use medicine to catch the growth of host cell usually, and those have the cell of novel gene can express the albumen that has drug resistance, survive in selection.The medicine that the example that these dominance are selected uses has Xin Meisu (Southern and Berg (1982) J.Molec.Appl.Genet.1,327), mycophenolic acid (Mulligan and Berg (1980) Science 209,1422) or Totomycin (Sugden etc. (1985) Mol.Cell.Biol.5,410-413).3 examples that more than provide application bacterial gene under eucaryon control is given the resistance to corresponding medicine Xin Meisu (G418 or rough gentian element), xgpt (mycophenolic acid) or Totomycin respectively.

Whether " transfection " refers to DNA is imported host cell, and DNA just expresses like this, no matter be functional expression; DNA can also be as the element outside the karyomit(e), or is incorporated in the karyomit(e) and duplicates.Whether no matter mention, the employed method of transfection host cell is Graham and van derEb (1973) Virology 52 here, the coprecipitation of calcium phosphate method that 456-457 is used.The other method of transfection is electroporation, DEAE-dextran method, lipofection and biolistics (Kriegler (1990) GeneTransfer and Expression:A Laboratory Manual, Stockton Press).

Host cell can be with expression vector transfection of the present invention, is cultivating on traditional substratum of evoked promoter, screening transformant or amplification gene so that be suitable for through revising.Host cell is cultivated on 50: 50 at F12: DMEM (Gibco), adds not added with antibiotic of glutamine.Culture condition, for example temperature, pH and like that are the conditions that is used for the host cell selective expression before those, will come into plain view for those of ordinary skill.

Following examples are only represented present known enforcement optimal mode of the present invention, but should not be construed as limiting the invention.This specification sheets quote all deliver thing and patent is all done as a whole reference in the lump here, just look like each to deliver that thing or patent use all be specific and independent explanation reference in the lump.

Embodiment 1

Use resistance to detect carrier and carry out phenotypic drug sensitivity and resistant proof

Use as the described means of PCT international application no PCT/US97/01609 and the method for filing on January 29th, 1997 and implement phenotypic drug sensitivity and resistant proof, reference in the lump here.

In these experiments, can use isolating viral RNA in the virion that exists the blood plasma of the individuality that infects from HIV corresponding to the fragment that comes from patient of hiv protease and reversed transcriptive enzyme coding region, come from patient's fragment by reverse transcription-polymerase chain reaction (RT-PCR) amplification, perhaps the parent who detects carrier DNA by antagonism clones the mutant strain of each wild-type HIV-1 type of point mutation system.The separation of use classical way enforcement viral RNA (for example, RNAgents TotalRNA Isolation System, Promega, Madison WI or RNAzol, Tel-Test, Friendswood, TX).The RT-PCR program divided for two steps, use retroviral reversed transcriptive enzyme (Moloney MuLV reversed transcriptive enzyme (Roche Molecular Systems for example, Inc., Branchburg, NJ), or avian myeloblastosis virus (AMV) reversed transcriptive enzyme, (Boehringer Mannheim.Indianapolis, IN)) viral RNA is copied into cDNA, then use heat-stable DNA polymerase amplification cDNA (for example, Taq (Roche Molecular Systems, Inc., Branchburg, NJ), Tth (Roche Molecular Systems, Inc., Branchburg, NJ), PrimeZyme is (from Thermus brockianus, Biometra, separate among the Gottingen, Germany)) or as " long PCR " operate described with the coupling of heat-stabilised poly synthase (Bames, WM., (1994) Proc.Natl.Acad.Sci, USA 91,2216-2220) (for example, the high-fidelity PCR system (Taq+Pwo) of expansion, (Boehringer Mannheim, Indanapolis, IN) or GeneAmp XL PCR Kit (Tth+Vent), (Roche Molecular Systems, Inc., Branchburg, NJ)).

Be used for the segmental primer that amplification " detection " comes from patient, ApaI primer (PDSApa) and AgeI primer (PDSAge), the site sequence that comprises ApaI and AgeI identification, they are incorporated into 5 ' and 3 ' end of PCR product, and this PCT international application no PCT/US97/01609 in filing on January 29th, 1997 has description respectively.

Mixing " detection " comes from the segmental resistance of patient to detect carrier is that description according to the PCT international application no PCT/US97/01609 of filing on January 29th, 1997 makes up, use viral RNA as template and oligonucleotide PDSApa and PDSAge as primer, prepare the DNA cloning product of 1.5kB by RT-PCR, then with ApaI and AgeI or isoschizomer PINAI digestion.In order to ensure detecting the representative sample that the corresponding plasmid DNA of carrier contains the HIV virus quasispecies that exists in the given patient blood plasma with the synthetic resistance, to detect the independently transformant merging of a large amount of (＞100) that obtain in the vector construction in given resistance, be used for the preparation of plasmid DNA.

The packaging expression vector of coding amphophilic MuLV 4070A env gene product can detect in resistance and produce resistance detection vector virus particle in the vector host cell, and it can effectively infect human target cell.Except that the env gene, the resistance of all HIV genes of encoding detects carrier and is used for transfection packaging host cell (detecting vector host cell in case the host cell of transfection is meant as resistance).The packaging expression vector and the resistance of coding amphophilic MuLV 4070A env gene product detect the carrier coupling, can detect in resistance and produce communicable false type resistance detection vector virus particle in the vector host cell.

Use the packing host or by human embryos kidney cell line 293 (Cell Culture Facility, UCSan Francisco, SF, CA) or Jurkat leukemia T cell strain (Arthur Weiss.UC SanFrancisco, SF, CA) the target host cell of Zu Chenging detects carrier with resistance and implements resistant proof.

Use two kinds of host cell types to detect carrier and implement resistant proof with resistance.Resistance detects the vector virus particle and is produced by first host cell (resistance detection vector host cell), it is by preparing with resistance detection carrier and packaging expression vector transfection packaging host cell, then resistance detection vector virus particle is used to infect second host cell (target host cell), the expression that can measure indicator in this cell.

The resistance that structure comprises function fluorogene box detects carrier, and detects the carrier DNA transfection host cell with resistance.The resistance that comprises the reversed transcriptive enzyme that comes from patient and proteolytic enzyme sequence detects carrier has susceptibility or resistance to the antiretroviral preparation, for example efabirenz, non-nucleoside reverse transcriptase inhibitor and proteinase inhibitor.No matter whether there is proteinase inhibitor, produce resistance detection vector virus particle by resistance being detected vector virus particle transfection host cell, under the condition of efabirenz or non-nucleoside reverse transcriptase inhibitor or drug level increase, be used to infect the target host cell.The two compares the luciferase live vol that produces in the target host cell that infects when luciferase live vol that produces in the target host cell that infects under medicine exists and medicine do not exist, detect 50% (inhibition concentration 50% of fluorescein when suppressing not have medicine, IC50) needed medication amount detects as drug resistance, the per-cent inhibition of log10 drug level is determined the value of IC50 by drawing medicine.

Host cell is inoculated in the culture dish of 10 cm diameters, inoculates after several days and detects vector plasmid DNA and the transfection of env expression carrier with resistance.Transfection is finished with the calcium phosphate precipitation program, and the cell culture medium that contains the DNA precipitation agent 1 to 24 hour after transfection changes fresh substratum into.After the transfection 1 collected to 4 days and comprised resistance detection vector virus particulate cell culture fluid, filtered the filter membrane of 0.45mm before 80 ℃ of storages.EIA method (the SIAC that the content of HIV capsid protein (p24) is described by manufacturers in the cell culture fluid of collecting; Frederick MD) determines.Before the infection, target cell (293 and 293/T) is inoculated in the cell culture fluid, uses self simulation transfection (not containing DNA) or comprises cells transfected nutrient solution that the resistance that does not have the env expression plasmid detects vector plasmid DNA and implement contrast and infect.Metainfective 1 to 3 day or removed substratum in more days, add cell pyrolysis liquid (Promega), the fluorescein activity (Fig.3) of analysis of cells lysate in each hole.The inhibition effect of medicine is calculated with following formula:

% luciferase inhibition=1-(the RLUluc[medicine] ÷ RLUluc) * 100

RLUluc[medicine wherein] be medicine relative light unit of uciferase activity in the cells infected when existing, RLUluc is medicine relative light unit of uciferase activity in the cells infected when not existing.The value of IC50 obtains by sigmoid curve, and curve is to produce from draw the data of medicine to the per-cent inhibition of log10 drug level.Medicine suppresses curve and sees (Fig.3).

Embodiment 2

Phenotype susceptibility and genotype correlation analysis

The sensitivity analysis of patient HIV sample phenotype

Make up resistance detection carrier as describing among the embodiment 1.In phenotype analytical, measure resistance and detect carrier, or derive from the clone that resistance detects the carrier storehouse, so that accurately with the sensitivity levels of quantitatively determining the antiretroviral drugs hurdle, this antiretroviral drugs hurdle may comprise several class members, resembles nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitor (NNRTIs) and proteinase inhibitor (PRIs).This medicine hurdle can enlarge when new medicine or novel drugs target are arranged.Check all detection of drugs susceptibility patterns, and compare that patient samples can further detect the genotype relevant with observed susceptibility pattern and change with known susceptibility pattern.

Patient HIV sample gene type analysis

Resistance detects carrier DNA s, no matter is storehouse or clone, can analyze by embodiment 2 described any genotype methods.In one embodiment of the invention, utilization viral RNA purifying, RT/PCR and ABI chain terminator automatic sequencing are determined patient HIV sample sequence, definite sequence compares with the control sequence that exists in the database, or if possible, beginning before with treatment, patient's sample compares.Genotype detection is to find out the sequence different with contrasting or treat presequence, and the dependency between it and the observed phenotype.

The phenotype sensitivity analysis of point mutation

The resistance that is included in specific sudden change under wild-type (to the medicaments insensitive) genetic background of qualification by structure detect that carrier estimates that observed genotype changes and drug susceptibility phenotype patterns of change between dependency.Sudden change can be mixed separately, and/or other known think can regulate HIV to the drug resistance of certain drug or medicine group susceptibility sudden change mix in the lump.To suddenly change by the method for any widely known rite-directed mutagenesis and to introduce resistance and detect carrier.In one embodiment of the invention, use the method for large primer PCR to carry out rite-directed mutagenesis, the resistance of using above-mentioned phenotype sensitivity analysis detection to comprise specific sudden change or sudden change group then detects carrier, and wild-type (to the medicaments insensitive) resistance that susceptibility collection of illustrative plates and the heredity that does not have specific sudden change limit is detected carrier, and the two compares, can be owing to the specific sudden change that is incorporated in the resistance detection carrier to the observed variation of antiretroviral drugs phenotype susceptibility pattern that is detected.

Embodiment 3

The dependency of phenotype susceptibility and gene type assay: the D4T resistance that links with multiple drug resistance (MDR) sudden change

From patient 96-136,97-240, the resistance of 98-955 and 98-960 detects the phenotype analytical of carrier

From being labeled as 96-136,97-240 makes up resistance and detects carrier in the patient samples of 98-955 and 98-960 according to embodiment 1 described method.With comprising that the medical compounds of d4T has carried out the treatment of different time, patient 955 and 960 history of medications are not clear before the patient 136 and 240.Use the separation of viral RNA and the fragment that comes from patient that the RT/PCR preparation comprises the virus sequence of all proteolytic enzyme and reversed transcriptive enzyme 1-313 amino acids, the fragment that comes from patient is inserted in the indicator, be labeled as RTV-136 thereby make up, RTV-240, the resistance of RTV-955 and RTV-960 detects carrier.Utilization phenotype sensitivity analysis detects these RTVs carriers, so that accurately and determine the sensitivity levels on antiretroviral drugs hurdle quantitatively.The antiretroviral drugs hurdle comprises known a few group membership, comprise efabirenz (AZT, 3TC, d4T, ddI, ddC and abacavir (Ai Bokai furan)), non-nucleoside reverse transcriptase inhibitor (draw furan pyridine (delavirdine) and Viramune (nevirapine)) and proteinase inhibitor (indinavir, nelfinavir, ritonavir and saquinavir).Measure the IC50 of every kind of detection of drugs, check patient virus is to the susceptibility of every kind of medicine, and with known susceptibility model relatively, observe in these RTV carrier storehouses each and contrast the susceptibility that compares 4dT with wild-type and all reduce.Check that further the genotype that patient samples may be relevant with observed d4T susceptibility pattern changes.

Determine the genotype of patient RTVDNAs

Analyze RTV DNAs by ABI chain terminator automatic sequencing, with nucleotide sequence and wild-type differentiation B HIV-1 (HIV Sequence Database Los Alamos, identical sequence comparison NM), the inspection nucleotide sequence sequence different with control sequence.RTV-136 is at M41L, D67N, and V75I, F116Y, Q151M, M184V, there is sudden change in T200A and T215Y site, and all sudden changes of RTV-136 mix with wild-type and mutating acid in each site and exist.RTV-240 is at A62V, S68G, V75I, F77L, F116Y, E138A, there is sudden change in Q151M and M184V site, and RTV-955 is at E6D, K20R, V35I, A62V, D67N, T69D, V75I, F77L, K101E, K103N, Y115F, F116Y, Q151M, I167V, Y181C, M184V, G190A, 1202V, R211K, F211K, F214L, there is sudden change T215V and K219Q position, exist with wild-type and mutating acid mixing in each site 101,103,181 and 190 sudden changes.RTV-960 is at A33I, T39A, M41L, E44D, D67N, T69D, K103N, Q151M, M184I, G190A, L210W, R211K, T215Y, D218E, there is sudden change T240K and A288S position, at A62V, V75I, F77L, description can cause the wide spectrum crossed resistance to efabirenz before F116Y and the sudden change of Q151M position, is also referred to as multiple drug resistance (MDR) sudden change.All these RTV DNAs comprise one or several these MDR sudden changes.In addition, some RTV DNAs have and AZT resistance (M41L, D67N, L210W, T215Y, and K219Q), ddC resistance (T69D), non-nucleoside reverse transcriptase inhibitor resistance (K101E, K103N, Y181C, with G190A), the relevant sudden change of 3TC resistance (M184V), or do not find in the past sudden change (E6D, K20R, the A33I of characteristic, T39A, E44D, S68G, Y115F, I167V, E138A, G196A, 1202V, T215V, D218E, and T240K).Known that at V35I R211K and the sudden change of F214L position are the polymorphisms of HIV wild-type (to medicaments insensitive) mutant strain.

Rite-directed mutagenesis

Structure comprises Q151M and suddenlys change separately, or with the adjusting HIV that generally acknowledges the resistance of the V75I drug resistance sudden change associating sudden change of efabirenz susceptibility is detected carrier.The large primer PCR method of use rite-directed mutagenesis will be suddenlyd change and be introduced resistance detection carrier.(Sakar?G?andSommar?SS(1994)Biotechniques?8(4)，404-407)。Use the resistance that above-mentioned phenotype sensitivity analysis detection contains Q151M sudden change (Q151M-RTV) to detect carrier, the result is detected carrier relatively with the heredity restriction resistance that in 151 sites is wild-type, compare with wild-type, change has taken place to the phenotype susceptibility pattern of efabirenz, d4T in Q151M-RTV.Under other wild-type background (being that Q151M suddenlys change separately), Q151M-RTV is lower to the susceptibility of d4T than wild-type contrast RTV, also observes the noticeable change to AZL ddC and ddI susceptibility in Q151M-RTV.Also the Q151M sudden change is incorporated among the RTV that comprises the V75I sudden change, will causes AZT susceptibility is increased and d4T susceptibility is reduced in the V75I sudden change adding Q151M background.V75I sudden change does not separately influence d4T susceptibility, and causes AZT susceptibility is increased.Also made up and comprise separately sudden change and unite the RTVs carrier of sudden change with V75I of A62V, these RTVs compare with wild-type RTV that they are constant to d4T susceptibility, yet the two susceptibility to AZT has slight increase.

Embodiment 4

The dependency of phenotype susceptibility and gene type assay: with insert relevant 4dT resistance in reversed transcriptive enzyme 69 amino acids

From patient 97-621,97-285,98-690, the resistance of 98-770 and 98-771 detects the phenotype analytical of carrier

According to embodiment 1 described method from being labeled as 97-621,97-285,98-690 makes up resistance and detects carrier in the patient samples of 98-770 and 98-771.Patient samples 285 and 690 is the series of samples that obtained in 6 months at interval from same patient, and with comprising that the medical compounds of d4T has carried out the treatment of different time, patient 621 history of medications is not clear before the patient 285/690,770 and 771.

The patient history that the viral sample of 69SSX insertion is arranged

Patient VL#770 (JCW) uses the AZT single therapy at first, and then ddC joins in the prescription, and these 2 kinds of medicines approximately used 8 months.15 months afterwards treatment is unclear, and then patient VL#770 (JCW) has treated about 2 years half time with AZT and 3TC coupling.Patient VL#285/690 (JWA) used the AZT single therapy about 1 year 09 months, then changed ddI single therapy 8 months over to, and he got back to the AZT single therapy again 1 year 03 months, had then treated nearly 2 years with ddC.Treatment becomes AZT and nearly 1 year of 3TC coupling, then becomes the d4T/3TC/NFV coupling about 9 months.Patient VL#771 (BMN) uses AZT at first, ddC, and PPI, and RTV coupling treatment, 3TC then joins in this coupling medicine and changes over to till d4T and the RTV up to treatment, and this treatment has continued about 6 months, then becomes 3TC/d4T/ddI and RTV coupling treatment.Have an appointment and do not receive treatment in 6 months, then accept AZT, 3TC and ddI treatment, combination therapy then is converted to 3TC, d4T and IDV.Patient VL#1057 (DHW) used the AZT single therapy about 3 years, then became the ddI single therapy 1 year, had do not receive treatment in about 8 months, then treated 1 year with AZT and 3TC, continues the treatment with AZT, but 3TC is changed to d4T and PRI, added IDV.Then short-term becomes d4T again 3TC, and up to by NNRTI, DEL replaces.Use viral RNA separation and RT/PCR preparation to comprise the fragment that comes from patient of the virus sequence of all proteolytic enzyme of coding and reversed transcriptive enzyme 1-313 amino acids, the fragment that comes from patient is inserted the indicator virus vector, indicate RTV-621 so that make up, RTV-285, RTV-690, the resistance of RTV-770 and RTV-771 detects carrier.Utilization phenotype sensitivity analysis detects these RTVs carriers, so that accurately and determine the sensitivity levels on antiretroviral drugs hurdle quantitatively.The antiretroviral drugs hurdle comprises known a few group membership, comprise efabirenz (AZT, 3TC, d4T, ddI, ddC and abacavir (Ai Bokai furan)), non-nucleoside reverse transcriptase inhibitor (draw furan pyridine (delavirdine) and Viramune (nevirapine)) and proteinase inhibitor (indinavir, nelfinavir, ritonavir and saquinavir).Measure the IC50 of every kind of detection of drugs, check patient virus is to the susceptibility of every kind of medicine, and with known susceptibility model relatively, observe in these RTV carrier storehouses each and contrast the susceptibility that compares 4dT with wild-type and all reduce.Check that further the genotype that patient samples may be relevant with observed d4T susceptibility pattern changes.

Measure the genotype of patient RTV DNAs

Use ABI chain terminator technology to measure the nucleotide sequence of resistant proof carrier automatically.The gained nucleotide sequence compares with the conserved sequence (New Mexico Loews Ah Georg Lammers HIV sequence library) of wild-type branch B HIV-1, finds out the sequence different with control sequence.RTV-621 contains M41L, A62V, T69SS, V75M, M184V, L210W and T215Y.The existing wild-type of nucleotide sequence that RTV-621 last 41,62 and 75 each site occur also has the nuclear of sudden change.RTV-285 contains following sudden change: M41L, A62V, T69SSA, L74V, A158S, I178M, M184V, T200A, L210W and T215Y.RTV-690 contains following sudden change: K20R, M41L, A62V, T69SSG, V75M, K102M, K103R, V179I, M184V, T215Y, V241L, L283I and E297I.The existing wild-type of the nucleotide sequence of 158 and 181 liang of each site of site also has mutant.RTV-770 contains following sudden change: V21I, M41L, T69SSG, V75M, K102M, K103R, V179I, M184V, T215Y, V241L, L283I and E297R.The existing wild-type of nucleotide sequence that arbitrary site occurs in 69 and 75 liang of sites also has mutant.RTV-771 contains following site mutation: E67D, V35I, M41L, T69SSS, V75M, I135V, Q174R, D177E, M184V, T299I, L210W, R211K, T215Y, R284K, A288S and E291D.The existing wild-type of nucleotide sequence that arbitrary site occurs in 174 and 200 liang of sites also has mutant.Near 69 of reversed transcriptive enzymes or its, the nucleic acid of all these resistant proof carriers all has extra amino acid to insert unusually.Here this unusual insertion is designated as T69SSX, X represents glycine (G), Serine (S) or L-Ala (A).In the past the A62V sudden change was described as one of multi-drug resistance sudden change (MDR).This multiple medicines thing sudden change necessarily acts on the wide spectrum crossed resistance performance for NRTI class medicine described in the embodiment 3.In addition, all these resistant proof carriers all have some ensemble de catastrophes relevant with the certain drug resistance.The ensemble de catastrophes relevant: M41L, D67N, L210W, T215Y and K219Q with the AZT-resistance; The ensemble de catastrophes relevant: L74V, L74I, T69D with the ddC-resistance; With the relevant ensemble de catastrophes of NNRTI class medicine-resistance: K103N, Y181C and G190A; The sudden change relevant with the 3TC-resistance has M184V: unidentified other sudden change perhaps: V75M, A158S, K20R, V21I, K102M, V179I, V241L, 1283I, E297R, E6D, Q174R, D177E, R284K, A288S, E291D.Sudden change at V35I, K103R, I135V, D177E, I178M, V179I, T200A/I, R211K and F214L belongs to the polymorphism of observing on HIV wild-type variant.

Inverse transition

Use the method that inverse transition is made in common name, further identify the effect of T69SSX sudden change in the d4T-resistance.Clone in the RTV-285 storehouse, its reversed transcriptive enzyme contains following sudden change: M41L, A62V, T69SSA, L74V, A158S, I178M, M184V, T200A, L210W and T215Y.From this storehouse, isolate a functional clone.Use the rite-directed mutagenesis means with 69 trisomes " SSA " single Threonine " T " that suddenly change specifically.This reverse mutation contains all other sudden change of inserting appearance on the 285-1 clone except that 69 SSA trisomes.The 285-1 muton all shows as remarkable increase to the resistance of d4T and AZT, and d4T increases by 3 times, and AZT increases by 30 times.Evaluation is isolating single clone from the RTV-770 storehouse, has obtained T69SSA and insert the further evidence that sudden change acts in the NRTI-resistance.Two classes clone appears in the RTV-770 storehouse.The first kind contains following sudden change: V21I, M41L, K102M, K103R, V179I, M184V, T215Y, V241I, L283I and E297R; The another kind of whole sudden changes that contain the first kind also contain T69SSG and V75M simultaneously.Those second class sudden changes that have 69 and 75 sudden changes all significantly increase d4T and AZT susceptibility, and d4T has increased by 4 times, and AZT has increased by 30 times.Make up the resistant proof carrier, only contain the carrier of T69SSA sudden change, perhaps also contain other medicines resistant mutation or the sudden change that can regulate HIV to NRTI class drug susceptibility under a cloud simultaneously.With use the large primer PCR method and carry out rite-directed mutagenesis, to the resistant proof carrier import sudden change (Saker G and Sommar SS (1994) Biotechniques 8 (4), 404-407).Use the method for above-described phenotype sensitivity test, the resistant proof carrier T69SSA-RTV that contains mutation T 69SSA is tested, the resistant proof carrier that gained result and another genetic background are clear and definite compares.The 69th site of this resistant proof carrier is " T ", is wild-type.Compare with wild-type resistant proof carrier, T69SSA-RTV also observes T69SSA-RTV and changes the susceptibility generation of AZT is slight and significant the susceptibility decline twice of d4T.

In addition the T69SSA sudden change is incorporated on the resistant proof carrier that contains V75I.V75I is introduced the T69SSA background to be caused AZT susceptibility is increased and d4T susceptibility is descended.V75I sudden change does not separately act on d4T susceptibility, but causes that AZT susceptibility increases.

In addition, also the T69SSA sudden change is incorporated on the resistant proof carrier that contains A62V.A62V suddenlys change separately to the not effect of susceptibility of all tested reverse transcriptase inhibitors class medicines.A62V is introduced the T69SSA background to the not influence of d4T susceptibility, but to the susceptibility of AZT then significantly descend (6 times).

Made up a resistant proof carrier that contains A62V, T69SSA and V75I.Triple mutant is to the susceptibility performance very small decline (2 times) (as T69SSA sudden change separately) of AZT, to the then slightly increase (less than 2 times) of susceptibility of AZT.

Make up some resistant proof carriers and contain T69SSA insertion sudden change, contain the various combination of AZT-resistant mutation (M41L, A62V, T215Y) and 3TC-resistant mutation (M184V) simultaneously.The resistant proof carrier that contains sudden change in M41L, T69SSA and T215Y site all has significant reduction to the susceptibility of d4T and AZT, has reduced by 5 times and 160 times respectively.Add the A62V sudden change to this background and make the susceptibility to d4T and AZT further descend, be respectively 10 times and above 1000 times.M184V occurs for the not influence of d4T susceptibility, but cause that AZT susceptibility increases.

Make up a resistant proof carrier, contain sudden change M41L, T69SSA, T215Y and L210W.Introduce sudden change L210W to a resistant proof carrier that contains M41L, T69SSA and T215Y triple mutant, cause the resistance of AZT that substantial reduction (greater than 1000 times) takes place.Relative therewith, M41L-T69SSA-T215Y-RTV has reduced by 140 times to the susceptibility of AZT.If compare with M41L-T69SSA-T215Y-RTV, L210W is to other not influence of NRTI class drug susceptibility.Compare with M41L-T69SSA-T215Y-RTV, the M41L-T69SSA-T215Y-L210W-RTV drug susceptibility has reduced by 3 times to ddC, ddI has been reduced by 3.5 times, 3TC has been reduced by 17 times, d4T has been reduced by 10 times, abacavir has been reduced by 14 times, all is slight change.Relative M41L-T69SSA-T215Y-RTV, the L2110W sudden change does not have adjection to the susceptibility of NNRTI class medicine, DEL (0.3 times) and NEV (0.42 times).And M41L-T69SSA-T215Y-RTV also shows slight increase to the susceptibility of DEL and NEV.

Make up a resistant proof carrier and contain following four site mutation: M41L, A62V, T69SSA and T215Y.This carrier, that is M41L-A62V-T69SSA-T215Y-RTV, the sensitivity phenotype is as follows: substantial decline (greater than 1000 times) takes place in the susceptibility to AZT, susceptibility to ddC is in wild-type level (2.1 times), susceptibility to ddI slightly descend (3 times), susceptibility to 3TC slightly reduces (8 times), to the susceptibility of abacavir slightly descend (10 times).This carrier has all increased the susceptibility of NNRTI class medicine, DEL (0.2 times) and NEV (0.8 times).

In addition, also the L210W sudden change is incorporated on another carrier.This carrier contains other four site mutation: M41L, A62V, T69SSA and T215Y.This carrier, that is M41L-A62V-T69SSA-T215Y-L210W-RTV, susceptibility performance to different pharmaceutical is as follows: substantial reduction (greater than 1000 times) takes place in the susceptibility to AZT, susceptibility to ddC slightly descend (2.1 times), susceptibility to ddI shows as slight reduction (3 times), susceptibility to 3TC has moderate reduction (22 times), and the susceptibility of d4T is had moderate reduction (13 times), and the susceptibility of abacavir is also had moderate reduction (16 times).This carrier has shown increase to the susceptibility of NNRTI class medicine, DEL (0.2 times) and NEV (0.6 times).The sensitivity phenotype of resistant proof carrier M41L-A62V-T69SSA-T215Y-L210W-RTV is similar in appearance to lacking L210W but contain the resistant proof carrier in other four mutational sites.

The T215Y sudden change is imported the carrier that contains A62V and T69SSA two sudden changes.Import T215Y and cause this carrier that substantive reduce (1000 times) are taken place the susceptibility of AZT, compare with A62V-T69SSA-RTV and reduce by 1000 times.The latter has only reduced by 7 times to the susceptibility of AZT.Observation is to the susceptibility of other inhibitor, and the result is similar to the carrier that does not have the T215Y sudden change.A62V-T69SSA-T215Y-RTV shows wild-type level (1.3 times) to the susceptibility of ddC, susceptibility to ddI slightly descend (2.5 times), susceptibility to 3TC has moderate decline (15 times), susceptibility to d4T slightly descends (7 times), and is slightly following to (10 times) to the susceptibility of abacavir.This carrier is to NNRTI class medicine, and the susceptibility of DEL (0.3 times) and NEV (0.6 times) increases.

In addition, the L74V that also will suddenly change imports the carrier contain A62V and T69SSA.To being tried the sensitivity test result of inhibitor, to get the result similar to observation post on the resistant proof carrier that does not contain the L74V sudden change, and similar to measured result on the carrier that contains T215 and A62V and T69SSA sudden change.Maximum variation is that the susceptibility that observes on A62V-T69SSA-L74V-RTV AZT is returned to the wild-type level.Carrier A 62V-T69SSA-L74V-RTV shows the horizontal susceptibility of wild-type (1.8 times) to ddC, ddI shown the horizontal susceptibility of wild-type (1.9 times), susceptibility to 3TC slightly descend (2.5 times), to the susceptibility of d4T slightly down with (1.5 times), to the susceptibility of abacavir slightly descend (3 times).This carrier all increases the susceptibility of NNRTI class medicine, DEL (0.4 times) and NEV (0.3 times).

Make up the resistant proof carrier, contain sudden change M41L, T69SSA, L210W and V75M.The V75M that will suddenly change imports the carrier that contains other four sudden changes (M41L, T69SSA, L210W and T215Y), compares with the carrier that does not have the V75 sudden change, to the not influence of drug susceptibility of carrier.Substantial decline (greater than 1000 times) takes place to the susceptibility of AZT in M41L-T69SSA-T215Y-L210W-V75M-RTV, susceptibility to ddC slightly descend (2.9 times), susceptibility to ddI slightly descend (3.5 times), susceptibility moderate to 3TC reduces (17 times), the susceptibility moderate of d4T is descended (11 times), the susceptibility of abacavir is taken place to fall (19 times) under moderate.This carrier all increases the susceptibility of NNRTI class medicine, DEL (0.25) and NEV (0.5 times).

Embodiment 5

Set up the cognation between sensitivity phenotype and the gene type assay: the d4T sudden change relevant with the complex combination of AZT-resistant mutation.

Make up the resistant proof carrier and carry out phenotype analytical from patient 98-757,98-844,98-937,98-964 and 98-966

Described in example 1, make up the resistant proof carrier from patient's sample 98-757,98-844,98-937,98-964 and 98-966.All these patients accepted in the past that the time do not wait comprises the pharmacological agent of d4T in class.Isolated viral RNA carries out reverse transcription-pcr, obtains one section fragment from the patient.This fragment comprises the virus sequence of proteins encoded enzyme total length and reversed transcriptive enzyme 1-313 position.This patient fragment of originating is inserted a virus vector that contains reporter gene, obtain some resistant proof carriers, be designated as RTV-757, RTV-844, RTV-937, RTV-964 and RTV-966 respectively.Use a kind of phenotypic assay method, on these resistant proof carriers accurately quantitatively its to the sensitivity levels of one group of antiretroviral drugs.This group antiretroviral drugs comprises some medicines (AZT, 3TC, d4T, ddI, ddC and abacavir), some NNRTI class medicines (delavirdine and nevirapine) and some the PRI class medicines (indinavir, nelfinavir, ritonavir and saquinavir) that is commonly referred to as the NRTI class.Each trial drug is measured its half-inhibition concentration IC50.Check the susceptibility of patient's virus, and compare with known drug susceptibility model to each medicine.On each these RTV storehouse, observe the drug susceptibility of d4T, compare to wild-type contrast RTV decline has all taken place.Further check patient's sample, find out the genotype relevant and change with viewed d4T susceptibility model.

Measure the genotype of patient RTV DNA

Use ABI chain terminator technology to measure the nucleotide sequence of resistant proof carrier automatically.The gained nucleotide sequence compares with the conserved sequence (New Mexico Loews Ah Georg Lammers HIV sequence library) of wild-type branch B HIV-1, finds out the sequence different with control sequence.RTV-757 contains V35I, D67N, T69D, K70R, V106A, V108I, L109V, Y181C, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I and R284K.106, the existing wild-type of nucleotide sequence of 108 and 109 each sites appearance also has the nucleotide sequence of sudden change.RTV-884 contains following sudden change: M41L, D67N, T69D, I135V, L210W, T215Y and K219N.RTV-937 contains following sudden change: P1L, P9R, K20R, V35T, K64Y, D67N, K70R, V75M, G99R, I135V, K173E, Y188L, R211H, T215F, D218E and K219Q.RTV-964 contains following sudden change: M41L, K43E, D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E and K219N.RTV-966 contains following sudden change: K20R, T39D, M41L, E44D, D67N, L74V, A98S, V118I, 1135T, K166R, K173T, M184V, G196E, L210W, R211K, T215Y, D218E, K219R, L228H, V245E, K277R, T286A, A288T and V293I.The nucleic acid of all these resistant proof carriers all contains one or several sudden changes; Sudden change (the M41L related before these sudden changes comprise with the AZT-resistance, D67N, L210W, T215Y/F and K219Q/R), the sudden change related (T69D and L74I/V) with the ddC-resistance, sudden change (the V106A related with NNRTI class drug resistance, V108I, Y181C/I and Y188L), the sudden change related (M184V) with the 3TC-resistance, perhaps former still unidentified sudden change (P1L, P9R, K20R, T39D, K43E, E44D, K64Y, V75M/S, G99R, L109V, V118I, K173E/T, I202T, R211H/T, D218E, K219E, H221Y, L228H, L283H, R284K and A288).Known is polymorphisms of HIV wild-type (medicaments insensitive) in these site mutation of V35I/T, A98S, 1135V/T, K166R, G196E, T200A, R211K, F214L, V245E, K277R, T286A and V293I.

On these patient's samples, cause sudden change that d4T susceptibility descends and unclear.Some patient's sample, it is identical to contain the sudden change that many and patient find on one's body; Yet the susceptibility that these patients do not show d4T reduces.

Rite-directed mutagenesis

Make up the resistant proof carrier with the rite-directed mutagenesis means, contain five (M41L, D67N, K70R, L210W, T215Y and K219Q) or six sudden changes that (M41L, D67N, K70R, L210W, T215Y and K219Q) is relevant with the AZT-resistance.Mensuration is to the sensitivity phenotype of NRTI class medicine.Observe at these AZT (75-180 doubly) and the susceptibility of d4T (2 times) are significantly descended.

Embodiment 6

Identify that HIV-1 reversed transcriptive enzyme amino acid changes, prediction is to the reaction of efabirenz

Phenotype that HIV-1 reversed transcriptive enzyme 69 amino acids change and the cognation between the genotype

In one embodiment of the invention, use following method and estimate the effect that HIV-reverse transcriptase protein 69 amino acids change.These methods comprise: (i) gather biological specimen from the hiv-1 infected individuals; Whether (ii) analyze the nucleic acid of biological coding HIV-1 reversed transcriptive enzyme undergos mutation at 69 bit codons.(iii) d4T susceptibility decline river AZT susceptibility reduces, 69 bit codon mutations be correlated with (T69SSX); This sudden change can Individual existence, perhaps is in the background (for example, M41L, A62V, D67N, K70R, L74V, V75I/M, T215Y/F, K219Q) of other NRTI-resistant mutation.

Biological specimen comprises that whole blood, blood moiety comprise peripheral blood lymphocytes (PBMC), serum, blood plasma (with preparations such as different antithrombotics such as EDTA, citric acid-dextran, heparin), tissue slice, cerebrospinal fluid (CSF) or other cell, tissue or body fluid.In another embodiment of the present invention, can directly from biological specimen, separate HIV-1 nucleic acid (geneome RNA) or reverse transcriptase protein, perhaps carry out later in separation and purification virus particle from biological specimen.Whether analyze HIV-1 reversed transcriptive enzyme 69 amino acids and suddenly change, available following several methods, for example directly the viral nucleic acid of coding reversed transcriptive enzyme is identified if carrying out, perhaps direct identifying virus reverse transcriptase protein itself.Available following listed method is determined reversed transcriptive enzyme 69 amino acids: utilize classical or new-type protein sequencing technology, antibody epitope recognition technology or other specific combination albumen or compound, directly identify reverse transcriptase protein; In addition, can also identify that HIV-1 reverse transcriptase protein encoding sequence amplified production determines HIV-1 reverse transcriptase protein 69 amino acids.HIV-1 nucleic acid is increased, can carry out with the following method, comprise reverse transcription-pcr, NASBA, SDA, RCR or 3SR.Operator with common skill just will know these methods.Whether nucleic acid 69 bit codons of analysis of encoding HIV-1 reversed transcriptive enzyme undergo mutation, can be directly to nucleic acid sequencing, comprise many primer extensions-chain termination method (Sanger, ABI/PE and Visible Genetics) or splitting of chain method (Maxam and Gilbert), perhaps nearest other sequencing technologies of developing, for example time-of-flight mass spectrometry (TOFMS) (MALDI-TOF), mass spectroscopy (Sequenom, Gene Trace Systems).In addition, the nucleotide sequence of mensuration coding 69 amino acids can be used various hybridizing methods and carry out, for example gene chip hybridization sequencing (Affymetrix), line probe assay (LiPA; Murex) and differential hybridization (Chion).

In a preferential embodiment of the present invention, prepare the resistant proof carrier from biological specimen and carry out sensitivity phenotype's test, analyzing HIV-1 reversed transcriptive enzyme 69 amino acids is wild-type or mutant.In one embodiment, gather plasma sample, purified virus RNA carries out reverse transcription amplification and goes out one section fragment from the patient (PDS).This section fragment coding virus protease and reversed transcriptive enzyme zone.Then, connect and bacterium transforms, this patient fragment insertion of originating is contained the virus vector of reporter gene, so obtain a resistance test carrier via DNA.Separation resistance test carrier from bacterial cultures carries out the phenotype sensitivity test as described in example 1 above.The patient's sample that contains T69SSX sudden change/insertion is carried out the phenotype sensitivity test, the results are shown in Figure5.From the HIV-1 proteolytic enzyme and the reversed transcriptive enzyme zone nucleotide sequence in the patient source that patient's sample 621,690,770,771 obtains, use fluoroscopic examination chain termination cycle sequencing (ABI/PE) and measure its sequence.This method is used to measure the conservative nucleotide sequence (representing accurate the genus) of the various HIV-1 variant mixture combined sequence of representative in the individual patients sample, and is used for measuring the nucleotide sequence of single variant.

The phenotype sensitivity maps of patient's sample and directed mutants show d4T susceptibility significantly decline and the increase of AZT-susceptibility.The two is all relevant with following sudden change: Serine, Serine, Serine (SSS) that coding HIV-1 reversed transcriptive enzyme is 69, the sudden change of Serine, Serine, L-Ala (SSA) or Serine, Serine, glycine (SSG) encode series; And the sudden change of some subclass of above-mentioned site.These sites before comprising the sudden change (41,67,70,74,75,184,210,215 and 219) relevant with NRTI class drug resistance or in these patients, observe but before the site (1,6,9,20,21,33,39,43,44,64,68,99,109,115,118,138,158,167,173,174,177,179,196,202,211,218,221,228,240,241,283,284,288,291 and 297) of never identifying.

69 contain the patient's sample that inserts sudden change, and its phenotype sensitivity maps shows AZT, 3TC, ddC, ddI, d4T and abacavir.

The phenotype of HIV-1 reversed transcriptive enzyme 62,75,77,116 and 151 amino acid mutations and genotypic cognation

The phenotype sensitivity maps and the directed mutants of patient's sample show: if HIV-1 reversed transcriptive enzyme 62,75,77,116 and 151 these sites, perhaps the subclass in these sites contains following amino acid 62V, 75I, 77L, 116F or 151M, then to NRTI class drug susceptibility significantly descend (AZT, ddC, ddI, 3TC, d4T and abacavir).Additional mutations occurs 41,67,69,184,210,215 and 219 and can further modify (increase or reduce) susceptibility NRTI class medicine.

Sudden change relevant with the AZT-resistance before setting up in the HIV-1 reversed transcriptive enzyme is to the phenotype of d4T resistance and the cognation between the genotype

The phenotype sensitivity maps and the directed mutants of patient's sample show: if the former and significantly reduced site of AZT-resistance appearance sudden change (41,67,70,210,215 and 219), and in the past with the subclass (74 of the susceptibility reduction related locus of other NRTI class medicine, 75,184) or with aforementioned measure on patient's the virus obtain before and have the sudden change identified relevant site (1,6,9,20,21,33,39,43,44,64,68,99,109,115,118,138,158,167,173,174,177,179,196,202,211,218,221,228,240,241,283,284,288,291,297).

Embodiment 5

Measure the genotype of patient RTV DNA

Use ABI chain terminator technology to measure the nucleotide sequence of resistant proof carrier automatically.The gained nucleotide sequence compares with the conserved sequence (New Mexico Loews Ah Georg Lammers HIV sequence library) of wild-type branch B HIV-1, finds out the sequence different with control sequence.RTV-757 contains V35I, D67N, T69D, K70R, V106A, V108I, L109V, Y181C, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I and R284K.106, the existing wild-type of nucleotide sequence of 108 and 109 each sites appearance also has the nucleotide sequence of sudden change.RTV-884 contains following sudden change: M41L, D67N, T69D, I135V, L210W, T215Y and K219N.RTV-937 contains following sudden change: P1L, P9R, K20R, V35T, K64Y, D67N, K70R, V7bM, G99R, I135V, K173E, Y188L, R211H, T215F, D218E and K219Q.RTV-964 contains following sudden change: M41L, K43E, D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E and K219N.RTV-966 contains following sudden change: K20R, T39D, M41L, E44D, D67N, L74V, A98S, V118I, I135T, K166R, K173T, M184V, G196E, L210W, R211K, T215Y, D218E, K219R, L228H, V245E, K277R, T286A, A288T and V293I.The nucleic acid of all these resistant proof carriers all contains one or several sudden changes; Sudden change (the M41L related before these sudden changes comprise with the AZT-resistance, D67N, L210W, T215Y/F and K219Q/R), the sudden change related (T69D and L74I/V) with the ddC-resistance, sudden change (the V106A related with NNRTI class drug resistance, V108I, Y181C/I and Y188L), the sudden change related (M184V) with the 3TC-resistance, perhaps former still unidentified sudden change (P1L, P9R, K20R, T39D, K43E, E44D, K64Y, V75M/S, G99R, L109V, V118I, K173E/T, I202T, R211H/T, D218E, K219E, H221Y, L228H, L283H, R284K and A288).Known is polymorphisms of HIV wild-type (medicaments insensitive) in these site mutation of V35I/T, A98S, I135V/T, K166R, G196E, T200A, R211K, F214L, V245E, K277R, T286A and V293I.

Rite-directed mutagenesis

Embodiment 6

The phenotype sensitivity maps and the directed mutants of patient's sample show: if the former and significantly reduced site of AZT-resistance appearance sudden change (41,67,70,210,215 and 219), and in the past with the subclass (74 of the susceptibility reduction related locus of other NRTI class medicine, 75,184) or with aforementioned measure on patient's the virus obtain before and have the sudden change identified relevant site (1,6,9,20,21,33,39,43,44,64,68,99,109,115,118,138,158,167,173,174,177,179,196,202,211,218,221,228,240,241,283,284,288,291,297).The table of the phenotype relevant with the specific sudden change that imports RTV

Observed average sensitivity in containing the resistance test carrier based on HIV of specific sudden change

Property reduction multiple (detection duplicate subnumber)

Claims

1. method of estimating patient's nucleoside reverse transcriptase antiretroviral therapy validity that HIV infects comprises:

(a) patient who infects from HIV gathers plasma sample; With

(b) determining whether plasma sample comprises is coded in the nucleic acid that 69 bit codons have the hiv reverse transcriptase of sudden change/insertion; Wherein Tu Bian existence is with relevant to the reduction of d4T susceptibility.

2. the process of claim 1 wherein sudden change/insertion encoding serine-Serine-L-Ala at 69 bit codons.

3. the process of claim 1 wherein sudden change/insertion encoding serine-Serine-glycine at 69 bit codons.

4. the process of claim 1 wherein sudden change/insertion encoding serine-Serine-Serine at 69 bit codons.

5. the process of claim 1 wherein that reversed transcriptive enzyme has additional mutations at 41 and 215 bit codons.

6. the method for claim 5, wherein at 41 bit codon mutations coding leucine (L), 215 bit codon mutations coding tyrosine (Y).

7. the process of claim 1 wherein that the patient of HIV infection is just accepting the treatment of antiretroviral preparation.

8. method of estimating patient's antiretroviral therapy validity that HIV infects comprises:

(a) patient who infects from HIV gathers biological sample; With

(b) determining whether this biological sample comprises is coded in the nucleic acid that has the hiv reverse transcriptase of one or more codon mutations in the codon that is selected from 62,75,77,116 and 151; Wherein Tu Bian existence is with relevant to the reduction of d4T susceptibility.

9. the method for claim 8, wherein 62 bit codons of sudden change coding Xie Ansuan (V).

10. the method for claim 8, wherein 75 bit codons of sudden change coding Isoleucine (I).

11. the method for claim 8, wherein 77 bit codons of sudden change coding leucine (L).

12. the method for claim 8, wherein 116 bit codons of sudden change coding tyrosine (Y).

13. the method for claim 8, wherein 151 bit codons of sudden change coding methionine(Met) (M).

14. the method for claim 8, wherein the patient of HIV infection is just accepting the treatment of antiretroviral preparation.

15. the method for claim 8, wherein this reversed transcriptive enzyme be selected from 41,67,210,215,219,184,69 and 215 or their one or more codons of combination in have additional mutations.

16. the method for an evaluate candidate HIV antiretroviral drugs compound biological effectiveness comprises:

(a) will contain the originate resistance test carrier of a fragment and a reporter gene of patient and import host cell, this patient fragment of originating further comprises one again in one or the sudden change of several codons being selected from 62,75,77,116 and 151; With one in one or the sudden change of several codons being selected from 41,67,210,215,219,184 and/or 69;

(b) cultivation is from the host cell of step a;

(c) expression of examining report gene in the target host cell;

(d) comparison step (c) the two reporter gene expression of surveying when not adding antiretroviral candidate medicine implementation step (a)-(c);

Wherein, at step (a)-(c); Step (b)-(c); Or there is the antiretroviral candidate medicine of test concentrations in the step (c).

17. a method of assessing HIV antiretroviral candidate drug compounds biological effectiveness comprises:

(a) import one in host cell and have the originate resistant proof carrier of a fragment and a reporter gene of a patient, this patient fragment of originating further comprises the sudden change of the sudden change/insertion of one 69 bit codon and 41 and 215 bit codons;

(b) host cell in the culturing step (a);

(c) expression of examining report gene in the target host cell;

18. resistant proof carrier, fragment and a reporter gene of containing one section HIV patient source, this patient fragment of originating further is included in the reversed transcriptive enzyme that 69 bit codons have a sudden change again, and wherein, the expression of reporter gene depends on this patient fragment of originating.

19. resistant proof carrier, comprise one section HIV patient originate fragment and a reporter gene, this patient fragment of originating further comprises a reversed transcriptive enzyme in the sudden change that is selected from one or several codon in 62,75,77,116 and 151 again, wherein, the expression of reporter gene depends on this patient fragment of originating.

20. the method for claim 5, wherein, reversed transcriptive enzyme has an extra sudden change at 210 bit codon places.

21. the method for claim 20, wherein, the sudden change coding colors propylhomoserin (W) at 210 bit codon places.

22. the method for claim 5, wherein, reversed transcriptive enzyme has an extra sudden change at 62 bit codon places.

23. the method for claim 22, wherein, the sudden change coding Xie Ansuan (V) at 62 bit codon places.

24. the method for claim 22, wherein, reversed transcriptive enzyme has an extra sudden change at 210 bit codon places.

25. the method for claim 22, wherein, at the sudden change coding colors propylhomoserin (W) of 62 bit codons.

26. the method for claim 20, wherein, reversed transcriptive enzyme has an extra sudden change at 75 bit codon places.

27. the method for claim 26, wherein, at the sudden change encoding serine (S) or the methionine(Met) (M) of 75 bit codons.

28. the process of claim 1 wherein that reversed transcriptive enzyme has an extra sudden change at 62 and 215 bit codon places.

29. the method for claim 28, wherein, at the sudden change at 62 bit codon places coding Xie Ansuan (V), and at the sudden change coding tyrosine (Y) at 215 bit codon places.

30. the process of claim 1 wherein that reversed transcriptive enzyme has an extra sudden change at 62 and 74 bit codon places.

31. the method for claim 30, wherein, at the sudden change at 62 bit codon places coding Xie Ansuan (V) and at the sudden change coding Xie Ansuan (V) at 74 bit codon places.