CN102154356B - Hybrid immunodeficiency plasmid containing human immunodeficiency virus (HIV)-1 circulating recombinant form (CRF) 01-AE subtype reverse transcriptase (RT) gene, virus and use - Google Patents
Hybrid immunodeficiency plasmid containing human immunodeficiency virus (HIV)-1 circulating recombinant form (CRF) 01-AE subtype reverse transcriptase (RT) gene, virus and use Download PDFInfo
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Abstract
The invention relates to the technical field of biology and discloses a hybrid immunodeficiency plasmid containing HIV CRF01-AE subtype reverse transcriptase (RT) gene, a hybrid immunodeficiency virus RT-simian-human immunodeficiency virus (SHIV) /AE, and use thereof in construction of a monkey model of AE subtype RT-SHIV. The hybrid immunodeficiency virus RT-SHIV/AE is formed by substituting a gene fragment of CRE-01AE subtype HIV-1 for the gene of SIVmac239 on a genome framework of the monkey immunodeficiency virus SIV mac239, carries the RT gene of the AE subtype HIV-1 and can infect peripheral blood mononuclear cell (PBMC) of rhesus monkey in vitro and replicate efficiently. The monkey model of acquired immune deficiency syndrome (AIDS), which is constructed by infecting rhesus monkey with the hybrid immunodeficiency virus RT-SHIV/AE, is suitable to be used for studying the inv-vivo infection process, pathogenic characteristics and pathogenesis of HIV in Chinese patients and screening and evaluating anti-reverse transcriptase inhibitors.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of hybridization immune deficiency plasmid, viral RT-SHIV/AE and application in making up AE hypotype RT-SHIV monkey model thereof that contains HIV-1 CRF 01-AE hypotype RT gene.
Background technology
AIDS; Be called AIDS (Acquired Immune Deficiency Syndrome again; AIDS) be by human immunodeficiency virus (Human immunodeficiency virus; HIV) the one type of communicable disease that mortality ratio is high that causes, very big to the people's of country in the whole world especially Asia and the Africa Development life and health security threat.The global HIV-1 that is popular at present is divided into three groups, and M group, O group and N group can be divided into 10 hypotypes of A～J again in the M group, and the gene dispersion ratio between each hypotype is 20%～35%, simultaneously, also has tens of kinds of circulation recombinant types existence.HIV-1CRF 01-AE subtype virus was found in Thailand in 1989 at first, mainly popular diffusion in the crowd of spreading through sex intercourse, and wide-scale distribution in the addicts, having changed B hypotype infection in the past is main popular general layout.In recent years, CRF 01-AE subtype virus spreads unchecked day by day, is becoming the most popular HIV-1 virus subtype of China.
(Highly Active Anti-Retroviral Therapy, invention HAART) is a major progress on the AIDS preventing and controlling road to highly active antiretroviral therapy.Most patient can suppress duplicating of HIV-1 virus through the treatment of HAART, makes the carrying capacity of virus be in a low-level state, and the application of this therapy can reduce mortality ratio and the quality of life that improves patient.Contain three types of antiretroviral medicines among the HAART, be respectively:
(1) Nucleotide approximation RTI (NRTIs).Contain the Nucleotide that lacks 3-OH among the NRTIs, the Nucleotide through this 3-OH of lacking combines with the RT gene in the process of rt, thereby the formation of blocking dna chain makes the rt procedure failure.
(2) non-nucleoside reverse transcriptase inhibitor (NNRTIs).NNRTIs is a kind of and reversed transcriptive enzyme bonded noncompetitive inhibitor, mainly aspect chemosynthesis, plays a role and blocking dna synthetic.
(3) proteinase inhibitor (PIs).PIs combines inhibitory enzyme activity with viral protein enzyme catalysis gene, cause amyloid protein precursor can not cracking with form mature virion.
Early stage HAART scheme comprises a kind of proteinase inhibitor and two kinds of Nucleotide approximation RTIs.After non-nucleoside reverse transcriptase inhibitor is succeeded in developing, just be widely used in the HAART scheme.The rt therapy of main flow mainly comprises the combination of two kinds of NRTIs and a kind of NNRTI or PI now.
Hide in the process of HAART treatment and remaining virus is still duplicated continuing; In body, do not reject; And long periods of treatment can cause the resistance sudden change of HIV virus; Will cause the bounce-back of virus load and the failure of treatment in case produce sudden change, the sudden change of HIV virus drug resistance is a major cause that causes HARRT treatment failure.Carry out rational evaluation for the resistance sudden change of better research virus and to the validity of medicine, need select for use suitable animal model to study.There is a lot of advantages in animal model in the antiviral resistance problem of research HIV-1; Use suitable animal model can study virus, comprise that the different cells subgroup is to the virus replication characteristics of the reaction of virus infection, immunoselection pressure, different loci etc. in the intravital situation of duplicating of people.
HIV and SIV belong to the Retroviridae lentivirus, and belong to primates slow virus group, concern the closest.Rhesus monkey infected monkey immunodeficiency virus SIV and people's infected by HIV-1 exist similarity, and it is also similar with AIDS to develop the symptom that then, and these characteristics can make SIV/ rhesus monkey animal model be used for the research of anti-reverse transcription treatment.Yet; Also there are some restrictions in the rhesus monkey taint with SIV as a curative animal model; The similarity of the RT of SIV and the RT of HIV-1 is 60%; On for the susceptibility of medicine and non-nucleoside reverse transcriptase inhibition, exist difference, subject matter be HIV with SIV in the susceptibility of reversed transcriptive enzyme RT gene pairs medicine different.
External scientist obtains embedded virus RT-SHIV through the genetic manipulation means with the RT gene of the RT gene replacement simian immunodeficiency virus SIV of human immunodeficiency virus HIV and solves this problem, and this embedded virus RT-SHIV includes the main target spot-HIV RT gene of anti HIV-1 virus medicine.The rhesus monkey animal model of being set up by RT-SHIV can carry out the screening of validity to antiretroviral drugs, and deep research and understand manyly about virus resistance in the therapeutic process, and the continuing of residual virus such as duplicates at problem.
Existing RT-SHIV virus is all carried the RT gene of HIV-1B hypotype; The B hypotype is mainly popular the America and Europe; This RT-SHIV is fit to the American-European treating AIDS problem of research, and the HIV-1 epidemic strain of China is mainly the CRF-BC hypotype, CRF-AE hypotype and B ' hypotype; It is main wherein having with CRF 01-AE hypotype again; The homology of HIV-1B hypotype and CRF 01-AE hypotype RT gene has only 90.36%, and the difference in a lot of resistances site is particularly arranged, so the RT-SHIV of B hypotype is not suitable for the AIDS-treating medicine research to Chinese patient.
In order to study AIDS in China treatment problem with the RT-SHIV animal model, need to make up the RT-SHIV that comprises Chinese epidemic strain RT gene.And up to the present; Also there is not a kind of RT-SHIV to make up in the world to HIV-1CRF 01-AE hypotype; Therefore make up a kind of SHIV of the CRF of carrying 01-AE hypotype HIV-1RT gene, the SHIV/ rhesus monkey model that can simulate the HIV-1 infection is significant with the screening RTI to the pathogenesis of studying AIDS to Chinese patient.
Summary of the invention
The object of the present invention is to provide a kind of hybridization immune deficiency plasmid of the HIV of containing CRF01-AE hypotype pol gene; The nucleotide sequence of said HIV CRF01-AE hypotype pol gene is shown in SEQ ID No.1; As preferably, said hybridization immune deficiency plasmid nucleotide sequence is shown in SEQ ID No.2.
The present invention also provides the hybridization immune deficiency that contains said hybridization immune deficiency plasmid preparation virus.In embodiment of the present invention; Utilize said hybridization immune deficiency plasmid to prepare hybridization immune deficiency virus strain RT-SHIV/AE; This virus is carried the pol gene RT of HIV-1CRF 01-AE hypotype; Ability target cell infection and the ability that has Infection in Vitro China rhesus monkey mononuclearcell PBMC and duplicate, but infected monkey is set up the AIDS monkey model, is used for the research of AIDS-treating medicine.This virus is monkey-human immunodeficiency virus of containing HIV CRF 01-AE hypotype RT gene, is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 31st, 2010, and deposit number is CGMCC No.4510.
Another object of the present invention provides said hybridization immune deficiency plasmid, hybridization immune deficiency virus is directed against the application in the RT-SHIV monkey model of HIV-1CRF 01-AE hypotype in preparation.
The present invention utilizes recombinant gene, on pathogenic SIVmac239 gene framework, the RT gene of HIV-1CRF 01-AE is replaced a kind of hybrid virus strain RT-SHIV/AE of SIVmac239 corresponding base thereby structure.In the specific embodiment of the invention; The RT gene of HIV-1CRF 01-AE is from the HIV-107CN.HN031 strain that is separated in Hainan AIDS patient's body; Its gene order is at (the National Center For Biotechnology Information of the U.S. state-run biotechnology information center; NCBI) gene pool, genbank accession number are FM251973.Through this strain of sequential analysis proof is HIV-1CRF 01-AE hypotype, and its RT gene is complete.
The application contriver goes out to remove the fragment of RT gene through the intussusception pcr amplification from p239spsp5 ', again this fragment is connected into the T carrier, and called after T-OE confirms not to undergo mutation through order-checking.From HIV-1 virus liquid, amplify the RT gene, the RT gene is connected in the T-OE carrier called after T-OE-RT.Purpose fragment on the T-OE-RT carrier is cut in the respective segments of replacing SIVmac239 total length plasmid through Pac I and BsrGI enzyme; Just be built into the monkey-human immunodeficiency virus's plasmid that carries HIV-1CRF 01-AE hypotype RT gene, called after RT-SHIV/AE total length plasmid.
Use three restriction enzyme EcoR I, Hind III and Kpn I enzyme are cut said RT-SHIV/AE total length plasmid, and find: EcoR I can not cut this plasmid by enzyme; Hind III cuts into three sections to plasmid, and length is respectively 9608bp, 3025bp and 583bp; Kpn I cuts into five sections to plasmid, and length is respectively 4524bp, 3855bp; 3191bp, 823bp and 823bp; Different with original SIVmac239 total length plasmid, the enzyme of SIV plasmid is cut the result and is: EcoR I can not cut this plasmid by enzyme, and Hind III cuts into two sections to plasmid; Length is respectively 12630bp and 583bp; Kpn I cuts into five sections to plasmid, and length is respectively 4524bp, 3855bp; 3516bp, 823bp and 495bp.Simultaneously; The PCR primer of use HIV-1RT gene specific can detect the positive products of 1659bp the cleer and peaceful viral supernatant from the RT-SHIV/AE transfection; On the SIVmac239 plasmid transfection cleer and peaceful viral supernatant then can not, prove successfully to make up the correct RT-SHIV/AE total length plasmid of sequence.
With said RT-SHIV/AE total length plasmid transfection 293T cell; Collect the complete virion that supernatant just obtains to have infective hybridization immune deficiency viral RT-SHIV/AE according to the invention; Hybridization immune deficiency viral RT-SHIV/AE according to the invention is monkey-human immunodeficiency virus of containing HIVCRF01-AE hypotype RT gene; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 31st, 2010, deposit number is CGMCCNo.4510.
Carry the cell of different sources with hybridization immune deficiency viral RT according to the invention-SHIV/AE Infection in Vitro; Comprise TZM-bl and CEMx174 cell; Identify the biological characteristics of recombinant virus, the result finds that hybridization immune deficiency viral RT-SHIV/AE according to the invention has the target cell infection ability; The TZM-bl cell generation swelling of its infection, endochylema are granular and change, and the after birth edge is irregular, and RT-SHIV/AE can make the CEMx174 cytogamy form synplasm, proves that this virus has the cytopathic ability that makes.
Through in Chinese rhesus monkey PBMC cell, cultivating the recombinant virus that obtains high titre, monitor the variation of SIV core protein P27 level in the nutrient solution, to judge the replication of recombinant virus, the result shows that virus has Infection in Vitro and replication.
Hybridization immune deficiency viral RT-SHIV/AE according to the invention has following advantage:
1) carries representative CRF 01-AE hypotype HIV-1 reversed transcriptive enzyme (RT);
2) has the ability of target cell infection;
3) has the ability of Infection in Vitro monkey PBMC and efficient replication.
Hybridization immune deficiency viral RT-SHIV/AE according to the invention is derived from human immunodeficiency virus HIV and simian immunodeficiency virus SIV, carries the reversed transcriptive enzyme RT of the main epidemic strain HIV-1CRF01-AE hypotype of China.The AE hypotype RT-SHIV/ monkey model that uses this hybrid virus to set up can be used for studying course of infection, pathogenic characteristic, pathogeny and immunoreation etc. in the virus of AIDS body, especially for detecting and the screening RTI, has a good application prospect.
Biomaterial preservation explanation:
Classification name: RT-SHIV/AE; Monkey-human immunodeficiency virus of containing HIV CRF 01-AE hypotype RT gene; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 31st, 2010; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, and deposit number is CGMCC No.4510.
Description of drawings
Fig. 1 is a RT-SHIV/AE gene structure synoptic diagram;
Fig. 2 is a RT-SHIV/AE total length plasmid gene structural representation;
Fig. 3 is that RT-SHIV/AE total length plasmid enzyme restriction is identified figure;
Swimming lane 1:DL10000marker;
Swimming lane 2-4: clone 1;
Swimming lane 5-7: clone 2;
Swimming lane 8-10: clone 3;
Swimming lane 11-13: clone 4;
Swimming lane 14-16: clone 5;
Swimming lane 17-19: clone 6;
Swimming lane 20-22:SIVmac239;
Swimming lane 23:DL5000marker;
Swimming lane 24:DL2000marker.
Fig. 4 is pcr amplification product 1.5% gel electrophoresis figure of RT-SHIV/AE plasmid transfection 293T cell and CEMx174 cell RT-SHIV/AE transfection liquid;
Swimming lane 1:DL2000marker;
Swimming lane 2:RT-SHIV/AE transfection 293T supernatant;
Swimming lane 3:RT-SHIV/AE transfection CEMx174 cell conditioned medium 1;
Swimming lane 4:RT-SHIV/AE transfection CEMx174 cell conditioned medium 2;
Swimming lane 5:SIVmac239 transfection 293T supernatant.
Fig. 5: RT-SHIV/AE infects 100 * light microscopic figure of TZM-bl cell;
A is for infecting 48 hours TZM-bl cell of RT-SHIV/AE; B is the TZM-bl normal cell.
Fig. 6: RT-SHIV/AE infects 100 * light microscopic figure of CEMX174 cell;
A is for infecting the 13rd day CEMx174 cell of RT-SHIV/AE, and the arrow indication partly is a sick cell; B is the CEMx174 normal cell.
Embodiment
The invention discloses a kind of said hybridization immune deficiency plasmid, hybridization immune deficiency virus and the application in making up AE hypotype RT-SHIV/ monkey model thereof, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention and application are described through preferred embodiment; The related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order to make those skilled in the art understand technical scheme of the present invention better, the present invention is done further detailed description below in conjunction with specific embodiment.
The specific culture of embodiment 1:RT-SHIV/AE full-length clone, prepare in a large number and identify
The RT-SHIV/AE gene is on the genome framework of SIVmac239; The corresponding gene that replaces SIVmac239 with the portion gene fragment of the HIV-107CN.HN031 strain that is separated in the AIDS patient's body of Hainan; RT-SHIV/AE gene structure synoptic diagram is seen Fig. 1; The part of white comes from HIV-107CN.HN031 among the figure, and the part of black comes from SIVmac239.(the genbank accession number is No.FM251973 to said HIV-107CN.HN031 strain gene order for National Center For Biotechnology Information, gene pool NCBI) in the U.S. state-run biotechnology information center.Through this strain of sequential analysis proof is HIV-1CRF 01-AE hypotype, and its RT gene is complete, and its nucleotide sequence is shown in SEQ IDNo.1.
Preparation RT-SHIV/AE total length plasmid is cut the purpose fragment on the T-OE-RT carrier (comprising the HIV-1RT gene) in the respective segments of replacing SIVmm239 total length plasmid through Pac I and BsrG I enzyme, is built into RT-SHIV/AE total length plasmid.RT-SHIV/AE total length plasmid gene structural representation is seen Fig. 2, and indicating CRF 01-AE RT fragment among the figure is the RT gene that comes from HIV-107CN.HN031, and rest part is all the other genes of SIVmac239 and carrier.RT-SHIV/AE total length plasmid nucleotide sequence is shown in SEQ ID No.2.RT-SHIV/AE total length plasmid is used for follow-up cell experiment, keeps the stability of plasmid simultaneously, avoids two ends LTR to recombinate.
The purpose plasmid prepares in a large number: contain the genomic cDNA clone of total length SHIV; Because two ends have viral LTR LTR; When in host e. coli, increasing in a large number; Homologous recombination and disappearance very easily take place in plasmid, must the strict stability of using special culture condition could as far as possible keep plasmid.Adopting defective recombinant inhibition type bacterial strain E.coli JM109 is the host bacterium, 30 ℃, and be not higher than 180rpm and be no more than the specific culture condition of 14h, can keep the stability of plasmid basically.In view of the singularity that contains total length SHIV geneome plasmid, had better not in intestinal bacteria, preserve, in order to avoid losing of plasmid occur.In the time of need increasing in a large number to plasmid, transform with the JM109 bacterial strain again, single bacterium colony that picking is fresh is cultivated.
Because zooperal needs; When a large amount of preparations do not contain endotoxic SHIV plasmid; Adopt following method to carry out: the SHIV plasmid is transformed the JM109 bacterial strain, 30 ℃ of dull and stereotyped cultivations after 18～20 hours, picking list colony inoculation contains in the LB liquid nutrient medium of ammonia benzyl to 5mL.30 ℃, 180rpm concussion was cultivated after 12～14 hours, get the 1.5mL bacterial culture fluid and carry out plasmid and carry for a short time, enzyme cut identify errorless after, remaining 3.5mL bacterial culture fluid all is inoculated into 1L contains in 2 * YT liquid nutrient medium of ammonia benzyl.30 ℃, 180rpm concussion were cultivated after 12 hours, got that the 1.5mL bacterial culture fluid is once more for a short time to be carried, enzyme cut identify errorless after, with reference to Qiagen company specification sheets, the bacterium liquid of 1L is carried out a large amount of preparations of plasmid with Endofree Plasmid Giga Kit.Step is following:
(1) with bacterium liquid in 4 ℃, the centrifugal 15min of 6000rpm, supernatant discarded is resuspended in deposition in the STE solution of 500ml ice precooling, 4 ℃, the centrifugal 15min of 6000rpm, supernatant discarded;
(2) bacterial sediment is resuspended among the Buffer P1 of 125ml, and vibration does not occur to there being the deposition agglomerate, adds the Buffer P2 of 125ml; Gently put upside down centrifuge tube for several times, leave standstill 5min under the room temperature, treat that cracking fully adds the Buffer P3 of 125ml in the back; Put upside down centrifuge tube for several times, fully mixing;
(3) split product behind the mixing is poured in the filter (Mega-Giga Cartridge), left standstill more than the 20min under the room temperature, open vacuum pump pressurization suction filtration; After treating that the liquid suction filtration finishes; Add the Buffer FWB2 of 50ml, stir the deposition agglomerate gently, proceed suction filtration with aseptic glass stick;
(4) the Buffer ER of adding 30ml in lysate puts upside down mixing, and ice bath 30min treats that solution clarification back adds the good preparative column of Buffer QBT balance of using 75ml in advance;
(5) treat lysate fully through behind the DNA preparative column, with the Buffer QC wash-out impurity of 600ml, treat the whole mistake of liquid posts after, with the elutriant Buffer QN eluted dna of 100ml;
(6) collect the DNA elutriant, add the Virahol of 70ml, behind the abundant mixing, in 12 ℃, the centrifugal 50min of 10000rpm;
(7) carefully remove supernatant, with 10ml 70% washing with alcohol deposition, discard the ethanol washing lotion, centrifuge tube is upside down in seasoning on the paper handkerchief;
(8) the physiological saline solution DNA of adding 5ml-10ml;
(9) concentration of spectrophotometric determination DNA is 1 μ g/ μ l (or 0.4 μ g/ μ l, 40ng/ μ l) according to the plasmid concentration dilution of measuring for final concentration ,-20 ℃ of preservations.
The result obtains the RT-SHIV/AE total length plasmid of 2.25mg, uses three restriction enzymes (EcoR I, Hind III and Kpn I) enzyme to cut RT-SHIV/AE total length plasmid, and enzyme is cut the product electrophorogram and seen Fig. 3; The result shows: EcoR I can not cut this plasmid by enzyme, and Hind III cuts into three sections to plasmid, and length is respectively 9608bp; 3025bp and 583bp, Kpn I cuts into five sections to plasmid, and length is respectively 4524bp; 3855bp, 3191bp, 823bp and 823bp; The enzyme of SIVmac239 total length plasmid is cut the result: EcoR I can not cut this plasmid by enzyme, and Hind III cuts into two sections to plasmid, and length is respectively 12630bp and 583bp; Kpn I cuts into five sections to plasmid, and length is respectively 4524bp, 3855bp; 3516bp, 823bp and 495bp.
Use the RT gene in the PCR method specific amplification plasmid, sequencing finds that plasmid keeps stable, guarantees carrying out smoothly of subsequent experimental.
Embodiment 2:293T cell cultures and plasmid transfection
In order to obtain hybrid virus strain RT-SHIV/AE, use the total length plasmid transfection 293T cell of embodiment 1 preparation, obtain to have infective complete virion.
The 293T continuous cell line is half attached cell, and growth is very fast, when cell state is good, when in containing the DMEM perfect medium of 8% foetal calf serum, cultivating, will pass 1 generation (1 bottle of biography is 3 bottles) in average 48 hours.After cell covers with bottle; Gently supernatant of culture medium is inhaled and go, add the mixture slaking liquid (pancreatin: EDTA=1: 3) peptic cell, microscopically observation of pancreatin and EDTA; Treat that cell begins to become bowlder; Rapidly Digestive system is sopped up, add fresh DMEM perfect medium and gently cell is dispelled, 1 bottle of cell is divided into 3 bottles.37 ℃ of 5%CO
2Cultivate in the incubator.
The height of transfection experiment success or not and transfection efficiency is influenced by several factors, mainly with to be used for the quality that cells transfected is growth conditions, DNA, the transfection reagent of selecting for use and concrete working method relevant.Because the SHIV full-length clone quantity that obtains is more, in first round transfection experiment, use 6 orifice plates to carry out cell cultures and obtain virion, concrete steps are following:
(1) with 10% complete DMEM substratum the 293T cell is spread in 6 orifice plates (5 * 10
5/ hole), incubated overnight, cell density reaches 70%, and purpose is in order to cultivate the long period.
(2) every hole does not contain antibiotic complete DMEM substratum with 2ml and changes liquid, continues to cultivate 2 hours.
(3) 5 μ g DNAs are diluted in the DMEM substratum of 650 μ l serum-frees, antibiotic-free, gently mixing.
(4) 10 μ l Lipofectamine 2000 reagent (before the use mixing) gently are diluted in the DMEM substratum of 650 μ l serum-frees, antibiotic-free, incubated at room 5 minutes.
(5) Lipofectamine 2000 reagent (TV is 1.3ml) with DNA and the dilution of dilution mix, mixing gently, and room temperature 20 minutes, note: solution may muddiness, but can not influence transfection.
(6) directly mixed solution is added respectively in every porocyte, shake the orifice plate mixing gently, 5%CO
2, cultivated 5 hours for 37 ℃.
(7) get 2% fresh perfect medium of 2ml and change liquid, 5%CO
2, cultivated 40 hours for 37 ℃.
(8) collect every hole supernatant, after filtering with 0.45 μ M filter, measure the SHIV virus titer, directly be used for TZM infection experiment or frozen in liquid nitrogen with SIV P27 antigen detecting agent box.
For transfection RT-SHIV/AE total length plasmid obtains virus, we have cultivated the 293T cell, and the 293T cell is derived from by 293 cells, expresses the SV40 large T antigen simultaneously, and the plasmid that contains SV40 replication origin and promoter region can duplicate.Transient transfection 293T cell is the easy way of expressing protein and packaging virus.The growth conditions of 293T cell is directly connected to the efficient of packaging virus and transfection, should select passage number few as far as possible, cultivates short cell of total time.See that from cellular form the observation stereoscopic sensation by force and the form good cell is produced viral rate or transfection efficiency is all very high down for phase microscope.In this research; We select passage number few; The short 293T cell of incubation time carries out transfection experiment, obtains result preferably, and the transfection supernatant detects high-caliber SIV P27 antigenic content; SIV P27 antigenic content proves that greater than 32ng/mL transfection goes out the hybrid virus strain RT-SHIV/AE of ability high level expression.
Said hybrid virus strain RT-SHIV/AE is monkey-human immunodeficiency virus of containing HIVCRF01-AE hypotype RT gene; Be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 31st, 2010; The address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.4510.Embodiment 3: hybrid virus strain RT-SHIV/AE according to the invention infects TZM-bl cell and viral TCID
50Titration
Hybrid virus strain RT-SHIV/AE according to the invention has infectivity, and the light microscopic figure (100 *) that RT-SHIV/AE infects the TZM-bl cell sees Fig. 5.A is the pathology figure of TZM-bl cell infection RT-SHIV/AE after 48 hours, and the arrow indication partly is a sick cell, sick cell generation swelling, and endochylema is granular to be changed, and the after birth edge is irregular; B is the TZM-bl normal cell.RT-SHIV/AE infect the CEMX174 cell light microscopic figure (100 *) see Fig. 6, A is for infecting the 13rd day CEMx174 cell of RT-SHIV/AE, the arrow indication partly be a sick cell, virus can make CEMx174 cytogamy formation synplasm; B is the CEMx174 normal cell.
TZM clone is attached cell also, and the speed of growth is slower.In containing the DMEM perfect medium of 10% foetal calf serum, cultivate, go down to posterity when cell covers with bottle.Gently supernatant of culture medium is inhaled and go, with pancreatin cell is all digested, centrifugal 5 minutes of 800rpm thoroughly removes pancreatin.With substratum that cell is resuspended, dispel gently, divide bottle.TCID50 to SHIV virus single loop infectious cycle (48 hours cultivate) in the TZM-bl cell measures, and 2.5 times of relative luminescence units (RLU) are decided to be the Cutoff value.
(1) SHIV-KB9 virus is taken out back 4 ℃ from liquid nitrogen and slowly melt,, get 100 μ l viral dilution liquid and add in 96 well culture plates, establish four holes and repeat with 2 times of virus dilution of complete DMEM substratum.
(2) 10ml contains the preceding 30 μ lIndinavir of replenishing of perfect medium experiment of 45 μ g/ml DEAE.
(3) one bottle of TZM-bl cell of digestion is centrifugal, with 3ml perfect medium suspension cell, gets the 1ml cell suspending liquid, and add 3ml perfect medium and 8ml and contain 45 μ g/ml DEAE perfect mediums, mixing, every hole adds 100 μ l.
(4) seal around the flat board with sealing film, 37 ℃, 5%CO
2Hatched 48 hours.
(5) remove substratum, 200 μ l, 1 * PBS washes one time.Add 50 μ l CCRL lysates, room temperature 400rpm vibration 30 minutes.Whether cracking is complete for observation of cell under the mirror.
(6) cell pyrolysis liquid is transferred in the black 96 hole reading plates (Optiplate-96F), avoided bubble to produce.
(7) lucifuge every hole in 96 hole reading plates adds 50 μ l luciferase substrates, and mixing gently vibrates.
(8) with machine (1420Multilabel Counter) on the 96 hole reading plates, the counting fluorescence intensity.
(9) half cell infection dosage TCID
50Can use Reed-Muench method formula to calculate:
TCID
50=CPE＞50% viral dilution degree+(＞50% percentage ratio-50)/(percentage ratio of 50% percentage ratio-＜50%)
The functional TAT albumen of TZM-bl cell expressing activates the expression of luciferase reporter gene, and the virus quantity of cells infected is directly proportional with the uciferase activity of cell generation, and does not have the uciferase activity of infective virus cell very poor.Detect the infectivity of hybrid virus strain RT-SHIV/AE according to the invention according to this principle; The result finds that the fluorescence intensity of cell contrast is 638RLU; After the dilution in 1: 2 of RT-SHIV/AE transfection supernatant; The fluorescence intensity of inoculation TZM-bl cell greater than the cutoff value of 2.5 times of cell contrasts, explains that RT-SHIV/AE virus according to the invention can infect the TZM-bl cell between 2500-4000RLU; This virus is the virus with target cell infection ability, and has similar biological with parent's SIVmac239 strain.On this basis, we have further measured the TCID of transfection supernatant
50, through calculating, we make up in the plasmid transfection supernatant contains 500TCID
50The virus of/mL.
Embodiment 4: hybrid virus strain RT-SHIV/AE infection of Chinese rhesus monkey according to the invention
Experiment purpose: utilize hybrid virus strain RT-SHIV/AE according to the invention to have special biological characteristics and advantage, set up a kind of novel RT-SHIV/ monkey model.
Animal is selected: select 36 monthly ages, 2 of China rhesus monkeies, raise with the commercialization expanded pellet diet.Body weight 3-5kg.No abnormal before the experiment through health check-up, get rid of the infection of simian immunodeficiency virus (SIV), monkey rt D C-type virus C (SRV-1,2,5) and monkey T lymphatic I C-type virus C (STLV-1) through serology indirect immunofluorescence antibody test procedure (IFA) inspection.
Virus infection and sampling: infect the viral liquid of every monkey injection 2mL through the hind leg vein.Before infection, gathered monkey venous blood 6mL in 3,7,10,14,17,21,28,35,42,49,56,70 and 84 days with the infection back, 3mL EDTA anti-freezing is used for virus load and CD4
+/ CD8
+PH-value determination pH; The 3mL anticoagulant heparin is used for virus and separates.
Gather heparin anti-coagulating, whole blood sample to be checked is done 5 times of dilutions of series on 24 orifice plates, contains specimen amount 200 μ l, 40 μ l, 8 μ l, 1.6 μ l, 0.32 μ l, 0.064 μ l after making every Kongzui.Every hole adds 2 * 10
5/ ml CEMx1740.4ml supplies every hole 1.6ml with containing 10% Ox blood serum 1640 at last.The used substratum of whole blood virus titer contains IL-210U/ml.The IL-2 amount can suitably reduce after 3 days.Put 37 ℃ of 5%CO
2Incubator changes liquid next day once.Change liquid weekly twice later on.Before changing liquid, observing every hole has not pollution and CPE at every turn.Observed for 4 weeks continuously, the virus titer of CPE for this sample still can appear in high dilution whole blood, with TCID
50/ ml representes.
The PBMC proviral DNA detects:
According to the requirement of product description, get EDTA anticoagulated whole blood 300 μ l, with Wizard Genomie DNA Purification Kit (Promega, A1120) genomic dna of extraction peripheral blood lymphocyte.
The RT-PCR amplified reaction carries out on Eppendorf PCR appearance, uses the precious biological Takara One StepRNAPCR Kit (DRR024A) in Dalian, and this test kit has adopted the single step reaction method; The operation of RNA-cDNA-PCR carries out in same reaction system; ThermoScript II by the AMV source is cDNA from the RNA reverse transcription, and by AMV-Optimized Tag amplification cDNA, total reaction volume is 20 μ l again; Include: 10 * One Step buffer, 2 μ l (10mmol/LTris-Cl, pH8.3; 50mmol/L KCl); 5mmol/L Mgcl
21mmol/L dNTP Mixture; 16U RNase Inhibitor; 2U AMV RTase XL; The Tag enzyme of 2U; Each 0.4mol/L of RT-PCR upstream and downstream primer; The viral RNA of 2 μ l is as template.Cycling condition is 60 ℃ of 30min (* 1), 95 ℃ of 5min (* 1), 95 ℃ of 15s (* 50), 60 ℃ of 1min (* 50).This product of taking turns is as the template of nest-type PRC.
Nest-type PRC is reflected on the Eppendorf PCR appearance and carries out, and total reaction volume is 20 μ l, includes: 10mmol/L Tris-C1, pH8.3; 50mmol/L KCl; 1.5mmol/L MgCl
20.2mmol/L 4 * dNTP; 0.5U the Tag enzyme; Each 20pmol/L of nest-type PRC upstream and downstream primer; The RT-PCR amplified reaction product of 2 μ l is as this template of taking turns.Cycling condition is 94 ℃ of 2min (* 1), 94 ℃ of 15s (* 40), 58 ℃ of 30s (* 40), 72 ℃ of 30s (* 40), 72 ℃ of 6min (* 1).
The mensuration of plasma viral load:
Use viral RNA carrying capacity in real-time fluorescence quantitative PCR (Real-time PCR) the method monitoring monkey blood plasma.
Rneasy Mini kit (QIAGEN, 74106) extracts viral RNA in the blood plasma, uses Quantitect SYBR Green RT-PCR Kit (Qiagen, 204243) to measure plasma viral RNA carrying capacity at Roche LightCycler fluorescent quantitation appearance, and primer is respectively:
2f：GTA?ACT?ATG?TCC?ACC?TGC?CAT?TA，
2r：CAG?CCT?CCT?CGT?TTA?TGA?TGT。
Reaction system is:
Amplification program is 50 ℃ of 2min (* 1), 60 ℃ of 30min (* 1), and 95 ℃ of 10min (* 1), 95 ℃ of 15s (* 40), 60 ℃ of 1min (* 40) measure fluorescent signal, 45 ℃ of insulations.
CD4
+/ CD8
+Value and CD4
+The mensuration of T cell absolute number:
The EDTA anticoagulation of 50 μ L is added in 12 * 75mm Falcon pipe, and after 15 minutes, adds 10% hemolysin haemolysis 10 minutes in the effect of room temperature lucifuge with antibody.The back is washed 2 times with PB S, and 1% Paraformaldehyde 96 is fixed and differential count on Calibur FACS appearance.The index that we analyze comprises: CD4
+, CD8
+Percentage ratio and CD4
+/ CD8
+Ratio.CD4 according to cells were tested by flow cytometry
+/ CD8
+Ratio, routine blood test and blood smear result calculate CD4
+T cell absolute number.
Experimental data proof RT-SHIV/AE can duplicate in Chinese rhesus monkey body, but evening appears in viremia, the weak point of holding time, and aggregate level is lower, still needs further monkey body to go down to posterity and improves the flexibility of virus to animal.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (3)
1. hybridization immune deficiency plasmid that contains HIV CRF01-AE hypotype pol gene, the nucleotide sequence of said HIV CRF01-AE hypotype pol gene is shown in SEQ IDNo.1.
2. hybridization immune deficiency plasmid according to claim 1, its nucleotide sequence is shown in SEQ ID No.2.
3. a hybridization immune deficiency that contains HIV CRF01-AE hypotype pol gene is viral, and its deposit number is CGMCC No.4510.
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| Deng W et al.Molecular epidemiological tracing of HIV-1 outbreaks in Hainan island of southern China.《AIDS》.2009,第23卷(第8期),977-985. * |
| K Uberla et al.Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.《Proc Natl Acad Sci U S A》.1995,第92卷(第18期),8210-8214. * |
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