CN101564408A - Medicinal composition containing korean ginseng for make hiv-1 have 5ltr/gag genetic defect - Google Patents

Abstract

The present invention relates to a medicinal composition which uses Korean Ginseng as active ingredient and is used for make HIV-1 have 5LTR/gag genetic defect. The medicinal composition of the present invention can induce 5'LTR and/or gag gene in HIV-1 to be defect by administration to HIV infection individual. Therefore, the medicinal composition of the present invention can be used for preventing and treating AIDS.

Description

Contain koryo insam be used to make HIV-1 have the pharmaceutical composition of 5 ' LTR/gag genetic flaw

Technical field

The present invention relates to a kind of contain as the koryo insam of effective ingredient be used to make HIV-1 have the pharmaceutical composition of 5 ' LTR and/or gag genetic flaw.

Background technology

HIV-1 (HIV (human immunodeficiency virus)-1) is acquired immune deficiency syndrome (AIDS) (AcquiredImmunodeficiency Syndrome; Be designated hereinafter simply as " AIDS ") the cause of disease.HIV-1 and HIV-2 belong to the lentiviridae (lentivirinae) of Retroviridae (retroviridae), and the infectious virus body has RNA (+) chromosome that is formed by two identical strands.During the HIV-1 infection cell, by the reverse transcriptase of above-mentioned RNA (+) coding, RNA is converted to double-stranded DNA, and this DNA is inserted in the chromosome of host cell, forms provirus.Under the pro-Virus State, virus is bred by transcribing viral gene production necessary proteinic mRNA in duplicating and assembling.

HIV-1 genome (9.2kb) constitutes (with reference to Fig. 8) by structural gene (structural gene) and tat, rev, nef, vif, the episome (accessory gene) of vpu, vpr etc. and the long terminal repeat (long terminal repeat sequence (LTRs)) at two ends of gag, pol, env.Gag and pol protein are decomposed to form presoma protein, and env protein becomes gp160 precursor protein (precursor protein), resolves into gp120 and gp41 in assembling process.Do not have symptom during initial infected by HIV, perhaps show as the symptom that resembles flu, virus latent infection (latent infection) in cd4 t cell, macrophage, microglia etc. is in latency for a long time.Antibody 3-20 week after infection to gp120 occurs, and after this can diagnose to have or not infection.Through incubation period of 2-10, induce variation in the immune system, before the immunodeficiency symptom occurring, show as prodromal stage symptom (AIDS related complex (ARC)-fatigue, have a fever, lose weight, suffer from diarrhoea).Make the quantity of cd4 t cell be reduced to below the 200/ul because HIV infects, show the symptom of immunodeficiency this moment, thereby be defined as AIDS clinically.

Koryo insam (Panax ginseng C.A.Meyer (Panax schinseng Nees)) belongs to Araliaceae (Araliaceae) Panax's perennial perennial root herbaceous plant, is used for the auxiliary treatment of various diseases over thousands of years in the various countries, East Asia.Up to now many pharmacological evaluation show that koryo insam has following activity: cholesterol reducing, anti peroxidation of lipid, bring high blood pressure down, increasing blood flow, expansion of cerebral vascular, promote cardiac function, anti-arrhythmia, antithrombotic, the chronic renal failure therapeutic effect, immunoregulation effect, memory reinforcing, promote the brain metabolism, compressive resistance, antioxidation, anti-aging effects, antiulcer and inhibition gastric secretion, anti-diabetic, detoxifcation, increase hepatocyte enzyme, treatment asthma, anti-inflammatory, analgesic activity, the treatment anemia, promote reproductive performance, reduce the blood determining alcohol, antiallergic, anticancer etc.

In addition, koryo insam and Radix Ginseng Rubra (Red Ginseng) are distinguished according to processing method, and so-called koryo insam is called trepang with dried koryo insam under raw situation, and undried koryo insam is called the water ginseng.Radix Ginseng Rubra is by koryo insam being steamed and drying, make that moisture is below 14%, promoting dark brownization reaction to form the form of the hard that is shown as dark umbrinaceous color in preparation process, and remain circular koryo insam.Therefore, koryo insam is difficult to long preservation under the state of the water ginseng that contains the moisture about 75%, cause that owing to microbial contamination the various enzymes that corruption or koryo insam self contain make the koryo insam composition decompose in circulation status; And Radix Ginseng Rubra has by moisture minimizing behind the dry processing, can prevent antibacterial, mouldy and contamination by micro, and volume and weight reduces the advantage of preserving easily, transporting.Because as the quantitative approach of the saponin of the representative actives of koryo insam and the development of quality management, thereby can in the course of processing, suppress the decomposition of saponin to greatest extent.Except saponin, can also further generate maltol, ginsenoside Rh2's etc. effective ingredient.That is, the main component of koryo insam is a saponin, and Radix Ginseng Rubra is the koryo insam that does not change effect after the processing.

Radix Ginseng Rubra especially has sedation and excitation for nervus centralis; act on blood circulation and have prophylaxis of hypertension and arteriosclerotic effect; in addition; hemoposieis and blood glucose value are reduced; the protection liver; it is effective to sexual function and reproductive function indirectly to act on hormonal system, has antiinflammatory and antitumor action, have the defence lonizing radiation effect, play a part to protect and soft and smooth skin.In addition, in the effect of Radix Ginseng Rubra, importantly increase for defence capability, make the effect that health more easily adapts to from the excessive tear disease of the various illeffectss of surrounding, various pressure etc. as adapting to plain (adaptogen) effect.So far, to the composition and the pharmacological effect of Radix Ginseng Rubra, Korea S has had a lot of researchs both at home and abroad, has reported that especially Radix Ginseng Rubra has antioxidant activity (Network オ Application ョ Application Off Application な ど, high Korea Radix Ginseng Hui Chi, 24 (1), 29-34,2000).

In addition, before the inventor after deliberation the HIV-1 infected patient is carried out independent koryo insam (KRG, Korean red ginseng) treatment (referring to list of references 1-4), later also usefulness koryo insam and azidothymidine AZT (ZDV in 1991, zidovudine) treatment (referring to list of references 5) and 1997 later and with the treatment (HAART, highly active antiretroviral drug therapy) (referring to list of references 6) of koryo insam with very strong efficient antiretroviral drugs.Through during the clinic application (referring to list of references 1-7), show significant reverse dependency relation (referring to list of references 8) between the tempo (progression rate) of KRG treatment and acquired immune deficiency syndrome (AIDS) (AIDS).There are inverse relationship in the aberration rate of HIV-1 gene and KRG treatment.And the KRG treatment can delay the drug-fast development to azidothymidine AZT.According to the inventor's result of study, the characteristic of other of KRG treatment and therapy or patient etc. is irrelevant, the minimizing that can slow down cd4 t cell.Find after having analyzed the combined therapy of KRG and HARRT, medicament-resistant mutation does not take place, can carry out consistent virus by this combined therapy and regulate (referring to list of references 7).

People (the long-term nonprogressor that the inventor's research institution (cohorts) does not worsen in this explanation disease between long-term; LTNPs) or long term survival person (long-term surviviors; LTSs) reason of the slow progress of visible acquired immune deficiency syndrome (AIDS).Recently, the inventor has made report (referring to list of references 9) to the ratio and the relatedness between the KRG picked-up time of the extensive defective of the nef gene of 10 LTS continuing to accept the KRG treatment.Compare the product that increases in these 10 long term survival persons' the nef gene, the result is in the product of gag zone and the amplification of 5 ' LTR zone, and the base sequence of provirus total length is to show two bands (band) (wild type and the band (band) that does not shorten) at high proportion.

The extensive defective of nef gene for (referring to list of references 10-18) among the LTNP has been carried out many researchs, still, has only reported the dependency of extensive defective in gag zone and 5 ' LTR zone up to now in several occasions.More particularly, reported one defective (referring to list of references 19) in three amino acid code in the gag gene of a LTS who has infected the HIV subtype C.In other research, two of the philtrum of 24 not treatment experience show as the defective of 16bp and the little defective of 9bp (referring to list of references 20) in 5 ' LTR.But, not about the report of the possible contact between the genetic flaw of keeping slow progress that the state that do not worsen between long-term or HIV infect and 5 ' LTR and gag zone.

List of references:

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Summary of the invention

The inventor gives Radix Ginseng Rubra for a long time with the 5 ' LTR among the affirmation HIV-1 and the defective of gag gene by the patient who infection is had HIV-1, thereby finishes the present invention when the Therapeutic Method of research AIDS.

The purpose of this invention is to provide a kind of contain as the koryo insam of effective ingredient be used to make HIV-1 have the pharmaceutical composition of 5 ' LTR and/or gag genetic flaw.

In order to realize purpose of the present invention, the invention provides a kind of contain as the koryo insam of effective ingredient be used to make HIV-1 have the pharmaceutical composition of 5 ' LTR and/or gag genetic flaw.

Describe the present invention below in detail.

The present invention is characterised in that the picked-up of having found out koryo insam can make have 5 ' LTR/gag gene among the HIV-1 of close ties to produce defective with virus grow and the AIDS cause of disease.

Under the preferable case, the invention is characterized in that described koryo insam is a Radix Ginseng Rubra.

In one embodiment of the invention, in order to determine between the generation of the extensive defective (g Δ) in 5 ' LTR and gag zone and KRG picked-up, whether have relatedness, the inventor is from KRG (altogether 13,364 ± 5,364g) 10 long term survival person (the long-term surviviorss of treatment more than 12 years; LTSs) and not picked-up or absorb on a small quantity koryo insam (altogether 1,436 ± 1, checked among 8 long term survival persons (matched group) 027g) around 1,125 base sequence in this 5 ' LTR and gag zone.Among above-mentioned 10 LTS, obtain 189 PCR products in 80 peripheral blood lymphocytes (PBMC) sample, 71 (37.6%) in 44 (55%) among 80 PBMC and 189 the PCR products show extensive defective (g Δ) altogether.The PBMC of discovery 55% and 37.6% PCR product in 10 LTS, still, the value of 8 matched group LTS is respectively 30.3% and 14.8%.Such difference has statistical significance (P＜0.05, and P can be 0).In addition, the dcc gene ratio of not taking 28 general matched group patients of Radix Ginseng Rubra is that benchmark is respectively 13.3% and 8.3% (reference example 3, Fig. 1 to Fig. 3) with PBMC and PCR product.

Therefore, can induce 5 ' LTR/gag gene among the HIV-1 to produce defective, thereby can in the prevention of AIDS and treatment, work by give Radix Ginseng Rubra to individuality.

The Radix Ginseng Rubra that is used as the effective ingredient of pharmaceutical composition of the present invention can prepare by known method.

Generally speaking, Radix Ginseng Rubra is through carrying out the steaming and decocting stage that steam steams and the ginseng of the water after the steaming and decocting is carried out exsiccant drying stage making to water ginseng (moisture about 75%) under certain temperature condition.The Radix Ginseng Rubra that makes is like this carried out heating extraction, make red ginseng extract, this red ginseng extract is concentrated, can make the Radix Ginseng Rubra concentrated solution.

In addition, among the present invention, above-mentioned red ginseng extract or concentrated solution can use separately, also can add more than one the carrier, excipient or the diluent that pharmaceutically allow, use after carry out preparationization according to the method for routine.Above-mentioned " pharmaceutically allowing " is meant that the physiology goes up permission, is meant when human body is carried out administration, generally can not cause the anaphylaxis of the intestines and stomach obstacle, dizziness etc. or reaction similarly.In addition, the Radix Ginseng Rubra of the present invention of aforesaid preparationization can carry out administration by suitable route of administration.Suitable route of administration can be an oral administration.

Preferably carry out preparationization according to method well known in the art with the form of the preparation that is used for oral administration of powder, granule, tablet, pill, sugar coated tablet, capsule, liquid preparation, gel, slurry agent, suspension etc.For example, preparations for oral administration be by with active component with after solid excipient mixes, after its pulverizing and adding proper assistant, be processed into granulate mixture, thereby can obtain tablet or sugar coated tablet.Appropriate excipients for example can be the saccharide that comprises lactose, glucose, sucrose, Sorbitol, mannitol, xylitol, erythritol and maltose alcohol etc., the starch based that comprises corn starch, wheaten starch, rice starch and potato starch etc., the cellulose family that comprises cellulose, methylcellulose, sodium carboxymethyl cellulose and hydroxypropyl emthylcellulose etc. comprises the filler of gelatin, polyvinylpyrrolidone etc.In addition, according to circumstances also can add the disintegrating agent that comprises crospolyvinylpyrrolidone, agar, alginic acid or sodium alginate (Sodium Alginate) etc.In addition, pharmaceutical composition of the present invention can also contain deflocculant, lubricant, wetting agent, spice, emulsifying agent and antiseptic etc.In addition, the Radix Ginseng Rubra as the effective ingredient of pharmaceutical composition of the present invention can be commercially available.The Red Ginseng capsule that has used Korea Ginseng Corporation to sell in one embodiment of the invention.

Pharmaceutical composition of the present invention can be used for mammal, comprises that especially people's animal carries out administration.Preferably there is the patient of HIV-1 to carry out administration to infection with effective dose.Above-mentioned " effective dose " is meant for making the nef gene among the HIV-1 produce the resultful amount of defective in external or organism.

The effective dose of pharmaceutical composition of the present invention need be considered the multiple factors such as severity, diet and excretion rate of route of administration, treatment number of times, the age for the treatment of necessary individuality, body weight, health status, sex, disease, and those skilled in the art can determine suitable effective dosage according to specific purposes.Under the preferable case, the effective dose that contains the pharmaceutical composition of Radix Ginseng Rubra of the present invention, the male is the 50-100 mg/kg/day, the women is the 20-70 mg/kg/day.

Pharmaceutical composition of the present invention can continue medication behind diagnosis of case and delay the AIDS advancing of disease.

In addition, among the present invention, the defective of above-mentioned 5 ' LTR and/or gag gene can be defined as have the premature termination codon (premature stop codon), lack start codon (initiation codon).

In one embodiment of the invention, from the amplified production that 10 LTS generate, observe as non-methionine start codon, premature termination codon that G to A hypermutation different (hypermutation) causes and the genetic flaw (embodiment 4) of little defective.In each patient and 10 LTS, defective, repetition or insertion big or small incomplete same.Even in 8 matched group LTS, also observed the genetic flaw (Fig. 4) that premature termination codon, non-methionine start codon cause.

Under the preferable case, 5 ' LTR of the defective that produces by method of the present invention and/or the sequence that the gag gene can be expressed as: the GenBank accession number is EF 370172, and EF 370173, EF 370175, and EU 047612, and EF 370184, EF 370186, and EF 370187, and EF 370188, EF 370191, and EF 370192, and EF 370193, EF 370196, and EF 370197, and EF 370199, EF 370201, and EF 370200, and EF 370202, EF 370205, and EF 370207, and EF 370211, EF 370213, and EF 3702115, and EF 3702116, EF 3702118, and EF 370219, and EF 370221, EF 370223, and EF 370224, and EF 370225, EF 370226, and EF 370227, and EF 370228, EF 370231, and EF 370235, and EF 370237, EF 370239, and EU 047647, and EF 370245, EF 370250, and EF 370251, and EF 370252, EF 370253, EF 370256, and EF 370259, and EF 370262, EF 370261, EF 370265, and DQ 295196, and EF 370275, EF 370276, EF 370277, and EF 3702778, and EF 370279, EF 370282, EU 047673, and EF 370283, and EF 370285, EF 370286, EF 370287, and EF 370288, and EF 370292.

As mentioned above, pharmaceutical composition of the present invention can induce HIV-1 to have 5 ' LTR and/or gag genetic flaw by the HIV infected individuals is carried out administration.Therefore, the present invention can be used for prevention and the treatment of AIDS.

Description of drawings

Fig. 1 represents not carry out antiretroviral therapy, gives the KRG of minimum or does not give 8 matched group LTS (Fig. 1 k～r) and accept 10 LTS (figure of the variation of the cd4 t cell number of Fig. 1 a～j) of the KRG administration of maximum of KRG.Give to utilize bar (bar) to represent during the Radix Ginseng Rubra.Three PCR reaction is accompanied by the amplification of a sample, and the sample that uses in the sequence of 5 ' LTR and gag gene is represented with downward arrow with to upward arrow in the bottom respectively with the sample of expression 5 ' LTR and the extensive defective of gag gene.In two groups, the cd4 t cell number reduced after 10 years.Thus, except the KRG dosage, there is not marked difference between two groups.

Fig. 2 is the figure of the sequence analysis of the 5 ' LTR of expression from the peripheral blood lymphocytes that 3 LTS that make a definite diagnosis at first obtain and gag.The product that the electrophoresis of separation by 1% agarose gel increases.PCR product from patient 87-05,89-17,90-05 is represented with road (lane) 1-10,11-18,19-31 respectively.Patient 87-05 have 4 kinds of wild types (wild type, road 1,3,6,9), with 3 repeat band ( road 2,7,10) and five such short being with of the short band of two wall scrolls ( road 4,8).Patient 89-17 represents 7 short bands ( road 11,12,13,14,16-18), and wherein, road 14 is short bands (14%).Patient 90-05 has 7 short bands ( road 19,22,23,24,26,29,30), and wherein, road 22,24,26,29,30 shows as a short band (71%).

(M: labelling, WT: wild type band (wild type band))

Fig. 3 is the relatively figure of 3 groups of extensive defect frequencies of expression.The ratio of representing the peripheral blood lymphocytes sample of extensive defective, KRG LTS (55%) is not only than other 28 patients (13.3%) height, and than 26.7% height of 9 matched group LTS.And to the ratio of the male PCR product of extensive defective, not only 28 patients (8.3%) than other are high significantly to have given 10 LTS (37.6%) of KRG, and 13.9% high significantly than 8 matched group LTS.The wife who also comprises 87-05 and 90-50 among 28 patients.And, even their part does not surpass 10 years after being diagnosed as HIV-1, also may become LTS.

Fig. 4 is the figure that the proteinic anticipation aminoacid sequence of representational part gag among 10 LTS of HIV-1 has been infected in expression.The non-proteinic common aminoacid sequence of gag that makes progress the patient from the U.S. is presented at the most above-listed (referring to list of references 23), and the anticipation sequence of 10 LTS is as follows.The same section of point (dot) expression sequence, bar (bar) expression part defective or the extensive defective except that the variation part.In addition, the premature termination codon represented in question mark.Aminoacid sequence is represented by a literal code.The source of each sequence is confirmed by each patient's coding (initial two digits represents that HIV makes a definite diagnosis year, and the two digits after the hyphen is illustrated in the inherent number that Korea S distributes to each patient).The date of the sample of GenBank number correspondence separately as shown in Figure 7.Can't see 7 kinds of base sequences that in the gag gene, do not contain defective among Fig. 7.

Fig. 5 is the table that the clinic feature of 18 LTS that HIV-1 is arranged is infected in expression.Except last year, the degree of complying with of the patient 91-20 among the LTS and 90-05 are best equally, and his actual KRG dose buys by the individual and reached 22, more than the 422g.Patient 91-22 and 91-23 are infected HIV-1 owing to accepted blood that 91-20 provides.Except LTS 87-05, all patients are for infecting the Korea S branch (subclade) that HIV-1 hypotype B is arranged.

Fig. 6 is the feature of extensive defective of expression 18 LTS and 28 non-KRG administration patients' 5 ' LTR/gag gene.

Fig. 7 is that expression is compared the table of the frequency of the extensive defective of 5 ' LTR/gag gene with the extensive defective of nef gene.

The specific embodiment

The present invention will be described in more detail below by embodiment.But following examples are to be used for example that the present invention is illustrated, and the present invention is not subjected to the qualification of following examples, can carry out various modifications and change.

Embodiment 1

Experimental subject and KRG treatment

Seminar comprises: 10 long term survival person (LTSs of picked-up maximum KRG; Long-termsurviviors), 8 matched group LTS, 28 HIV infected patients that do not absorb KRG.

In this research, 10 LTS have been described in detail (referring to list of references 9) in the research before the inventor.Above-mentioned 10 LTS were all made a definite diagnosis between 1987 in March, 1996, in this research, except a patient (93-04), did not all give any antiretroviral therapy to other patients.This patient (93-04) has used ZDV (daily dose 200mg or 300mg) discontinuously in year August in April, 1993 to 1997.

8 matched group LTS are selected by the inventor's research institution (cohorts).Being assigned to patient among the above-mentioned matched group LTS was diagnosed as to infect before 1996 HIV-1 is arranged, they are under the situation of not carrying out strong antiretroviral drugs treatment, used minimum dose (1,526 ± 1, KRG 183g) or do not use KRG (patient 89-05 does not use KRG fully) fully.A unique patient 91-23 has used ZDV from a year August in April, 1993 to 1999.Patient 89-05 has gone to other country in 1997, therefore delete from research sample of the present invention.In 8 matched group LTS, comprise two donees (recipients, patient 91-22 and 91-23) that accepted long term survival person 91-20 blood transfusion.Patient 89-26 and 92-23 resist picked-up KRG.Patient 90-01 is for having carried out providing with compensation for 83 times the blood plasma supplier of blood plasma.This patient 90-01 is made supplier O (referring to list of references 21) by note.Therefore, when 10 high KRG picked-up LTS and 8 matched group LTS were compared, the most basic feature (baseline characteristics) was identical.

In other words, between 10 LTS and 8 the matched group LTS, except the intake difference of KRG, basic progression of disease speed (progression rate) or cd4 t cell number do not have marked difference.

Other 28 matched group patients have been investigated in addition.This 28 philtrum also comprises the wife of 87-05 and 90-50.Part among these 28 patients is the long term survival person.But they were made a definite diagnosis after 1997, therefore, and in the person's group that is not included in the long term survival.Each participant provides letter of consent.

To infection have the patient of HIV-1 carry out KRG treatment be since the second half year in 1991 the NIH's of Korea S.The Red Ginseng capsule that the KRG that uses in the experiment sells as Korea Ginseng Corporation.To the dosage on the one of each patient's KRG, the male is 5.4g (taking three times the capsule of each six 300mg in one day), and the women is 1.8g.Each patient's KRG administration time as shown in Figure 1.

The mean dose of the Radix Ginseng Rubra that 10 LTS take is for being 13,364 ± 5 during 155 ± 28 months, and the mean dose of the Radix Ginseng Rubra that 364g, 8 matched group LTS take is for being 1,436 ± 1 during 132 ± 29 months, 027g (Fig. 5).In 10 LTS and 8 matched group LTS, the annual dose of KRG respectively does for oneself 1,035g and 131g.In KRG absorption level, 7.9 times have statistical significance (P＜0.001).

Embodiment 2

Cd4 t cell number and blood plasma HIV-1RNA levels of replication (copy level)

At interval after 3 months to 6 months, each patient to embodiment 1 takes a blood sample, respectively with the peripheral blood lymphocytes in each sample and be combined with phycoerythrin (phycoerythrin, PE) and the antibody corresponding of Fluorescein isothiocyanate (FITC) with T4 antigen cultivate (Simultest reagent; BectonDickinson, San Jose, CA, USA).The level of cd4 t cell is measured by FACScan (Becton-Dickinson) flow cytometer (flow cytometry).(Roche Diagnostics, Branchburg NJ) measure (referring to list of references 7) to the HIV-1RNA concentration of blood plasma by Amplicor HIV-1Monitor kit.

10 LTS of Radix Ginseng Rubra and the cd4 t cell number of 8 matched group LTS have been taken in range estimation in per 6 months, and the result is same as shown in Figure 1.

The cd4 t cell number of accepting 10 LTS of maximum Radix Ginseng Rubra treatment dosage reduces to 245 ± 160/ul of measuring after 168 ± 29 months (a year reduce 22/ul, Fig. 1 a to Fig. 1 j) significantly from the 555 ± 208/ul that records at first.In addition, the cd4 t cell number of 8 matched group LTS reduces to 353 ± 297/ul of measuring after 132 ± 29 months (a year reduce 20/ul, Fig. 1 k to Fig. 1 r) from the 574 ± 222/ul that records at first.

Can confirm that the cd4 t cell number all reduced in a similar fashion in two groups in 10 years.

Embodiment 3

The amplification of part 5 ' LTR zone (region) and gag gene

Extract among the PBMCs that does not cultivate that proviral DNA obtains from embodiment 2.By utilized first round primer (first round primer) CE23 (5 '-tgtggatctaccacacacaaggctactt-3 '; 46 to 73; Serial number 1), 514 (5 '-tccagaatgctggtagggtatac-3 '; 1,617 to 1,640; Serial number 2), second take turns primer CE1 (5 '-cgagagctgcatccggagtacta-3 '; 297 to 319; Serial number 3), 512 (5 '-ctgcagcttcctcattgatggtc-3 '; 1,398 to 1,420; Serial number 4), third round primer 501 (5 '-gtgtggcctgggcgggactg-3 '; 380 to 399; Serial number 5) and 510 (5 '-gatgtaccatttgcccctgga-3 '; 1,202 to 1,222; Serial number 6) dual (few is triple) nido polymerase chain reactions (nested PCR), from No. 297 to 1, No. 421 (this and below, numbering derives from the sequence of HIV NL4-3) 1, the PCR product of 125bp is by each sample amplification (above-mentioned position from No. 297 to 1, No. 421 be 5 ' the LTR part of representative part and the part of gag gene).

Containing the maximum PCR reaction that each sample of negative control group once carries out is 4, and in order to increase the nef gene, and containing the maximum PCR reaction that each sample of negative control group once carries out is 5.Refining PCR product, directly ordering.PCR has pollution-free physical separation by the PCR product to monitor, and the sub-comparison of amino acid code that the physical separation of PCR product is carried out by BLAST retrieval, by manual alignment (manual alignment) and each formality of systematic analysis are carried out.

Experimental result is represented with meansigma methods ± standard deviation.Statistical significance carries out two tail t checks (Student ' s two-tailed t-test) with SPSS packageversion 12.0 and X 2 test (Chi-square test) is confirmed.

Experimental result is by becoming 189 PCR products (Fig. 1 a to Fig. 1 j) at different time points from 80 PMBC sample amplification that 10 LTS obtain.At most of time points, 10 all LTS show g Δ (Fig. 1) at 5 ' LTR/gag gene region.44 (55%) in 80 samples observe defective (Fig. 2).In 189 PCR products, 71 (37.6%) shows g Δ 5 ' LTR/gag.Wherein, the amplification of 23 (32.4%) only shows as a short band (short band), does not have wild type gene.The feature of g Δ 5 ' LTR/gag of 71 is summarized in Fig. 7.

5 PCR products (in January, 2007 from patient 87-05, July calendar year 2001 from patient 89-17, in October, 1993 from patient 90-05 and in February, 2006 and in May, 2004 obtain from patient 92-13) and 2 PCR products (Δ 86bp and the Δ 420bp from patient 90-50 acquisition in 2006 of obtaining from patient 90-05 in 1993 10) only contain g Δ (table 3) respectively in 5 ' LTR zone or in the gag gene.The part that defective in the remaining PCR product is positioned at the both-side ends of 5 ' LTR (contains 753 positions at least; Δ 37bp) and the start-up portion of gag gene (the Δ 29bp of No. 818 positions in HIV-1NL43).In other words, defective is more than the 66bp near the start codon of gag gene.

Therefore, be 36.5% (69/189) in the ratio of the g in 5 ' LTR zone Δ, be 34.9% (66/189) based on the ratio of the g Δ of gag gene.This illustrated the KRG administration to the influence of 5 ' LTR greater than influence to the gag gene.

Remove outside all otiose patient 91-20 of any sample and the 93-60, the original observed of the g Δ in 5 ' LTR and gag zone is early than the original observed of nef genetic flaw after KRG picked-up beginning.The interlude that begins to observe to initial g Δ from the picked-up of KRG is 26 months (19 months to 101 months).It is shorter that this observes required time than nef genetic flaw.

Patient 87-05 has infected HIV-1 hypotype B and the HCV from the blooc coagulation factor 9 of the pollution that produces abroad simultaneously.This patient begins to carry out the KRG administration in June, 1994.Though the little defective of 9bp is arranged in all amplified matters, in 10 LTS, has only this patient not show g Δ nef (referring to list of references 9).Surprisingly, the inventor has observed the g Δ in the initial useful sample that obtains in April, 1997, observed in 4 36 PCR products that obtain from 12 samples altogether 7 g Δs (Fig. 1 a to Fig. 1 j, Fig. 5).This patient does not show any symptom relevant with acquired immune deficiency syndrome (AIDS) since in April, 2007, this is do not survive through any antiretroviral therapy patient more than 20 years of Korea S's the first.

Patient 89-17 begins to carry out the KRG administration in December, 1991.But the degree of complying with for administration is not uniform.In July, 1993, a g Δ of report was an exception.Former because Δ 917bp by the 474bp of pol gene (in the opposite direction 4, of NL4-3 No. 296 to 4, No. 037) and gag gene (2,029bp to 1, dwell section 838bp) is replaced.This patient begins to carry out the HARRT treatment in February, 2002, and the cd4 t cell number is 513/ul in April, 2007.

Patient 90-05 is diagnosed as HIV-1 in February nineteen ninety and infects, and begins to absorb KRG in December, 1991.Treatment shows best degree of complying with to this patient for KRG in this research.Except 21 of supply, the KRG of 522g, this patient have also absorbed the KRG that oneself holds.The daily dose of KRG is 6.0g (one day twice, the capsule of each 10 300mg) under 95% degree of complying with.The original observed of g Δ compare January calendar year 2001 (the KRG administration begin the back 111st month, absorb KRG12 altogether, g Δ nef original observed 012g) take the morning 87 months (Fig. 6 a, 6b, 6c).Details such as Fig. 1 and shown in Figure 6 of the example of g Δ and 7 LTS of residue.

In addition, 88 PCR products of 33 PBMC samples that will obtain from 8 matched group LTS that append increase.13 g Δs (14.8% in the PCR product after the amplification) are present in 10 PBMC samples (altogether 30.3%).On PBMC sample and PCR product, 10 LTS compare 8 matched group LTS, and the g Δ (the former P＜0.05, latter P＜0.0001) that contains significantly high ratio (Fig. 3).

In addition, will increase at 60 PCR products that the clinic stage irrespectively never gives 30 PBMC samples obtaining among 28 patients of KRG.5 g Δs (8.3% in the PCR product after the amplification) are present among 4 PBMC (altogether 13.3%).

Embodiment 4

Genetic defect and little defective

Except extensive defective, the inventor has observed following genetic flaw, for example, and premature termination codon and little defective that non-methionine start codon, G to A hypermutation different (hypermutation) cause.In 10 LTS, patient 87-05 demonstrates the premature termination codon in 4 sequences (EU047601, EU047605, EU047607, EU047609), patient 89-17 also contains the isoleucine as premature termination codon and start codon (GenBank EF370193).Patient 90-18 has the insertion of 6bp in the gag gene, patient 92-13 has two repetitions (duplications) of 102bp (GenBank EF370252), at defective and G to the A hypermutation different (Genbank EF370256) of the 159bp in 5 ' LTR zone, this patient has the insertion (Fig. 4) of 6bp in 327 and 328 zones (GenBankEF370258) in addition.

In 8 matched group LTS, patient 91-22 has the isoleucine as start codon in premature termination codon or 3 sequences (GenBank EF370314, EU047654, EU047655).Patient 89-26 has two premature termination codons in a sequence (EU047635).

Embodiment 5

The frequency of different extensive defectives along with the cd4 t cell number

The inventor has analyzed the frequency of the extensive defective different along with the cd4 t cell number.Wherein, preferably the pcr amplification product of 10 LTS is counted below the 200/ul, 200-400/ul, divided into groups more than the 400/ul according to cd4 t cell.The frequency of extensive defective is 47.4% (9/19), 51.8% (14/27) and 61.8% (21/34) in having given 10 LTS of KRG, and this shows the some dependencies to the cd4 t cell number.But in 8 matched group LTS, extensive defective proportion is 44.4% (4/9), 20% (4/20), 50% (2/4), and 28 HIV-1 positive patients that do not give KRG in addition are 18.2% (2/11), 14.3% (1/7), 8.3% (1/12).

Discuss

The inventor pays close attention to the health of LTS or LTNP for a long time in Korea S, even its result does not exist useful host factor (host factor) the 32bp defective in CCR5 or Korean's HLA-B57, the inventor can observe the most LTS among 323 HIV-1 the infecteds that made a definite diagnosis in 31st since December in 1993.

More particularly, 9 HIV-1 infected patients were made a definite diagnosis in Korea S in 1987, and wherein three patients are still survived, and this three people all uses independent KRG treatment or also uses with HARRT.Long term survival person 87-05 is as one among these patients, and (Fig. 1 a) to begin to absorb KRG after in June, 1996.Two people of other among these patients accepted simultaneously from 1998 to 1999 HARRT and KRG the treatment, their cd4 t cell number increases to 1 of in December, 2004 from 231/ul in 1998,6/ul in 1999,052/ul, 2006 1,127/ul.

In addition, 22 HIV-1 patients were made a definite diagnosis in 1988, only had two patients (9%) that carry out the KRG treatment still to survive.Patient's HIV-1 hypotype is CRF02_AG, so this patient is not included in the research of the present invention, and still, it is among the above LTNP of 500/ul that this patient is assigned to the cd4 t cell number.The most of patients of making a definite diagnosis later in 1997 is carried out KRG treatment and HARRT treatment simultaneously, and there is not medicament-resistant mutation (drug resistance mutations) in they, and these patients show good reaction to this concurrent treatment method.

The inventor is by former research, and relevant between the KRG treatment of having reported the HIV-1 infected patient and the altofrequency of the extensive defective of nef gene (referring to list of references 9), KRG treats can slow down advancing of disease (referring to list of references 18).

Among the present invention, three PCR products from each sample are increased, although this lacks (referring to list of references 9) than four PCR products by nef gene amplification, but the inventor observes in 10 identical LTS, compare the frequency higher (P＜0.0001) of extensive defective (55% and PCR product of sample 37.6%) with nef defective (34.3% and PCR product of sample 18.8%) (referring to list of references 9).The defect frequency of 5 ' LTR/gag than the frequency of in the nef genetic flaw, confirming high 1.6 times/2.0 times.In 80 samples altogether, be applicable to the amplifications of nef gene before 61.13 (21.3%) in above-mentioned 61 samples all have extensive defective in two genes.Especially patient 87-05,89-17,90-05,90-50,96-51 in 5 ' LTR/gag gene than the frequency (P＜0.05) that in the nef gene, demonstrates significantly high extensive defective (Fig. 5).In addition, the ratio that demonstrates the sample of extensive defective confirms as 37.6% in 10 LTS that accept the KRG treatment, and this is obviously higher than 8 matched group LTS (14.8%) and 28 HIV-1 infected patients (8.3%).And, among the present invention, can confirm not have any contact (embodiment 5) between the extensive defect frequency and cd4 t cell number in any group of 8 matched group LTS or other 28 patients, find, can prove extensive defective and the KRG relation between absorbing according to this.

Above-mentioned table of discovery reveals the good comparison with the suitable dependency with respect to the cd4 t cell number (modest dependency) in 10 LTS.Patient 87-05, the 90-05,92-13, the 96-51 that accept to adhere among 10 LTS of KRG treatment have kept cd4 t cell number (Fig. 1 a, 1c, 1g, 1j) always, patient 90-50, the 91-20 that does not adhere in the period of the still opposite 1-2 recently, the very fast minimizing of cd4 t cell number (Fig. 1 e, 1f, 1i) of 93-60.As mentioned above, the cd4 t cell number that is caused by attitude reduces appearance (Fig. 1 b, 1d) in patient 89-17 and 90-18.Surpass 10 years long-time down without any the AIDS related symptoms, therefore, most of patients has the health status of not noting them or ignores the tendency of the picked-up of KRG.

Several pieces of nearest papers disclose the inherited characteristic of the total length virus sequence among the LTNP.Alexander etc. (referring to list of references 22) do not find defective in other gene except in the nef gene, and found premature termination codon, random defective (out-offrame deletions) and general defect.Uncommon pleomorphism (polymorphism) and the extensive defective of nef gene are found in 8 LTNP.In addition, Huang etc. (referring to list of references 23,24) has reported the hereditary detect of 5 ' LTR and gag gene among 8 LTS.In this research, obtain the total length virus sequence, only in a patient's 5 ' LTR and gag gene, found G to A hypermutation different (hypermutations).Recently, Calugi etc. has confirmed extensive defective (referring to list of references 25) in env gene (gene adjacent with the nef gene).Blankson etc. (referring to list of references 26) do not find any extensive defective in the base sequence in the HIV-1 total length of inhibitive factor (supressor) down at the quantity of viruses (viral loads) of the blood plasma RNA that lacks than 50copies/mL.

HIV-1 gene expression is regulated by the cis-acting regulatory element (cis-acting regulatory elements) of the several high preservations in the LTR of the calmodulin binding domain CaM that contains TATA box, transcription factor NF-kB and Sp1 (referring to list of references 27).Some composition of Radix Ginseng has suppressed NF-kB and the Ap-1 transcription factor activity (referring to list of references 28) in the human body cell strain.Compare the other parts of HIV-1, the LTR part more likely is subjected to the influence of KRG picked-up.

Among the present invention, in 10 LTS that comprise patient 87-05 (not showing the extensive defective of nef gene), observe the extensive defective of 5 ' LTR.And the size of observed each extensive defective is bigger, but only is positioned at intragenic single position.The inventor has confirmed the extensive defective of env gene (＞1000bp) (data are not shown) of 6 LTS (patient 90-05,90-18,90-50,91-20,92-13 and 96-51) when obtaining HIV-1 total length base sequence.

Therefore, do not consider the relation between KRG picked-up and the genetic flaw, although observation of the present invention only is a discovery in the various useful factor that obtains in research of the present invention, but result of the present invention provides preliminary data, and this preliminary data is meant in 5 ' LTR part and the gag extensive defective and the progression rates of HIV disease partly to be had significantly and get in touch (referring to list of references 1-9).How to influence the possible mechanism of LTS or LNTP about the viral gene defective, the inventor infers that genetic flaw is the result of the indirect immunomodulating (immune modulation) that causes of the picked-up of KRG.As everyone knows, koryo insam is media (referring to list of references 29-32) with the cell medium immunoreation (cell mediatedimmune response) by Th1 cytokine (cytokines), suppresses general immunocompetence state (generalized immune activation state) in HIV-1 infects.Yet other 28 patients that the LTS that does not give KRG compares infected by HIV still have higher 5 ' LTR/gag defective, so the KRG picked-up may not be unique key element.This discovery has proved the importance of the genetic flaw that is caused by the KRG picked-up in the life of LTS or LNTP prolongs.

The result of＜base sequence (Sequence DATA) 〉

Submitted to GenBank, and provided the GenBank accession number for 178 in 336 base sequences altogether: EF370172 to EF370349, EU047600 to EU047667, EU047670 to EU047676 and EU047681 to EU047693.

Industrial applicibility

Specifically, pharmaceutical composition of the present invention can be by inducing to the administration of HIV infected individuals HIV-1 has 5 ' LTR and/or gag gene defect. Therefore, pharmaceutical composition provided by the invention is passable Be used for prevention and the treatment of AIDS.

Sequence table

＜110〉Korea Ginseng Corp.

Univ Ulsan Found For Ind Coop

＜120〉contain koryo insam be used to make HIV-1 have the pharmaceutical composition of 5 ' LTR/gag genetic flaw

<150>KR10-2008-0037555

<151>2008-04-23

<160>6

<170>PatentIn?version?3.3

<210>1

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉first round primer CE23

<400>1

tgtggatcta?ccacacacaa?ggctactt

<210>2

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉first round primer 514

<400>2

tccagaatgc?tggtagggta?tac

<210>3

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉second take turns primer CE1

<400>3

cgagagctgc?atccggagta?cta

<210>4

<211>23

<212>DNA

＜213〉artificial sequence

<220>

＜223〉second take turns primer 512

<400>4

ctgcagcttc?ctc?attgatg?gtc

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the third round primer 501

<400>5

gtgtggcctg?ggcgggactg

<210>6

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the third round primer 510

<400>6

gatgtaccat?ttgcccctgg?a

Claims (6)

1, a kind ofly is used to make HIV-1 to have the pharmaceutical composition of 5 ' LTR and/or gag genetic flaw, it is characterized in that said composition contains the koryo insam as effective ingredient.

2, compositions according to claim 1 is characterized in that, described koryo insam is a Radix Ginseng Rubra.

3, compositions according to claim 1 and 2 is characterized in that, said composition is used for having the patient of HIV-1 to carry out administration to infection.

4, compositions according to claim 2 is characterized in that, with the dosage of 50-100 mg/kg/day the male is carried out administration, with the dosage of 20-70 mg/kg/day the women is carried out administration.

5, compositions according to claim 1 and 2 is characterized in that, described 5 ' LTR and/or gag genetic flaw are selected to be had the premature termination codon, lack start codon.

6, compositions according to claim 2 is characterized in that, described 5 ' LTR and/or gag genetic flaw are expressed as the sequence with following GenBank accession number: EF 370172, EF 370173, and EF 370175, and EU 047612, EF 370184, and EF 370186, and EF 370187, EF 370188, and EF 370191, and EF 370192, EF 370193, and EF 370196, and EF 370197, EF 370199, and EF 370201, and EF 370200, EF 370202, and EF 370205, and EF 370207, EF 370211, and EF 370213, and EF 3702115, EF 3702116, and EF 3702118, and EF 370219, EF 370221, and EF 370223, and EF 370224, EF 370225, and EF 370226, and EF 370227, EF 370228, and EF 370231, and EF 370235, EF 370237, and EF 370239, and EU 047647, EF 370245, and EF 370250, and EF 370251, EF 370252, and EF 370253, and EF 370256, EF 370259, EF 370262, and EF 370261, and EF 370265, DQ 295196, EF 370275, and EF 370276, and EF 370277, EF 3702778, EF 370279, and EF 370282, and EU 047673, EF 370283, EF 370285, and EF 370286, and EF 370287, EF 370288, and EF 370292.

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