CN101920022B - Specific anti-virus antisense nucleic acid medicament for avian influenza virus - Google Patents

Specific anti-virus antisense nucleic acid medicament for avian influenza virus Download PDF

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CN101920022B
CN101920022B CN2010101596521A CN201010159652A CN101920022B CN 101920022 B CN101920022 B CN 101920022B CN 2010101596521 A CN2010101596521 A CN 2010101596521A CN 201010159652 A CN201010159652 A CN 201010159652A CN 101920022 B CN101920022 B CN 101920022B
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nucleic acid
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antisense nucleic
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avian influenza
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韩健宝
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    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Abstract

The invention discloses a specific anti-virus antisense nucleic acid medicament for avian influenza virus. The active ingredients of the specific anti-virus antisense nucleic acid medicament for the avian influenza virus are four types of monomer antisense nucleic acid, and nucleotide sequences of the four types of monomer antisense nucleic acid are respectively a sequence 4, a sequence 7, a sequence 11 and a sequence 14 in a sequence list. The specific anti-virus antisense nucleic acid medicament for the avian influenza virus has the advantages of high-level bioavailability, stable physical and chemical properties, no toxic or side effect, no medicament tolerance, capacity of specifically killing the AIV, good anti-virus effect and low cost.

Description

The specificity antivirus antisense nucleic acid medicament of avian influenza virus
Technical field
The present invention relates to the specificity antivirus antisense nucleic acid medicament of avian influenza virus.
Background technology
Bird flu is a stud bird deadly infectious disease that is caused by orthomyxovirus section influenza A virus, and its clinical manifestation is respiratory system disease, egg drop reduction etc.Bird flu once in eruption and prevalence all over the world, caused the rapid decline of a fowl mortality and fertility performance, made aviculture suffer enormous economic loss.Continental rise poultry, aquatic bird and wild bird all can be infected, and to cause respiratory system and serious septicemia so that whole body MOFE, disease progression is fast, case fatality rate is high is principal character.
Bird flu will cause direct infection to the mankind as the hugest gene bank that the new strain of human influenza forms, some bird flu viruss (H7 type) morph, and the virus of variation can be in interpersonal propagation, at present, total 15 countries of China that comprise have broken out human and bird fluenza epidemic situation, cause 412 people to infect wherein 256 people's death altogether.In birds in infectious all bird flu viruss, the H5N1 bird flu virus has now possessed required all prerequisite of beginning that are very popular, and up to now promptly, H5N1 virus causes serious state of an illness number and death toll maximum, it has crossed over species infection barrier, has infected the mankind from birds.In case form effective interpersonal propagation, H5N1 virus will make a variation and become to cause the required characteristic of new round influenza epidemic situation.
At present, be badly in need of a kind of specificity antivirus medicine that possesses high-caliber bioavailability, stablizes the avian influenza virus of physicochemical property and highly effective and safe.
Summary of the invention
The purpose of this invention is to provide a kind of specificity antivirus antisense nucleic acid medicament that possesses high-caliber bioavailability, stablizes the avian influenza virus of physicochemical property and highly effective and safe.
The specificity antivirus antisense nucleic acid medicament of avian influenza virus provided by the present invention, its active component are four kinds of antisensenucleic acidses, and the nucleotide sequence of described four kinds of antisensenucleic acidses is respectively sequence 4, sequence 7, sequence 11 and the sequence 14 in the sequence table.
The specificity antivirus antisense nucleic acid medicament of avian influenza virus of the present invention, wherein: described four kinds of antisensenucleic acidses are any mass ratio.
The specificity antivirus antisense nucleic acid medicament of avian influenza virus of the present invention, wherein: the mass ratio of described four kinds of antisensenucleic acidses is 1: 1: 1: 1.
The specificity antivirus antisense nucleic acid medicament of avian influenza virus of the present invention, wherein: also comprise pharmaceutically suitable carrier or excipient.
The specificity antivirus antisense nucleic acid medicament of avian influenza virus of the present invention possesses high-caliber bioavailability, stablizes physicochemical property, has no side effect, and has no drug resistance, and can directly kill AIV by specificity, and antiviral effect is good, and is with low cost.
The specific embodiment
The following antisensenucleic acids of synthetic:
AM1：5’-CAUCCACAUCACUCUGCUGUU-3’；
AM2：5’-CCUGCCGUAGAUGGCCCUCUU-3’；
AM3：5’-AAGGCGAGGAUAAAUGCAUUU-3’；
AM4：5’-GCAACAACAAGAGGAUCACUU-3’；
ANP1：5’-AUUGUCAUACUCCUCAGCAUU-3’；
ANP2：5’-CUGUGACUUGGUCAUCAUCUU-3’；
ANP3：5’-AAUUCCCCUUUGACGCGUAUU-3’；
ANP4：5’-UUGAACAAGCCCAUCAUCAUU-3’；
APA1：5’-CACCGAGAGCCCAGUUUAAUU-3’；
APA2：5’-CGUUUUCCGCCUUCCUUCUUU-3’；
APA3：5’-GAAUGUGGUACUUGUAGCAUU-3’；
APA4：5’-UCUUUGUGAUACCAUUUUCUU-3’；
APB1：5’-GUCCAUCAACCGGGUUGAGUU-3’；
APB2：5’-UACCAUUCUCUUGGUCCUGUU-3’；
APB3：5’-GAAUGUGGUGCUUAUAGCAUU-3’；
APB4：5’-UUUCCCUAAGCUAGCCAUCUU-3’。
Influenza strain: H5N1, H9N2
Experiment condition: relate to carrying out at the BSL-3 laboratory of H5 hypotype, other carries out at the BSL-2 laboratory.
Cell line: Madin-Darby canine kidney(cell line) MDCK
Antisensenucleic acids: be 1.5 μ g/ml
Adopt the cytopathy series indices to measure the antisensenucleic acids antiviral activity.
Day1：
Bed board: the mdck cell of digestion early-stage preparations, centrifugal collection, cell counting is transferred to 1 * 10 with complete medium with cell concentration 5Individual/ml, spread 24 orifice plates, in 37 ℃ of CO2 gas incubator, cultivated 18-24 hour.
Day2：
The density of microscope observing cell treat that cell covers with 70 of orifice plate area～80% o'clock, and cell state is good.Culture fluid is removed in suction, and every hole adds 300 μ l and treats screening of medicaments, 10 holes of each medicine.Behind the incubation 1 hour, add 100 μ l influenza virus (infect than be 0.01), adsorb after 2 hours, after the virus of not adsorbing with the nutritional solution flush away, add complete medium again, at 37 ℃ and 5%CO 2Continue in the environment to cultivate.Also establish the normal cell matched group that does not add virus and antisensenucleic acids in the experimentation, add virus, do not add the antisensenucleic acids positive controls and add antisense nucleic acid medicament, do not add the negative control group of virus.
Day3-5：
Observe the protective effect of medicine pair cell, and the result is passed judgment on.
The result passes judgment on: matched group is set up and the cytopathy judging quota.
(1) normal cell matched group experimentation does not have pathological changes; Cytopathy appears in positive controls, and when experiment finishes 90% above cell generation pathological changes; Medicine negative control experimentation cell does not have pathological changes.
(2) cytopathy judging quota:
Cell do not have pathological changes-cytopathy below 25%+
Cytopathy is at 26%-50% ++ and cytopathy is at 51%-75% +++
Cytopathy is at 76%-100% ++ ++
Experimental group The selection result such as table 1.
The antiviral activity analysis of table 1. antisensenucleic acids on cell
Figure GSA00000085875100031
Figure GSA00000085875100041
Above-mentioned cell virus experiment analysis results shows that AM4, ANP3, APA3, APB2 are for preferred.
Oxicity analysis
Day1：
Bed board: the mdck cell of digestion early-stage preparations, centrifugal collection, cell counting is transferred to 1 * 10 with complete medium with cell concentration 5Individual/ml, spread 24 orifice plates, in 37 ℃ of CO2 gas incubator, cultivated 18-24 hour.
Day2：
The density of microscope observing cell treat that cell covers with 70 of orifice plate area～80% o'clock, and cell state is good.Culture fluid is removed in suction, and every hole adds 300 μ l and treats screening of medicaments, and every kind of medicine adds 10 holes.At 37 ℃ and 5%CO 2Continue in the environment to cultivate and continue to cultivate.Establish in the experimentation and only add the negative control group that PBS liquid does not have antisensenucleic acids.
Day3-5：
Observe the poisonous effect of medicine pair cell, and the result is passed judgment on result such as table 2.
Table 2. antisensenucleic acids oxicity analysis
Four kinds of antisensenucleic acids AM4, ANP3, APA3 and APB2 are mixed and made into antisense nucleic acid medicament of the present invention according to any mass ratio.
Be 1: 1: 1 with antisensenucleic acids AM4, ANP3, APA3 and APB2 according to mass ratio specifically: 1 antisense nucleic acid medicament that is mixed and made into is an example.
The antisense nucleic acid medicament stability analysis
High temperature: 105 ℃ of flowing steam high temperature, sterilization in 20 minutes does not influence its biological activity.
Extreme temperature: depositing for 50 ℃ did not influence its biological activity in 2 months.
Room temperature: depositing did not influence its biological activity in 24 months.
Low temperature: depositing for-20 ℃ did not influence its biological activity in 48 months.
Zoopery
Experimental animal: 160 of the SPF chickens of 20 ages in days are divided into 16 groups, 10 every group.
Virus: H5N1, H9N2
Embodiment
(1) efficacy test: experimental group is infected (infection toxic agent amount: 100 TCID50) with H5N1 or H9N2 poison, after 24 hours, difference each monomer antisensenucleic acids of intramuscular injection or nucleic acid drug of the present invention, dosage is 10 μ g, and establishes a counteracting toxic substances without the antisensenucleic acids positive controls with only set up usefulness antisensenucleic acids, counteracting toxic substances as negative control group not.Observe the situation of chicken every day behind the counteracting toxic substances, statistics chicken death number after 14 days (see Table 3, table 4).
Table 3, each monomer antisensenucleic acids and nucleic acid drug of the present invention therapeutic result of the test after to SPF chicken artificial challenge H5N1 poison
Figure GSA00000085875100062
Figure GSA00000085875100071
Table 4, each monomer antisensenucleic acids and nucleic acid drug of the present invention infect therapeutic result of the test behind the H9N2 to the SPF chicken
(2) the different dosing group is to chicken body inner virus inhibition test: chicken is at the injection antisense nucleic acid medicament after 72 hours and 168 hours, embrocate cloaca with the cotton swab of sterilization respectively, the cotton swab that will soak into secretions is put into HANK ' the S liquid that contains penicillin, streptomycin 2000U/ml, fully twist, shake, 8000 commentaries on classics/min, centrifugal 10min gets supernatant as inoculation material.Inoculate 10 age in days SPF Embryo Gallus domesticus respectively, cultivate the embryo alive of freezing to death behind the 5d, all measure blood clotting, with HA tire＞2log2 is judged to be chicken row virus-positive (seeing Table 5), the result shows that antisensenucleic acids of the present invention can obtain excellent curative.
Each monomer antisensenucleic acids of table 5. and nucleic acid drug of the present invention infect H5N1 to the SPF chicken, and H9N2 suppresses effect
Figure GSA00000085875100081
Sequence table
＜110〉Han Jianbao
＜120〉the specificity antivirus antisense nucleic acid medicament of avian influenza virus
 
<160>16
 
<210>1
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>1
cauccacauc?acucugcugu?u 21
 
<210>2
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>2
ccugccguag?auggcccucu?u 21
 
<210>3
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>3
aaggcgagga?uaaaugcauu?u 21
 
<210>4
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>4
gcaacaacaa?gaggaucacu?u 21
 
<210>5
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>5
auugucauac?uccucagcau?u 21
 
<210>6
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>6
cugugacuug?gucaucaucu?u 21
 
<210>7
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>7
aauuccccuu?ugacgcguau?u 21
 
<210>8
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>8
uugaacaagc?ccaucaucau?u 21
 
<210>9
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>9
caccgagagc?ccaguuuaau?u 21
 
<210>10
<211>21
<212>RNA
＜213〉artificial sequence
<400>10
cguuuuccgc?cuuccuucuu?u 21
 
<210>11
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>11
gaauguggua?cuuguagcau?u 21
 
<210>12
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>12
ucuuugugau?accauuuucu?u 21
 
<210>13
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>13
guccaucaac?cggguugagu?u 21
<210>14
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>14
uaccauucuc?uugguccugu?u 21
 
<210>15
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>15
gaauguggug?cuuauagcau?u 21
 
<210>16
<211>21
<212>RNA
＜213〉artificial sequence
 
<400>16
uuucccuaag?cuagccaucu?u 21

Claims (3)

1. the specificity antivirus antisense nucleic acid medicament of avian influenza virus, its active component is four kinds of antisensenucleic acidses, the nucleotide sequence of described four kinds of antisensenucleic acidses is respectively sequence 4, sequence 7, sequence 11 and the sequence 14 in the sequence table.
2. antiviral antisense nucleic acid medicament according to claim 1 is characterized in that: described four kinds of antisensenucleic acids mass ratioes are 1: 1: 1: 1.
3. antiviral antisense nucleic acid medicament according to claim 2 is characterized in that: also comprise pharmaceutically suitable carrier or excipient.
CN2010101596521A 2010-04-29 2010-04-29 Specific anti-virus antisense nucleic acid medicament for avian influenza virus Expired - Fee Related CN101920022B (en)

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