Detailed description of the invention
Fig. 1 is the application schematic diagram of the acquired immune deficiency syndrome (AIDS) immunization therapy instrument proposed according to the present invention.
Fig. 2 is the internal structure schematic diagram of the blood separator proposed according to the present invention.
Fig. 3 is the internal structure schematic diagram of the plasma separator proposed according to the present invention.
Fig. 4 is the internal structure schematic diagram of the depurator proposed according to the present invention.
In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, the other end through heparin and blood pump (2) with contain
The blood separator (3) having waste liquid outlet (5) is connected, and blood separator (3) exports (4), blood pump (6), circulation pipe through blood
Road (7) is connected with plasma separator (8), the plasma outlet port of plasma separator (8) through blood plasma pump (9) and blood vessel (10) with two
Depurator (11) in parallel is connected with depurator (12), the export pipeline (13) of two depurators and the blood of plasma separator (8)
Cell outlet pipeline (14) circulates through venous line (15) fluid confluence after converging.
Fig. 2 represents the internal structure of the blood separator (3) in Fig. 1.In Fig. 2, the tube wall of the inner chamber (2) of separator (1)
On have a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and be delayed at inner chamber (2), thus can be eliminated, energy
By in micropore (3), the mononuclear blood cell (5) of small size enter exocoel (6), then flow out through outlet (7), and then through Fig. 1
Shown plasma separator (8) isolates hemocyte and blood plasma.
Fig. 3 represents the internal structure of the plasma separator (8) in Fig. 1.In Fig. 3,1 is plasma separator, and 2 is that blood plasma separates
Device inner chamber, 3 is the micropore on plasma separator inner chamber tube wall, and 4 is the mononuclear blood cell that can not pass through micropore (3), and 5 is to pass through
The blood plasma chemical analysis of micropore (3), 6 is plasma separator exocoel, and 7 is blood plasma flow export, and 8 is that to have the blood of switchable valve thin
Born of the same parents export.
In Fig. 4,2,4 HIV antibody being respectively fixed in agar gel (6), CD4+T cell;1 is free HIV;3、
Being delayed at the conjugate in agar gel (6) after 5 respectively HIV and HIV antibody, CD4+T Cell binding, 7 is by agar
The large volume of HIV of gel (6) molecular sieve detention.
Below in conjunction with Fig. 1, Fig. 2, Fig. 3 and Fig. 4, the embodiment of the acquired immune deficiency syndrome (AIDS) immunization therapy instrument that the present invention proposes is made in detail
Thin description.
One, the preparation of acquired immune deficiency syndrome (AIDS) blood purification agent
(1) preparation of CD4+T cell strain
1, the source of primary lymphocyte
Have to sow by way of 1. frozen in the Infectious Diseases Lab Sample Storehouse that scientific research preserves lymphocyte strain (warp
Inactivation HIV totivirus is immune but is uninfected by the lymphocyte of HIV);2. buy the fresh White Blood Cells Concentrate in blood station, then went out
The lymphocyte that HIV strain of living is immune;3. the T lymphocyte series (strain) directly bought from businessman;4. preserve for scientific research
Cord blood lymphocytes cell (through inactivation HIV immunity);The most directly take from the peripheral blood lymphocyte of HIV-1 the infected (for certainly
Body), use Histopaque lymphocyte separation medium separation mononuclearcell (PBMC).
2, the preparation of CD4+T cell
1. main agents and instrument: CD4, CD8 immunomagnetic beads (Mei Tian Ni Bioisystech Co., Ltd of Germany);Isothiocyanic acid
Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company);Lymphocyte separation medium (Shanghai perseverance letter biochemistry
Reagent company limited);Ethylenediaminetetraacetic acid (EDTA), 0.2% Trypan Blue liquid (Shanghai raw work biotechnology service public affairs
Department);New-born calf serum (Hyclone company);MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany);
EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. separation (the density gradient of mononuclearcell (PBMC)
Centrifuging): aseptic take 20mL blood sample (500IU/mL2mL heparin sodium anticoagulant);PBS liquid, by hemodilution 2~3 times, fully mixes
After 6mL anticoagulant venous blood dropper is slowly superimposed on, along tube wall, the 10mL centrifuge tube having added 4mL lymphocyte separation medium
Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge;Being divided into 3 layers after Li Xin in pipe, upper strata is blood plasma and PBS liquid,
Lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, has one with mononuclearcell in upper, interface, middle level
The white cloud and mist layer narrow band being main is PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC and inserts another 50mL centrifuge tube
In, add 5 times and be centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandon supernatant 50mLPBS re-suspended cell, centrifugal
(350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new-born calf serum+2mmol/LEDTA, pH7.2)
2mL re-suspended cell, takes the cell (PBMC) that 15uL cell suspension adds on blood counting chamber in 4 block plaid of counted under microscope
Sum.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell suspension is divided equally to two 1.5mLEppendorf pipes,
Centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, the every 80uLBuffer of re-suspended cell contains cell number 107Individual, every 107Individual carefully
Born of the same parents add 20uLCD4MicroBeads or CD8MicroBeads, fully mix, and hatch 15min at 4~8 DEG C, wash with 1mLBuffer
Wash cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed on
In the magnetic field of MACS separator, rinse with 500uLBuffer, by 500uL cell suspension by detached dowel, use 500uLBuffer
Rinse detached dowel repetitive operation 3 times, collect effluent, containing non-CD4+T lymphocyte or non-CD8+T lymphocyte in effluent,
Taking out detached dowel in separator, with 1000uLBuffer pressure flush detached dowel, collect effluent, this is that CD4+T lymph is thin
(cell viability detects: take 15uL cell suspension before and after cell purification respectively molten with equal-volume trypan blue for born of the same parents or CD8+T lymphocyte
Liquid mixes, and the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates in 200 cells and lives
The percentage ratio of cell).
3, amplification in vitro CD4+T cell
Document is had to report the stimulant utilizing the monoclonal antibody of T cell surface C D3 molecule to grow, great Liang Pei as cell
After supporting the T cell that HIV sufferers separates, feed back as self therapeutic cells.But HIV is also with the cultivation of HIV cell
Breeding and at endogenous multiplication, the feedback of increment T cell result also in the feedback of increment HIV.The present invention is with SV40 and/or hTERT
Immortalization CD4+T cell, and with CD3 monoclonal antibody for cell growth stimulant, a large amount of amplification CD4+T cells.
With the method that CD3 monoclonal antibody is cell growth stimulant it is: by anti-CD49d McAb, (CD4+T cell contains simultaneously
CD3 molecule) it is coated on culture plate stimulation mononuclearcell (lymphocyte) growth, referred to as anti-cd 3 antibodies is coated method, can obtain
Well expanding effect, the lymphocyte that should expand in this way has been used for the second stage of clinical treatment of tumor and achieves certain
Curative effect.Foreign literature report [Shimizu etc.] has also cultivated the lymphocyte of 5 example full-blown AIDS patients, training by the method
Support and within 4 weeks, be achieved with the amplification of 1000 times, and in the cell mass of amplification, CD4+/CD8+T all can expand that (CD4+T cell is more in a large number
Substantially).Another kind is the dual anti-cross-linking method of AntiCD3 McAb/CD28, will dual anti-being crosslinking on taste pearl of AntiCD3 McAb/CD28 train as stimulant
Support HIV person's PERIPHERAL BLOOD MONONUCLEAR CELL (lymphocyte), substantial amounts of CD4+T cell, and the CD4+T expanded can be expanded
Cell has the ability of antagonism HIV, and in its incubation, virus is also below detection level, finds that this may be with CD28 afterwards
Providing secondary signal, it is relevant with chemotactic factor that selective induction secretes substantial amounts of Th1 cytokine, with the method amplification
CD4+T cell have been used for HIV person clinical treatment feed back, devoid of risk but effect is general.
With the method for hTERT immortalization CD4+T cell it is: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo-
HTERT and carrier pLXSNneo, connects hTERT and pLXSNneo separated through PCR amplification, gel electrophoresis with Ligation Mix
Digestion products, builds pLXSNneo-hTERT recon, converts DH5a competent cell blue or green with amplification, purification picking resistant to ammonia benzyl
Mycin bacterium colony extracting plasmid, imports with lipofection and passes on the T lymphocyte in logarithmic growth in vitro, makes recon with thin
The DNA of born of the same parents integrates, and the clone of positive recombinant that amplification culture screen through G418, screening cellular morphology, growth curve, dyeing
The test of body caryogram, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, thin
Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT immortality
The CD4+T cell changed.
With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4 DNA ligase
PcDNA3.1 (-) DNA with PCR amplification, the SV40LTag DNA that separates of agarose gel electrophoresis, structure SV40LTag-
PcDNA3.1 (-) recombiant plasmid, convert DH5a competent escherichia coli cell amp-R with amplification, purification picking
Bacterium colony extracting plasmid, imports the T lymphocyte of In vitro culture with lipofection, makes the DNA integration of recon and cell, with
The cell containing positive recombinant of G418 screening, passes on, amplification culture, screening cellular morphology, cell growth curve, chromosome
SV40 big T gene test in the test of caryogram, nude mice tumorigenesis, transfectional cell DNA, mrna expression product measure and determined dna sequence
Result meets immortalized cells characteristic person same or like with the primary cell CD4+T cell as SV40 immortalization.
1. with the concrete grammar of hTERT immortalization CD4+T cell
(I) extraction of hTERT: (i) enzyme action pClneo-hTERT:hTERT be positioned at the EcoRI of plasmid pClneo-hTERT with
Between SalI site, pLXSNneo vector multiple cloning site (MCS) restriction enzyme site Han EcoRI Yu XhoI.Commercially available purchase pCIneo-
HTERT plasmid, is dissolved in appropriate ultra-clean H2In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H2O, adds restriction
Property restriction endonuclease EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C heating 15min, inactivator, add 5uL electrophoresis sample-adding
Buffer (also can by add 0.5mol/L EDTA) terminates reaction, routinely after PCR method amplification hTERT, collect amplified matter with
Standby electrophoresis.(ii) hTERT electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours into and seals
Gel casting platform on, plug sample comb, after gelling is solid, from glue platform, removes envelope band, extracts comb, put into added with
Enough in the electrophoresis tank of electrophoretic buffer, buffer exceeds gel surface about 1mm, prepares with 10 appropriate × sample loading buffer
HTERT enzyme action sample, then adds sample in sample well with pipettor, and does suitable standard control thing simultaneously, connects electricity
Pole, make hTERT face south Ghandler motion move, then under the voltage of 1-10V/cm (80V) gel electrophoresis to being sufficiently separated hTERT fragment
During distance (30min), close power supply.(iii) hTERT purification and recovery: separate hTERT band from agarose: purple at long wave
Under outer light source, the gel-tape containing target hTERT fragment is cut in loading bag filter, in bag filter, add 2ml running buffer
Liquid, is allowed to submergence gel, and empties steam bubble, and bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), adds suitable
Amount buffer, by bag filter submergence (about 6-7mm), switches on power, and 150 volts of electricity are washed, and observes and treat that hTERT all moves under uviol lamp
Going out gel, change direction of an electric field and continue energising 1 minute, from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds
Entering 1.5 times of volume n-butyl alcohol, EB is removed in mixing extracting, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution,
So repeating secondary, add equal-volume phenol chloroform (2) and extract 2 times in the solution of lower floor hTERT, supernatant proceeds to another
1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol of addition in Eppendorf pipe, in 20 DEG C overnight, 12000g, 4 DEG C
Under be centrifuged 10 minutes, obtain hTERT precipitation, abandon supernatant, after adding 70% washing with alcohol 2 times, abandon dry ethanol, add 50 μ l TE and dissolve
hTERT.Additionally, can also be used with low melting-point agarose gel method, hTERT filter membrane inserted sheet method etc. by purpose hTERT fragment from gel
Separate, be purified.
(II) connection of hTERT Yu pLXSNneo carrier: take the hTERT composition (0.1-5 μ g) of the 9 above-mentioned purification of μ l, 1 μ
L10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15
DEG C incubation 24h, builds pLXSNneo-hTERT recon.
(III) pLXSNneo-hTERT recon purification, expand, identify: the preparation of (i) E. coli competent: its
Basic skills is ice-cold CaCl2Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses
CaCl2Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to greatly
Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16~20h
(such as bacillus coli DH 5 2), or the 16~20h overnight culture that 1ml is fresh, forward to one containing 100mlLB culture medium 1L or
In 500ml flask, cultivate about 2~3h (rotary shakers 200~300r/min) in 37 DEG C of violent shakings, survey every 20~30min
Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial
Upper placement 10~20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C
10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml
The 0.1mMCaCl of ice pre-cooling2Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn
Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual
Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling2Resuspended every part is sunk calmly, at this point it is possible to rapidly
Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby.(ii) with competence escherichia coli purification, amplification
PLXSNneo-hTERT recon: take 200 μ l from every kind of competent cell suspension with the sterile pipette tip of cooling and transfer to nothing
In the microcentrifugal tube of bacterium, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), rotates gently with mixed
Even content, places 30min in ice, centrifuge tube is put into pre-heating to the test tube rack in the circulator bath of 40 DEG C, places
90s~2min, does not shake test tube, quickly transfers in ice bath by pipe, makes cell cooling 1~2min, and every centrifuge tube adds 800 μ
LSOC culture medium, is warmed to 37 DEG C with water-bath by culture medium, is then transferred to by pipe on 37 DEG C of shaking tables, and incubation 45min makes antibacterial
Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, turn by proper volume (every 90mm flat board is up to 200 μ l)
The competent cell changed is transferred to, in the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, flat board is placed in room temperature
Absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, after 12~16h, may occur in which bacterium colony.(iii) screen, expand recon: use
Sterile toothpick or disinfection inoculation pin select single colony inoculation in LB culture medium aseptic for 5mL or rich medium (such as super meat
Soup or TB super broth culture medium) in, after overnight incubation, it is then added to 500mL containing LB culture medium (containing suitable antibiotic)
In 2L flask, cultivate to saturation (OD then at 37 DEG C600≈ 4, for improving yield, should use surface area relatively big and band deflection plate
Flask to increase venting quality as far as possible, shaking speed should be greater than 400r/min), in 4 DEG C, 6000g is centrifuged 10min, uses 4mL GTL
The resuspended precipitation of solution, and transfer in the high speed centrifugation pipe of a volume >=20mL that (bacterial precipitation can be at-20 DEG C or-70 DEG C
Indefinite duration preserves), add the GTE solution containing 25mg/mL lysozyme that 1mL newly joins, resuspended precipitation, place 10min in room temperature, add
Enter 10mL and newly join NaOH/SDS solution, and mixing, until liquid becomes homogeneous, limpid and thickness, is placed on ice gently
10min, adds 7.5mL acetic acid solution, is gently mixed with suction pipe until viscosity declines and formed big precipitation, places on ice
10min, in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant in another clean centrifuge tube, if having visible
Drift the isopropanol of 0.6 times of volume by several layers of filtered through gauze, can be added, reverse mixing, room temperature places 5~10min, in room
Temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, sucks ethanol, very
Empty dry (precipitation can be 4 DEG C of long-term preservations).(iv) qualification of recombiant plasmid and amplification: the single bacterium colony on picking plate, inoculation
In 3ml is containing 100ug/ml ampicillin LB culture medium, 37 DEG C, 250r/min shaking table is cultivated, collection culture after 14h, 4
DEG C, 10000r/min be centrifuged 5min, extract in a small amount and purification of Recombinant plasmid by test kit description;Double with EcoRI and HindIII
Enzyme action recombiant plasmid reaction system: each 0.5ul of restricted enzyme, 10 × buffer 2ul, recombiant plasmid 10ul, add water and supply
To 20ul, 37 DEG C of enzyme action 1h.Digestion products carries out 0.8% sepharose electrophoresis, time 30min under 80V voltage conditions, and gel becomes
As system is taken pictures;Measure the sequence of recombiant plasmid routinely;Recombiant plasmid, will be containing this matter after enzyme action, order-checking are identified accurately
The microbionation of grain in LB culture fluid, amplification cultivation, carry out heavy dose of plasmid by heavy dose of plasmid extraction test kit description
Extracting and purifying, ultraviolet spectrophotometer is standby after measuring plasmid concentration and purity.
(IV) CD4+T cell: from the present invention " preparation of (2) CD4+T cell " sampling preparation.
(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5~10nmol/L insulins, 20% hyclone
In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5~10nmol/L insulin,
Typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer;15% hyclone (FBS);1% penicillin
And streptomycin;PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, from
The heart, removes supernatant, standby.
(VI) pLXSNneo-hTERT recon imports T lymphocyte and amplification culture: make in 1.5ml microcentrifugal tube
Standby following solutions: pipe A, is dissolved in pLXSNneo-hTERT recon in 100 μ l serum-free mediums;Pipe B, by 20 μ l
Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, left at room temperature 45min, trains with serum-free
Nutrient solution washs above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-pLXSNneo-hTERT mixture
Culture fluid, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone concentration is 20ml/ to add 1ml serum-free medium
L), at CO2Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues to cultivate
20h, discards culture fluid, and changing concentration is 400mg L-1G418 culture fluid continue cultivate, after 8 days select living cells make expand
After cultivation, then strengthen G418 concentration to 800mg L-1, by can in the G418 environment of high concentration the cell of stable growth continue into
Row amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If cell increases
Slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches 14ml
In time, transferred in 75ml culture bottle, and every 2-3 week adds 5-10ml fresh culture.Cell is cultivated to 9-10 week (the about the 75th generation),
Still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, and dead cell is less than 10% (by reading
The scale of culture vessel judges the increase situation of cell quantity;Dead cell and living cells is differentiated by trypan blue staining.Because
Normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter intracellular;And the cell of loss of activity, born of the same parents
The permeability of film increases, and can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method be draw weekly a certain amount of
Suspension culture, mixes rearmounted room temperature 5~10 minutes, then makes cell sheet, under the microscope with Trypan Blue agent
Count 1000 total cellular score, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with cultivating algebraically
Increase and the prolongation of incubation time, the increase of cell quantity is slack-off, dead cell get more and more, until cell is not further added by,
Even dissolve, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes, abandon
After Qing, add 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, become cell to hang
(cell concentration is about 10 to supernatant liquid5/ml).Cryopreservation tube subpackage, 1ml/ manage, put-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen-
In 196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).
(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious
Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection
Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively4Cell is inoculated in 30 containing 15 FBS low sugar DMEM culture medium trainings
Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days
The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium
In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes
After curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof ";(iii) check
Chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then this cell is described
System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, if do not had
Having, there is not tumor feature in explanation cell line yet).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid
250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making,
G shows band post analysis karyotype;(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line
Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that hTERT integrates, expresses.(vii)
Determined dna sequence: sequenator detection routinely, shows hTERT gene order.In (v) transfectional cell DNA hTERT detection: as with
Immunohistochemical detection, in the nucleus of hTERT transfection, the visible a large amount of brown particles of dyeing, show that hTERT has been integrated into carefully
Intracellular;(vi) mrna expression product measures: takes the pcr amplification product of 100 μ l systems, reclaims test kit (Takara, day with gel
This) reclaim product, take 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining DNA and each 10 μ l of upstream and downstream primer surveys
Sequence.
(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, amplification culture meets forever after above-mentioned qualification
OEG cell characteristic the cell same or like with primary cell, take the difference that growth conditions is good, be in exponential phase
Cell from generation to generation, is performing centrifugal separation on (1200r/min, 6min), with the frozen stock solution 0.5~1ml re-suspended cell containing dimethyl sulfoxide, carefully
Born of the same parents' density is 5 × 105Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h;-20 DEG C, 2h;-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen and freeze
Depositing, the immortalization CD4+T cell bank building biological characteristics stable in this way is standby.
2. with the concrete grammar of SV40 immortalization CD4+T cell
(I) extraction of SV40 large T antigen DNA: (i) SV40DNA enzyme action: contain large T antigen gene from commercially available purchase
SV40 freeze dried powder or SV40 plasmid, be dissolved in appropriate H2In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL
H2O, adds restricted enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL
Electrophoresis sample loading buffer (also can by add 0.5mol/LEDTA) terminates reaction in case electrophoresis.(ii) SV40DNA electrophoresis: power taking
Swimming level agarose is made into 10% agarose gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample
Comb, removes envelope band after gelling is solid from glue platform, extracts comb, put in the electrophoresis tank added with enough electrophoretic buffers,
Buffer exceeds gel surface about 1mm, prepares DNA sample with 10 appropriate × sample loading buffer, then with pipettor by sample
Add in sample well, and do suitable DNA molecular amount standard control thing simultaneously, connect electrode, make the DNA Ghandler motion that faces south move, at 1-
Under the voltage of i0V/cm gel, electrophoresis is to when being sufficiently separated the distance of DNA fragmentation, closes power supply.(iii) separate from agarose
About 2600bp SV40 large T antigen DNA: (use long wave ultraviolet light source to prevent DNA from damaging under 300-360nm long wave ultraviolet light source
Wound) gel-tape containing target DNA fragments is cut in loading bag filter, in bag filter, add 2ml electrophoretic buffer, be allowed to
Submergence gel, and empty steam bubble, bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), adds appropriate amount of buffer solution
By bag filter submergence (about 6-7mm), switching on power, 150 volts of electricity are washed, and observe and treat that DNA all removes gel, change under uviol lamp
Direction of an electric field continues energising 1 minute, and from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of volumes
N-butyl alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats two
Secondary, in the solution of lower floor speech DNA, add equal-volume phenol chloroform (2) extract 2 times, supernatant proceeds in another Eppendorf pipe
Add 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, be centrifuged 10 minutes at 4 DEG C,
DNA precipitation, abandon supernatant, abandon dry ethanol after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.Additionally, can also be used with
Target DNA fragment is separated from gel, is purified by low melting-point agarose gel method, DNA filter membrane inserted sheet method etc..
(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take 9 μ l above-mentioned DNA composition (0.1-5 μ g),
10 μ l 2 × connection buffer, 1 μ l 10mmol/L ATP, T4 DNA ligase (20~500 sticky end unit) or large intestine bars
Bacterium DNA ligase, pcDNA3.1 empty carrier mix, and 15 DEG C of incubation 24h are built into SV40T/pcDNA3.1 recon.
(III) SV40T/pcDNA3.1 recon amplification, separate and identify: the preparation of (i) E. coli competent: its
Basic skills is ice-cold CaCl2Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses
CaCl2Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to greatly
Most coli strains, operating process is summarized as follows: one single bacterium colony of picking from 37 DEG C of fresh plate cultivating 16~20h
(such as bacillus coli DH 5 2), or the 16~20h overnight culture that 1ml is fresh, forward to one containing I00mlLB culture medium 1L or
In 500ml flask, cultivate about 2~3h (rotary shakers 200~300r/min) in 37 DEG C of violent shakings, survey every 20~30min
Amount OD600 value ≈ 0.4, aseptically transfers to one, in ice-cold 50ml polypropylene centrifuge tube, at ice by antibacterial
Upper placement 10~20min, is centrifuged with 4000r/min with SorvallGS2 rotary head (or the rotary head matched with its centrifuge tube) in 4 DEG C
10min, to reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, uses with 10ml
The 0.1mMCaCl of ice pre-cooling2Resuspended every part of precipitation, is put on ice, in 4 DEG C with SorvallGS3 rotary head (or its corresponding turn
Head) it is centrifuged 10min with 4000r/min, to reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace of final residual
Culture fluid flows to end, the 0.1M CaCl of every 50ml initial incubation thing 2ml ice pre-cooling2Resuspended every part is sunk calmly, at this point it is possible to rapidly
Cell being distributed into aliquot, freezes in liquid nitrogen ,-70 DEG C of storages are standby, hang from every kind of competent cell with the sterile pipette tip of cooling
Liquid respectively takes 200 μ l and transfers in aseptic microcentrifugal tube, should add in often pipe DNA or coupled reaction mixture (volume≤
10 μ l, DNA≤50ng), rotate gently to mix content, ice is placed 30min, centrifuge tube is put into pre-heating to 40 DEG C
Circulator bath in test tube rack on, place 90s~2min, do not shake test tube, quickly transfer to pipe, in ice bath, make cell
Cooling 1~2min, every centrifuge tube adds 800 μ lSOC culture medium, with water-bath, culture medium is warmed to 37 DEG C, is then transferred to by pipe
On 37 DEG C of shaking tables, incubation 45min makes bacteria resuscitation, and antibiotic-resistance marker's gene of expression plasmid coding, by appropriate bulk
The competent cell that long-pending (each 90mm flat board is up to 200 μ l) have converted is transferred to containing 200mmol/LMgSO4 and corresponding antibiotic
SOB culture medium on, flat board is placed in room temperature and is absorbed to liquid, be inverted plate, in 37 DEG C of cultivations, may occur in which after 12~16h
Bacterium colony.(ii) recon screening, expand and extract: select single colony inoculation in 5mL with sterile toothpick or disinfection inoculation pin
In aseptic LB culture medium or rich medium (such as super broth or TB super broth culture medium), after overnight incubation, add
In the 500mL 2L flask containing LB culture medium (containing suitable antibiotic), cultivate to saturation (OD then at 37 DEG C600≈ 4, for
Improving yield, surface area should be used relatively big and the flask of band deflection plate is to increase venting quality as far as possible, shaking speed should be greater than 400r/
Min), in 4 DEG C, 6000g is centrifuged 10min, by the 4mL resuspended precipitation of GTL solution, and transfers to the high speed of a volume >=20mL
In centrifuge tube (bacterial precipitation can preserve at-20 DEG C or-70 DEG C of indefinite duration), add that 1mL newly joins containing 25mg/mL lysozyme
GTE solution, resuspended precipitation, place 10min in room temperature, add 10mL and newly join NaOH/SDS solution, and gently mixing until liquid
Body becomes homogeneous, limpid and thickness, places 10min on ice, adds 7.5mL acetic acid solution, is gently mixed until viscous with suction pipe
Denseness declines and is formed big precipitation, places 10min on ice, and in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant
To the centrifuge tube that another is clean, if there being visible drift that the isopropyl of 0.6 times of volume by several layers of filtered through gauze, can be added
Alcohol, reverse mixing, room temperature places 5~10min, and in room temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs gently
Precipitation, the ofest short duration the most centrifugal, suck ethanol, and be vacuum dried (precipitation can be 4 DEG C of long-term preservations).(iii) recon
Identifying: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence escherichia coli, ibid method is with restricted interior
Cutting enzyme BamH I and carry out enzyme action, 10g/L agarose gel electrophoresis is identified, it is thus achieved that 2 bands of size about 2600bp and 5600bp, front
Person meets the size of SV40T fragment in GenBank.
(IV) CD4+T cell: from the present invention " preparation of (2) CD4+T cell " sampling preparation.
(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5~10nmol/L insulins, 20% hyclone
In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5~10nmol/L insulin,
Typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer;15% hyclone (FBS);1% penicillin
And streptomycin;PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, from
The heart, removes supernatant, standby.
(VI) importing of SV40T/pcDNA3.1 and amplification culture: prepare following solutions in 1.5ml microcentrifugal tube: pipe
A, is dissolved in SV40T/pcDNA3.1 in 100 μ l serum-free mediums (hyclone concentration is 20ml/L);Pipe B, by 20 μ l
Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, the underlying 45min of room temperature.Use serum-free culture
Liquid washs above-mentioned T lymphocyte 2 times.The training of 1ml serum-free is added in Lipofectamine-SV40T/pcDNA3.1 mixture
Nutrient solution, mixes gently, then drops in above-mentioned T lymphocyte, and (hyclone concentration is to be subsequently adding 1ml serum-free medium
20ml/L), at CO2Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues
Continuous cultivation 20h, discards culture fluid, and changing concentration is 400mg L-1G418 culture fluid continue cultivate, after 8 days select living cells
After making amplification culture, then strengthen G418 concentration to 800mg L-1, will can stablize the cell of growth in the G418 environment of high concentration
Proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If it is thin
Born of the same parents increase slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches
Transferring in 75ml culture bottle during to 14ml, every 2-3 week adds 5-10ml fresh culture.Cell cultivates about 6-8 week the (the about the 55th
Generation), still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, and dead cell is less than 10% (by reading
The scale taking culture vessel judges the increase situation of cell quantity;Dead cell and living cells is differentiated by trypan blue staining.Cause
For normal living cells, after birth structural integrity, it is possible to repel, make trypan blue can not enter intracellular;And the cell of loss of activity,
The permeability of after birth increases, and can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method be draw weekly a certain amount of
Suspension culture, mix rearmounted room temperature 5~10 minutes with Trypan Blue agent, then make cell sheet, at microscope
1000 total cellular score of lower counting, calculate dead cell and the percentage ratio of non-staining living cells of coloring).Hereafter along with cultivating generation
The increase of number and the prolongation of incubation time, the increase of cell quantity is slack-off, dead cell gets more and more, until cell no longer increases
Add, even dissolve, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes,
Abandon supernatant, add 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, become cell
(cell concentration is about 10 to suspension5/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen
In-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).
(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious
Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: take growth preferably transfection
Cell, makes cell suspension, through counting, takes 1.4 × 10 respectively4Cell is inoculated in 30 containing 15 FBS low sugar DMEM culture medium trainings
Support bottle.Taking 2 bottles of cells every day to count, calculate average, Continuous Observation is until cell quantity is decreased obviously, every after cultivating 3 days
The cell giving no count every 2 days changes liquid, uses same method to observe transfectional cell at hepato ZYME-SFM serum-free medium
In growing state.Result is with incubation time as transverse axis, and cell quantity is the longitudinal axis (logarithm), is depicted on semi-logarithmic scale and makes
After curve the growth curve of this cell, immortalized cell line is formed in typical " S " feature or " arched roof ";(iii) check
Chromosome: by analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY ", then this cell is described
System does not occur vicious transformation (to can use simultaneously and whether occur abnormal DNA colony in flow cytometry analysis cell line, as not having
Have, illustrate that tumor feature does not occurs in cell line).Chromosome karyotype analysis method is: by adding preheating in 5mL culture fluid
250ug/ml Colchicine 100ul, mixes rearmounted 37 DEG C of incubators 4 hours, by centrifugation, go supernatant, hypotonic, fixing, film-making,
G shows band post analysis karyotype;(iv) Flow cytometry: the cell ratio synthesizing, dividing in detection the 19th continuous cell line
Example, if its multiplication capacity substantially ratio does not builds the normal cell enhancing being, explanation is the result that SV40 large T antigen is integrated, expressed.
(viii) determined dna sequence: sequenator detection routinely, shows SV40 large T antigen DNA sequence.In (v) transfectional cell DNA
SV40 big T gene test: as with Immunohistochemical detection, dye in the nucleus of SV40 transfection visible a large amount of brown particles,
Show that SV40T antigen has been integrated into intracellular;Also T antigen expression in cell, wherein T antigen can be detected by RT-PCR method
Primer: forward primer (A4239) 5 '-GTT ATG ATT ATA ACT GTT ATG-3 ', downstream primer (S4496) 5 '-GAA
ATG CCA TCT AGT GAT-3’;The a length of 268bp of amplified production, amplification condition is 94 DEG C, 5min, it may be assumed that (94 DEG C, 1min;
55 DEG C, 1min ,-0.5 DEG C/circulation;72 DEG C, 1min) × 30, (94 DEG C, 30S;40 DEG C, 30S, 72 DEG C, 30S) × 15, expand body
System is 50 μ l:[Mg2+] 2mmol/L, dNTPs 200 μm ol/L, primer concentration 0.4 μm ol/L, Taq1U, template 5 μ l;Experimental group
With the cDNA of the 19th generation cell as template (carry out the synthesis of cDNA the first chain with reference to commercially available cDNA the first chain synthetic agent box, product-
20 DEG C of preservations);Negative control sets two, does template with the cDNA of sterilized water, primary cell respectively, and positive control is with SV40 DNA
(extract SV40 DNA with reference to SDS-proteinase-K pathway for template, because SV40 virus is without peplos, do not use SDS rupture of membranes, take 5 μ l and enter
Row 1.5% agarose gel electrophoresis detects, and remaining-20 DEG C save backup);(vi) mrna expression product measures: T antigen mRNA
RT-PCR product checks order: take the amplified production of 100 μ l systems, reclaims test kit (Takara, Japan) with gel and reclaims product, takes
2 μ l DNA solutions dilute 100 times, survey concentration, and remaining DNA and each 10 μ l of upstream and downstream primer checks order.
(VIII) SV40LT gene mediated CD4+T cell bank: screen and continue to pass on, amplification culture accords with after above-mentioned qualification
Close immortalized cells characteristic the cell same or like with primary cell, take that growth conditions is good, be in exponential phase
The cell of different generations, is performing centrifugal separation on (1200r/min, 6min), resuspended carefully with the frozen stock solution 0.5~1ml containing dimethyl sulfoxide
Born of the same parents, cell density is 5 × 105Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h;-20 DEG C, 2h;-70 DEG C, overnight, enter-196 DEG C of liquid
Nitrogen is frozen, and the CD4+T cell bank building biological characteristics stable in this way is standby.
(4) with the preparation of CD4+T cell identity function granule: can be by CD4 molecule, the CD4 molecule of gene recombinaton and similar
The molecule of function by conventional chemical coupling, crosslinking, affine absorption etc. be fixed on carrier make be coated with CD4 molecule
Grain, or directly take the replacement CD4+ cell application of intimate granule.It is thin that the CD4+T cell of the present invention represents other CD4+
Born of the same parents, prepare immortalization CD4+T cell including with additive method.
(2) preparation of HIV-1gp120 antibody
Antibody involved in the present invention can entrust specialty businessman to prepare, or directly buys, as Shanghai is auspicious from specialty businessman
Unit such as neat bio tech ltd and Shanghai Linc-Bio Science Co., Ltd. etc. all specialize in HIV-1gp120 antibody,
The preparation of the various antibody such as gp41 antibody and goat anti-human igg and sale.Method includes that hybridoma technology prepares monoclonal antibody, EB
Virus Transformation technology is prepared monoclonal antibody, hybridoma technology and is combined with Epstein-Barr virus transformation technology preparation monoclonal antibody and base
Because of engineered antibody, specifically it is listed below.
1, use lymphocyte Epstein-Barr virus to convert with hybridoma technology and combine and prepare HIV-1gp120 monoclonal antibody
Specimen origin have following several by way of: take lymphocyte strain frozen in Infectious Diseases Lab Sample Storehouse (through inactivation
HIV immunity and the lymphocyte of EBV transfection);Buy the fresh White Blood Cells Concentrate in blood station, then carried out inactivation HIV strain and exempt from
The lymphocyte of epidemic disease;Take from the cord blood lymphocytes cell (through inactivation HIV immunity) preserved as scientific research;Directly take from HIV-1
The peripheral blood lymphocyte (for self) of the infected self, uses Histopaque lymphocyte separation medium separation single core thin
Born of the same parents (PBMC), regulation concentration is 2 x 106Epstein-Barr virus (EBV) stock solution that rear addition is appropriate, is placed in 370C, 5%CO2 and cultivated
At night, preparing B cell to be hybridized, with the positive hole of ELISA method screening anti-HIV-1 outer membrane protein (gp120), transfer cell is to 24 holes
Plate continues to cultivate 2 weeks, repeats to measure anti-gp120 by ELISA method and confirms positive, continuously clone's secondary a large amount of amplification cultivation.
After heterogeneous with people Mus for positive cell strain myeloma cell's (being buied by Zhejiang University's siberian crabapple) is mixed (3: 1), add 1ml50%
PEG makes the two merge, and then re-suspended cell cultivated liquid in IMDM culture fluid, within second day, adds Peritoneal Cells of Mice (by Zhejiang
Jiang great Xue siberian crabapple is buied) as trophocyte, screen anti-gp120 antibody with ELISA after continuing to cultivate 3 weeks, select strong positive
Hole hybrid tumor cell amplification is cultivated, and repeatedly clones until obtaining stable cell line, cultivates with this cell line, prepares
HIV-1 antibody, uses ELISA detection kit, and by specification operates, and measures the Ig subclass of antibody, and surveys with conventional ELISA method
Determine titer and the specificity of antibody, select high specificity, antibody that titer is high.
2, use gene recombinaton HIV-1gp120 to combine hybridoma technology and prepare antibody
(1) reagent and recombinant antigen: relate to reagent: HIV-1gp120 genetic fragment, HIV-1gp120 antigen, HIV antigen
Nitrocellulose membrane bar is provided BamH I restriction endonuclease Xho I restriction endonuclease, nucleic acid coprecipitator, T4 DNA by Beijing Wan Tai Pharma Inc.
Ligase is purchased from precious biological company limited;Liagen glue reclaims test kit purchased from QIAquick company;RPMI 1640 dry powder is cultivated
Base is purchased from Gibco company;Top grade new-born calf serum is purchased from bright marine growth Engineering Co., Ltd;SupersignalWest Pico
Trial Kit, TMB Substrate Kit are purchased from Pierce company;Mice Ig subclass detection kit, freund adjuvant and PEG,
Hy-poxanthine, Thymidine and Aminoptem are purchased from Sigma company.HIV-antibody diagnosing reagent kit is purchased from Shanghai section
Biological company limited of China, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) labelling is purchased from doctor moral company limited.
Construction of recombinant plasmid and qualification: vector plasmid PEGX-4T-2 BamH I, Xho I enzyme action, T4DNA Ligase connects gp120
Genetic fragment, recombinant plasmid transformed enters E.colistrain XL1 blue, checks order.The abduction delivering of recombiant protein and qualification: weight
Group Plastid transformation enters in XL1-Blue escherichia coli, under IPTG derivant effect 25 DEG C, and 190r/min shakes, overnight, and 4
000r/min is centrifuged 10min, collects antibacterial, SDS-PAGE testing goal protein expression situation.Fusion protein purification and qualification: table
Reach product centrifugal collecting precipitation to hang through PBS, after 30W Ultrasonic Pulverization instrument cracking antibacterial, be centrifuged collection supernatant and filter, filter
Liquid AKTA PURIFYER100 protein purification instrument, GST column purification, obtain fusion protein GST-HIV, concentrates centrifuge tube and carries out dense
Contracting, S21 type biology spectrophotometer measurement concentration, purifying protein is identified by SDS-PAGE.
(2) animal immune: BALB/c mouse 6 week old, female, 4, before immunity, take mouse vein blood, separation serum stays and does
Negative serum.Mixed with equal-volume Freund's complete adjuvant by the GST-HIV fusion protein of 50-100 μ g, the injection of emulsifying pneumoretroperitoneum is exempted from
Epidemic disease mice.After initial immunity, booster immunization mice after using incomplete Freund's adjuvant and fusion protein emulsifying every 2 weeks, immunity
Dosage and approach are the same, repeat immunity 2-3 time, and the GST-HIV of last booster immunization direct lumbar injection 50-100 μ g melts
Hop protein.
(3) foundation of Detection of Monoclonal Antibody: third time immunity one week after tail vein blood, determines positive serum by square formation method
Best effort concentration and GST-HIV fusion protein be most preferably coated concentration.Operate as follows: according to 1: 1000,1: 500,1:
200,1: 100 four dilution factor, uses and is coated buffer dilution antigen, be longitudinally coated 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of bags
By overnight, wash 3 times, every minor tick 3 minutes.Positive serum and negative serum are made doubling dilution respectively by 1: 1000,
Laterally it is loaded onto the 10th hole, every hole 100 μ L, is placed in 37 DEG C of incubation 1h in wet box, wash 3 times, every minor tick 3 minutes.Enzyme mark resists
Body HRP-sheep anti-mouse igg makees 1: 10000 dilution, every hole 100 μ L, 37 DEG C of incubation 1h to specifications, washs 3 times.Add people now to join
OPD substrate solution, every hole 100 μ L, 37 DEG C of lucifuges reaction appropriate times, every hole adds 100 μ L stop buffers and terminates reaction, detects it
OD492 value.Positive hybridoma cell screening technique is set up.According to experiment condition and method in square formation method, melt with GST-HIV respectively
Hop protein is experimental group, and after abduction delivering, recombinant bacterium (containing plasmid pET-32a) albumen is matched group, screening positive clone.Operation
Step is as follows: with the suitableeest concentration envelope antigen that is coated in 96 hole ELISA Plate, and 100 μ l/ holes, 4 DEG C of refrigerators are coated overnight.Take out bag
Washed 3 times by adding cleaning mixture after plate, wash 3min every time;Every hole adds cell conditioned medium to be measured (1: 5 dilution) 100 μ L, 37 DEG C of temperature
Case hatches 50min, and washing 3 times, wash 3min every time afterwards;Every hole adds two and resists 100 μ l, 37 DEG C of incubation 30min, washes
Wash 3 times, wash 3min every time;Every hole adds the OPD substrate solution 100 μ L now joined, room temperature lucifuge reaction 10-15min;In every hole
Add 2mol/LH2SO4 stop buffer 100 μ l to be used for terminating reaction;Detection plate is placed in microplate reader survey OD492 value.Matched group
Setting up: positive controls is the positive serum of suitably dilution, negative control group is to resist with one to have the most dilution unrelated monoclonal antibody
Cell conditioned medium.Indirect ELISA the selection result judges.Often group detection OD492 value, with P (sample value)/N (negative value) >=2.0
It is judged to positive value.Screening positive clone standard: cell conditioned medium reacts with just screening group (fusion protein is coated after purification) and is positive,
React, with negative screening group (tropina containing pET-32a plasmid after induction), the detection hole being negative is positive simultaneously.
(4) cell merges: myeloma cell prepares: merge the myeloma taking out a pipe the last week in liquid nitrogen container frozen thin
Born of the same parents, are immediately placed in hot water and thaw.Adding appropriate complete culture solution after thawing, 1000r/min is centrifuged 3min;It is repeated 1 times.Will precipitation
Thing moves in Tissue Culture Flask, adds DMEM culture fluid, puts CO2 incubator and cultivates, within 3-4 days, once passes on or amplification culture,
Cell state is adjusted, it is ensured that before merging, cellular morphology is good, it is vigorous to grow in merging first 24 hours.Appropriate pancreatin is used before merging
Using centrifuge tube to collect after digestion, add appropriate basal medium in centrifuge tube, after beaing mixing gently, 1000r/min is centrifuged
5-10min, repeated washing cell 2 times.Splenocyte prepares: before fusion, takes a Balb/c mice, wins eyeball and take blood, blood-letting
Post-tensioning neck completely is put to death, and soaks in 75% ethanol.Taking-up layback is put and is fixed on dissection plate, takes spleen under gnotobasis, will
Spleen moves in plate.Then 10mL RPMI 1640 basal medium is added at plate, with flat mouth tweezers attrition crushing repeatedly
After, use asepsis injector repeatedly to aspirate piping and druming, make single cell suspension.After meter viable count, 1000r/min is centrifuged 10min,
Add the basal medium total cell of adjustment and count to 1 × 108~2 × 108Merge for cell.Cell merges: by splenocyte and bone marrow
Oncocyte is with 10: the ratio of 1-5: 1 adds in centrifuge tube, is mixed evenly, and 1000r/min is centrifuged 5min, supernatant discarded, strikes gently
Beat at the bottom of pipe to cell without granular precipitate, be repeated 2 times.Preheating centrifuge tube, aseptic bar after taking-up is rotated gently in 37 DEG C of water-baths
Under part, the 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s,
Afterwards the basal medium of the 25mL of preheating is also added drop-wise in centrifuge tube along tube wall in 3-5min, light during adding
Lightly rotating centrifugal pipe, is then statically placed in 37 DEG C of water-bath 10min, and 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T
Culture medium.Suitably it is inoculated in 96 well culture plates after mixing, is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.
(5) screening of positive clone strain: observe cell growth status in 96 well culture plates, only has hybridoma thin after 7-10 days
Born of the same parents can grow division, now discards HAT culture medium, changes complete medium.Cell clone growth area reaches 1/10 carefully
During hilum, go culture supernatant, cloned by the monoclonal antibody screening technique screening positive hybridoma cell set up before.Use improvement
The positive cell hole that indirect ELISA Preliminary screening is gone out by limiting dilution assay gradient limiting dilution assay continuously carries out 3-4 wheel
Sub-clone.Selection has the culture hole of the positive hybridoma cell strain that growth conditions is good, labelling cell strain growth under microscope
Position, size, use aseptic rifle head to draw cell clone in the position of mark in the new culture hole having complete medium, so
After successively doubling dilution count hole to below, 37 DEG C, cultivate about one week in 5%CO2 incubator, basis of microscopic observation cell grows
Situation, when cell clone covers with to hole floor space more than 1/10, takes cells and supernatant and carries out antibody inspection side.To testing result
Repeat next round dilution for the cell clone in positive culture hole to cultivate, repeat 2-3 wheel, after detection supernatant titer plateaus
Take out, proceed to culture bottle mass propgation.
(6) preservation of hybridoma cell strain and Secondary Culture: preserve and recover: preserve first 12 hours and adjust cell growth shape
State.Taking one bottle of growth vigorous, make cell suspension after the cell that form is good, suitably digestion, 1000r/m is centrifuged 5min, goes
Clear liquid, flicks and makes cell loose at the bottom of pipe, add 4 DEG C preserve 9 parts of complete culture solutions and 1 part of DMSO, subpackage cell cryopreservation tube,
1mL/ manages, and cryopreservation tube is put in liquid nitrogen container after taking-up and saved backup by-70 DEG C of refrigerator overnight.40 DEG C of left sides are got out before recovery
Right hot water, carefully takes out cryopreservation tube from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, after defrosting
1000r/m is centrifuged 5min, opens cryopreservation tube in superclean bench under aseptic condition, and the cell complete culture solution after thawing is washed
Washing once, be then centrifuged 5min, supernatant discarded at 1000r/m, cell precipitation moves into training after using complete culture solution the most resuspended
Support in bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Secondary Culture positive hybridoma cell passed for 10 generations continuously, used indirectly
The method of ELISA measures culture supernatant antibody titer, observes the change of titer, and whether observe this positive hybridoma cell strain can be steady
Determine secretory antibody.
(7) preparation of monoclonal antibody mouse ascites: low-speed centrifugal collects the hybridoma after cultivating, dilute through basal medium
Releasing cell number is 1 × 107/mL.Mouse peritoneal injection 0.2mL/ only, observes mouse ascites production, treats that abdominal part is bright after injection
Aobvious distension rises, and punctures abdominal cavity with syringe needle, and centrifuge tube collects ascites.Gather complete, mouse peritoneal is injected appropriate basal medium,
Every 2-3 days, same method took ascites again.The ascites collected, 10000r/m is centrifuged 5min, Aspirate supernatant, subpackage ,-20 DEG C
Preserve.
(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out with reference to " molecular cloning " method, half
Dry method transfers, and program is as follows: first with recombinant bacterium albumen after the CD4 fusion protein of purification and induction through 12%SDS-PAGE, and one
Group is used as comparison, and one group is used as transfer.Electrophoresis is complete, puts into Cathode buffer with an equal amount of 6 filter paper after being cut by glue
In;NC film is first used soak with ethanol 3-5min, is then placed in deionized water, big filter onesize with 6 again after 1-3min
Paper is put in anolyte together;With Cathode buffer, the minus plate of electrophoresis tank is smeared moistening, take out the filter in Cathode buffer
Paper and gel are successively placed on minus plate, extrude bubble gently.Again NC film in anode buffer liquid and 6 filter paper are taken from anolyte
Go out and be layered on successively on gel, extrude bubble gently.Finally cover electrophoresis tank positive plate gently.After switching on power, according to NC film
Area, 2mA/cm2 size of current, transfer 2h;After transfer terminates, glue dyes after taking out, after transfer membrane takes out, dilute with PBS
5% defatted milk powder released is closed, and 4 DEG C of refrigerators stand overnight.After closing overnight, discard confining liquid, wash 3 with lavation buffer solution
Secondary, each 5min.After washed, add the monoclonal antibody cell conditioned medium of 1: 10 dilution, jog on shaking table, room temperature reaction 60min.Abandon
Go one to resist, then wash 3 times with lavation buffer solution, each 5min.The condition groped according to Dot-ELISA, adds 1: 5000 dilution
The two of degree resist, jog on shaking table, room temperature reaction 50min.Discard two to resist, then wash 3 times with lavation buffer solution, each 5min.
The NC film having protein band is carried out labelling, adds DAB developer, terminate with deionized water rinsing anti-after reaction appropriate time
Should.Titer, with the CD4 fusion protein of purification for detection antigen, uses indirect ELISA method to measure hybridoma supernatant and list
The titer of anti-ascites.Monoclonal antibody subgroup identification selects mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody i.e. with kit measurement, presses
Operating according to test kit operating instruction, step is as follows: the fusion protein of CD4 after purification suitably diluted is coated in ELISA Plate,
Every hole 100uL, puts 4 DEG C of refrigerator overnight.The liquid that is coated in ELISA Plate is patted dry, then washes once with lavation buffer solution, 3min.So
After Hybridoma Cell Culture supernatant to be measured is added in hand-hole, every hole 100 μ L, put 37 DEG C of incubator incubation 30min.With washing after patting dry
Wash buffer and wash five times, 3min/ time.6 kinds of enzyme marker in this test kit are separately added in hole, every hole 100 μ L, are placed in 37
DEG C incubation 30min.Continuation lavation buffer solution washes five times, 3min/ time.Add after OPD substrate solution lucifuge develops the color 15 minutes and add eventually
Only liquid, detects OD492 value by microplate reader, and OD492 value substantially exceeds the type in other hole and is HIV-1gp120 monoclonal antibody Ig classification.
(9) HIV-1gp120 monoclonal antibody applies (specialty businessman can be entrusted to complete) after the most refined.
(4) preparation of HIV-1gp41 antibody
Preparation with HIV-1gp120 antibody
(5) preparation of goat-anti people-Ig
Being antigen by made HIV-1gp120 antibody and gp41 antibody, immune sheep prepares antibody.
1, laboratory animal: what immunity laboratory animal was selected is that animal institute of Zhejiang University hybridizes white goat, body weight 30 jin
The healthy between twenty and fifty ewe two of left and right, the front dyestuff of immunity smears the back animal, makes clear and definite labelling.Employing is done as everybody else does
The feeding manner of stable breeding, ensures that sheep has to appropriate motion every day, and between the lights, general half an hour of every time moving is left for movement time
The right side, the most healthy and strong, it is to avoid sheep overfertilization, increase the immunity of body, drinking-water abundance, and give appropriate concentrate,
Grass, Semen Maydis, wheat bran, Semen Tritici aestivi, vitamin etc. so that it is balanced in nutrition.Often check experiment sheep health status.
2, HIV-1gp120 antibody and gp41 antibody are antigen: HIV-1gp120 antibody prepared by the present invention and gp41 antibody
(IgG) concentration is respectively 2.5mg/mL (can be provided) by businessman, and before inoculation, mixing 0.1mL antigen, 1.9mL aseptic PBS, 2mL are not
It is standby that immunogen emulsion made by family name's (or incomplete) adjuvant completely.
3, the immunity of goat: choose two goats, is labeled as goat A, goat B, antigen inoculation (HIV-1gp120 antibody and
HIV-1gp41 antibody).Immunity position is front and back's groin, and often place's groin divides 2 some injections, a total of 8 injection points.Note
The mode of penetrating is subcutaneous injection, and every goat per injection 4mL immunogen, containing 250 μ g antigens;Each injection point injecting immune is former
0.5mL, containing about 32 μ g antigens.Front two goats of immunity are drawn blood 10mL respectively, are labeled as 0dP1 ,-20 DEG C of preservations;Immunity for the first time
I.e. 1, immunogen: 0.1mL antigen+1.9mL aseptic PBS+ Freund's complete adjuvant 2mL (CFA) is exempted from;Draw blood after immune 7 days 10mL,
Separate serum, be labeled as 7dP1, ELISA and detect serum titer, remain serum-20 DEG C preservation.Within 3~4 weeks, start second time immunity,
Two immunogens: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), draw blood after immune 7 days 10mL, separates
Serum, is labeled as 7dP2, ELISA and detects serum titer, remain serum-20 DEG C preservation.After immunization time is 6-8 week for the third time,
Three exempt from immunogen: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), and draw blood after immune 7 days 10mL, point
From serum, it is labeled as 7dP3, ELISA and detects serum titer, remain serum-20 DEG C preservation.If now serum titer does not reaches 1
∶106Above, then need to exempt from again once;If serum titer has reached 106Below then need not be immune again, draw blood the most week about
50mL, separates serum ,-20 DEG C of preservations.
4, prepared by serum: within after general each immunity 7~10 days, can detect in sheep jugular vein blood collection.By assistant Baoding
Animal so that it is keep stance, after cervical region cropping, aseptic cotton balls cleaning disinfection, searches out the blood sampling of jugular vein hand syringes,
Blood 5-10mL is taken by fixing for syringe position.Bioactivity is carried out after isolating serum.7~10 days after the third immunization, warp
The most desirable blood 30-50mL after bioactivity is qualified.Aseptically separate serum, in plate to be collected in or triangular flask
After blood coagulation, after clot being peeled off with bottle wall in gnotobasis (such as superclean bench) with aseptic dropper, put into 37
DEG C, 1~2h take out after put into 4 DEG C overnight, make serum fully separate out (can not freeze, otherwise produce haemolysis), by centrifugation precipitation point
Go out serum, put cryogenic refrigerator into and preserve.Before using must through signing qualified after again subpackage save backup.
5, the mensuration of antiserum titre: antiserum titre is to use ELISA assay method: be with being coated when being coated by sample
After liquid is diluted to the concentration of 1: 1000, in ELISA Plate, every hole adds 100 μ L, is then placed in aluminum box and puts in 4 DEG C of refrigerators overnight.
Take out the next morning to pat dry and be coated liquid, wash three times with PBST, every minor tick five minutes, pat dry enzyme with gauze for the last time
Target, adds the confining liquid of 10% serum, and every hole adds 100UL, puts into 37 DEG C, water-bath 1~2h.Take out the most again and pat dry closing
Liquid, with the every minor tick of PBST detersive enzyme target 3 five minutes, pats dry with gauze for the last time, adds 100 μ L/ holes one anti-(1: 2000
Dilution, dilutes with the PBST of 4% Ox blood serum, puts into 37 DEG C, water-bath 1~2h).Taking out and pat dry an anti-liquid, PBST washes
Wash ELISA Plate three times, every minor tick 5 minutes, pat dry ELISA Plate with gauze for the last time, add two anti-liquid (the anti-sheep of rabbit 1: 1000),
Every hole adds 100 μ L, puts into 37 DEG C, water-bath 1~2h.Take out again and pat dry the two anti-every minor ticks five of liquid PBST detersive enzyme target 3
Minute, patting dry with gauze for the last time, every hole adds 50 μ L substrate nitrite ions, and black out adds the H SO of 2M after developing the color 10-20 minute
Solution 50 μ L terminate reaction, after with microplate reader survey OD value (in half an hour).
Two, the preparation of acquired immune deficiency syndrome (AIDS) blood purification
1, the preparation of blood purification cell column
CD4+T cell prepared by the present invention, with physiological saline solution clean, 1000r/min be centrifuged, clean after with
1000r/min is centrifuged 5min, takes cell precipitation and loads the 200ml hydrostatic column that acrylate etc macromolecular material is made
In, it is fills up to more than 4/5, cell column is sealed standby.
2, the outfit of blood purification gel antibody
Gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti gp120 in mixed liquor
All it is fully tied with gp41 antibody, but surplus free gp120 and the gp41 antibody having sufficiently high titre, then will be containing combining
Type and the mixed antibody of sequestered gp120 antibody and the mixed antibody containing conjunction type and sequestered gp41 antibody again with etc.
Titer mixes, and is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies with the agarose C1-4B solution of gradient concentration respectively,
The titer making HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and the agarose concentration of correspondence
It is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, irrigates standby blood with equal equal-volume from low to high by antibody titer
Liquid daf molecule post.The filter opening that agar gel is formed reduces along with increasing of agarose concentration, depurator import department agar
Sugar concentration is low, and filter opening is just big, beneficially the association reaction of plasma perfusion and high titer antibody or cell and HIV;And exit is dense
Degree height, filter opening is the least, it is easy to detention HIV or Large molecular conjugates.Cleanser is combined with multiple special and non-specific HIV and removes
Composition, in order to avoid extraordinary strain is because of the futile treatment caused by immunity difference.
Formula one: by gp120 and gp41 antibody with play being incubated at 39~41 DEG C after 100 DEG C dissolve of carrier function
0.7%, the agarose C1-4B normal saline of 0.8%, 0.9%, 1.0%, 1.1% is made into the most corresponding 1: 700,1: 500,1
: 300, the gel antibody of the gradient titre of 1: 200,1: 100, take 10~15ml gel antibody successively by the paramount titre of low titre and add
Enter in blood purification cell column, it is desirable to the gel antibody being initially charged be cooled to 37 DEG C become semi-solid agar gel after the most then
Add next time, make blood purification cell column form antibody titer from high to low from top to bottom (from import to outlet) and from low to
The layer distributed of high agarose concentration, wherein with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120
Antibody, gp41 antibody and CD4+T cell are all fixed in gel the effect playing absorption HIV.
Formula two: by gp120 and gp41 antibody with rise carrier function after 100 DEG C dissolve, be cooled to 39~41 DEG C
After the agarose C1-4B of 1% content is made into the titre gradient of 1: 50~1000 by multiple proportions, Reperfu-sion blood purification cell column, make
Blood purification cell column forms antibody titer from high to low and agarose from low to high from top to bottom (from import to outlet)
The layer distributed of concentration, wherein resists with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120 antibody, gp41
Body and CD4+T cell are all fixed in gel the effect playing absorption HIV.
Formula three: indicate the titre being made into by multiple proportions of the different antibody prepared by HIV strain and correspondence thereof respectively
The valence value of gradient, such as HIV strain 1 titre 1:100 pipe, 1:200 pipe ...;HIV strain 2 titre 1:100 pipe, 1:
200 pipes ..., the rest may be inferred, then will infect strain 1 titre 1:100 pipe and 10ml39~the liquid agarose C1-of 41 DEG C of insulations
Put into blood purification cell column after 4B mixing, to be placed be cooled to semi-solid gel after, then will infect strain 1 titre 1:200 pipe with
The liquid agarose C1-4B mixing of 10ml39~41 DEG C of insulations, mixing liquid puts into the gel upper strata of blood purification cell column, according to this
Analogize.A, B, C totally 3 strain is had in actual applications by combination preparation, the HIV strain in such as somewhere period, preparation
Antibody has A strain 1:100 pipe, 1:200 pipe ...;B strain 1:100 pipe, 1:200 pipe ...;C strain 1:100 pipe, 1:200 pipe ...;Warp
After combination be exactly ABC strain 1:100 pipe, ABC strain 1:200 pipe ... ABC strain 1:1000 manage, then by ABC strain 1:1000 pipe with
Put into blood purification cell column after the liquid agarose C1-4B mixing of 10ml39~41 DEG C of insulations, to be placed be cooled to semisolid
After gel, then the liquid agarose C1-4B mixing by ABC strain 1:900 pipe with 10ml39~41 DEG C of insulations, mixing liquid puts into blood
Being cooled to semisolid gel upper strata in daf molecule post, the rest may be inferred, and centre can be spaced selection, the most then selects ABC
Strain 1:500 pipe mixes with the liquid agarose C1-4B of 10ml39~41 DEG C of insulations, and mixing liquid has put into blood purification cell column
Being cooled to semisolid gel upper strata, the consumption of liquid agarose C1-4B (can also be made by adjusting in 1ml~10ml
The blood purification cell column become, from import to going out interruption-forming gradient antibody content from low to high, in view of antigen and antibody only
Just can play immunoreation when proper ratio, form precipitation line and stop moving ahead of comparator antibody or antigen, so different HIV contains
When the blood plasma of amount is by cleanser, HIV just adsorbed detention in different aspects, and also the HIV strain of the present invention is permissible
The most almost all of infection strain of letter lid, and be combined with goat-anti Ig in adsorbent and unconjugated gp120 and gp41 antibody
Being all the HIV aglucon being fixed in advance in Agar Gel, HIV antibody is tied with HIV especially in conjunction with the HIV antibody having goat-anti HIV
Close the immune complex molecular weight that formed bigger, be entirely capable of being delayed in Agar Gel and be eliminated, add aperture about
For 85nm Agar Gel inherently can the detention a diameter of 100~HIV of 120nm, so inferring theoretically and having hardly
The HIV sufferers failed to respond to any medical treatment).
3, the making of blood purification
Blood purification cell column or blood purification are the cylinder that footpath, the end is little, footpath, top is big, or square, infundibulate, volume
Being 200~300ml, import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh;Footpath sieve mesh at the bottom of exit
Number is 2.0~5.0 mesh (2.5~5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), specifically can be made into 2.0 mesh,
7 kinds of different sizes of 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop 120 nanometers inhibition of HIV or
Bigger antibacterial;Liquid outlet arranges the cell strainer that mesh number is 100 mesh (being equivalent to 4 microns), may leach in order to stop
Cell;Relief area, the beneficially stability of system circulation it is provided with between liquid entrance and mesh screen.
Blood purification selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, activates benefit hardly
Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can pass through covalency,
The methods such as grafting, polymerization improve the structure of material, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress
Impact thus improve biocompatibility, reduce complication generation.Hydrophilic gel is added, by 2 methyl-prop at depurator inner surface
Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate membrane, by controlling wet-spinning procedure, can generate
CA/PMB30, CA/PMB80 and CA/PMB30-80, have higher blood and cell compatibility.Some had anticoagulation
Material be solidificated on the material of carrier or depurator inner surface, can suppress blood coagulation, improve biocompatibility, also can reduce
Heparin consumption, and likely realize no-rod tractor.As being aggregated in by heparin on polyacrylonitrile-polymine film, effect may
More preferably, and can reduce the anaphylaxis during purification, the polyacrylonitrile surface of solidifying shell polysaccharide and heparin covalent thing displays that
Good blood compatibility, and the activity of pseudomonas aeruginosa can be suppressed, reduce cell-cytotoxic reaction.Heparin covalent is combined
To polyether sulfone surface, both maintained the mechanical property of polyether sulfone, the anticoagulation function of depurator inner surface can have been improved again.At acetic acid
Covalent immobilisation linoleic acid film on fibrous membrane, maybe will be covalently bound to polyacrylic linoleic acid and be grafted onto polysulfone membrane surface, all may be used
To have more preferable histocompatibility and anticoagulant effect.Along with macromolecular material and the development of nanotechnology, with mankind's blood
The close material of endothelial tube in the near future it would appear that.
Three, the preparation of separator
(1) preparation of blood separator
1, preparation principle: 1. hemocyte, antibacterial, the molecular size of virus: in blood of human body, visible component (cell) is big
Little it is: normocyte is about 7 microns (μm), is the discoid cell of concave-concave;Leukocyte is divided into 5 kinds, and neutrophilic granulocyte is about
12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, and small lymphocyte 6-8 μm, with erythrocyte
Approximation, mononuclear cell is maximum, about 15-20 μm.Platelet is disc, diameter 1~4 microns to 7~8 microns, the blood of people
Platelet average diameter is 2-4 micron, thick 0.5~1.5 micron.The size of antibacterial is: the diameter of coccus about 0.75-1.25 μm it
Between, bacillus length is about in 2-5 μm, and spirillum is about 100-200 μm.Virus size with nanometer (nm) as unit [1cm=
10mm, 1mm=1000 μm, 1 μm=1000nm], between different virus, difference in size is very big, minimum such as the gemnivirus of plant
(Geminiviruses) diameter only 18-20nm, maximum animal poxvirus (Poxviruses) size reach 300-450nm ×
170-260nm, the longest as filamentous virus section (Filoviridae) virion size be 80nm × 790-14000nm, most
The diameter of single virus particle is at about 100nm, and HIV (human immunodeficiency virus) is 100-120nm (0.1-0.12 μm).2. HIV sufferers blood
Related compounds present in liquid: (gp120 with the CD4+ Cell binding of HIV cell surface is substantially for multinucleated giant cell
Long-pending HIV cell), gp120 cell (surface is with the presence of gp120 but with the HIV cell of individual cells), gene integration
Cell (integrate and have HIV double-stranded DNA, but the HIV that cell surface does not has gp120 is thin by HIV (human immunodeficiency virus) infection initial stage or incubation period
Born of the same parents), normal white cell (being uninfected by granulocyte that the individual cells of HIV exists, mononuclear cell, lymphocyte), erythrocyte, blood little
Plate, chemical analysis (protein, saccharide, lipid, electrolyte etc.), free HIV, antibacterial and other microorganisms.3. multinuclear is big and small
Born of the same parents are natural large volume cell;Gp120 cell and gene integration cell can make cell by the immunoreation of antigen and antibody
Coagulation is large volume many cells polymer;Free HIV can be changed into large volume composition by carrier granular/immunoreation.④
According to above-mentioned 3 points, can prepare can by individual cells but large volume cell or the blood separator of granule can not be passed through.5. select
The material of the selective adsorption function of apparatus, screens out the HIV cell in blood through the extracorporeal circulation of blood branch road of the present invention.
2, the material of blood separator and requirement: with the blood purification of the present invention, selects poly-vinegar non-woven fabrics, acetic acid fine
Dimension, absorbent cotton etc., it is desirable to good biocompatibility, hardly activating complement, do not cause inflammatory reaction and leukocyte, platelet, blood
Partial pressure of oxygen, the change of C3a, C5a.
3, the type and spec of blood separator: the profile of blood separator be prepared as column construction (with acetate fiber or
Filter element made by the materials such as absorbent cotton), the shape such as flat structure (making filter element with materials such as poly-vinegar non-woven fabrics);Aperture be prepared as 150~
250 μm, 50~150 μm, 15~40 μm, 8~15 μm, 5~8 μm, 3~5 μm, 1~2 models such as μm.
4, the application of principle of blood separator: select the separator of different model, principle according to the state of an illness of HIV sufferers
Upper first selection large aperture model does pre-sieving, the then model in selection of small aperture.Severe HIV sufferers often occurs serious
Opportunistic infection, containing different size of composition in blood.As the fungus containing especially big volume, spirillum, tumor cell and
His foreign body, then selecting aperture is 150~250 μm or the separator of 50~150 μm;As bitten carefully for the monokaryon of sieving HIV is huge
Born of the same parents, multinucleated giant cell, many cells polymer and be adsorbed with the particulate matter of HIV, and in order to replace easily by HIV
CD4+ cell, then select 15~40 μm, 8~15 μm, 5~8 μm, 3~5 models of μm etc.These several models approximate or are less than
The volume of single erythrocyte, neutrophilic granulocyte, small lymphocyte in blood, but erythrocyte, neutrophilic granulocyte and macrophage tool
There is the characteristic of amoeboid movement, can be by the micropore less than own vol.
(2) preparation of plasma separator
(1) preparation principle: prepare according to the molecular size of hemocyte and blood plasma components.Such as visible component in blood of human body
The size of (hemocyte) is: normocyte is about 7 microns (μm), is the discoid cell of concave-concave;Leukocyte is divided into 5 kinds,
Neutrophilic granulocyte about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, small lymphocyte 6-
8 μm, approximate with erythrocyte, and mononuclear cell is maximum, about 15-20 μm.Platelet is disc, diameter 1~4 microns to 7~8 microns
, the platelet mean diameter of people is 2-4 micron, thick 0.5~1.5 micron.
(2) material: can be selected for poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc., it is desirable to good biocompatibility, swash hardly
Live complement, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can be by altogether
Valency, be grafted, the method such as polymerization improves the structure of material, the regulation microinhomogeneities on surface, hydrophilic, minimizing be to blood coagulation and oxygen
Change stress impact thus improve sieving adequacy and biocompatibility, the generation of minimizing complication.
(3) type and spec: for the profile of separator, can prepare as filter element with the material such as acetate fiber or absorbent cotton
Become column construction, be prepared as the shapes such as flat structure with materials such as poly-vinegar non-woven fabrics as filter element;By hemocyte to be separated and blood
The molecular size of slurry composition determines aperture.Plasma separator stable in properties involved in the present invention, good biocompatibility, penetrating
Hollow fibre type filter made by the high molecular polymer that property is high, hollow-fiber film a diameter of 270~370 μm, and film thickness is 50 μm,
Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.This hole is only permitted blood plasma and is filtered, but can stop that all of cell becomes
Point.
Four, the component of acquired immune deficiency syndrome (AIDS) immunization therapy instrument
1, key member: (1) blood separator: screen out with multinucleated giant cell or many cells polymer for by volume size
The HIV cell that state exists, is i.e. used for removing endoglobar HIV;(2) plasma separator: be used for separating single blood thin
Born of the same parents and blood plasma;(3) blood purification: the HIV in adsorbed plasma.
2, additional member: include blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system,
The part compositions such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring.(1) blood pump (Blood Pump): be used for promoting blood
Circulating to maintain being smoothed out of blood purification treatment, usual blood pump part often has rotary test speed function, to monitor patient
Blood circumstance, therefore blood pump runner and flute pitch set and want accurately and it needs to often adjust, according to the feelings of bloody path pump line
Condition, is typically set as 3.2~3.3mm by spacing, can not be the most loose, and blood flow detection otherwise can be caused to be forbidden;Also can not be too tight, otherwise
Pipe breakage can be caused.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, uses
To continue injecting heparin in sieving pipeline (patient blood), owing to the blood of patient circulates and air contact, easily in vitro
There is blood coagulation phenomenon, use heparin pump to be possible to prevent the generation of blood coagulation.(3) sound pulse pressure monitoring: arterial blood pressure monitoring mainly in order to
The stopping state of dynamic monitoring blood separator micropore, additionally in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When
During blood flow deficiency, arterial pressure will reduce;When having blood coagulation, thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will
Raise;Venous pressure monitoring is used for monitoring the pressure of pipeline blood backflow, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow
When deficiency and venous return syringe needle come off, venous pressure will decline, if the distortion blocking of bloody path return duct or backflow syringe needle
When there is blocking, venous pressure will raise.(4) air monitering (Air Detector): be used for monitoring the air gas of blood pathway
Bubble, the principle of general ultrasonic listening, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble
Time, detecting system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent the generation of danger.
In a word, on the basis of key member of the present invention and additional member, Import computer regulates and controls and makes the people of operation
Property, the personalization for the treatment of, the safety of design, and modularity, automatically monitoring and regulation and control, liquid crystal display, judge voluntarily to warn
The blood purifying therapeutical instrument that the report micro computer such as reason and ring off signal processes.
Five, the connecting path of acquired immune deficiency syndrome (AIDS) immunization therapy instrument and using method
1, install: such as Fig. 1, with sterile working's connecting components, including blood separator, plasma separator, blood purification
Device and each circulation line.
2, aerofluxus: with physiological saline solution topping up separator, depurator and each circulation line, gets rid of separator, depurator
And gas in circulation line, bubble, go through, confirm without use after gas, bubble.
3, logical liquid: arterial blood line pipe (1) is connected the arteries of HIV sufferers, the row of going through the most again
Gas is the most complete, and liquid stream is the most unobstructed, and avoids managing the pollution of interior flow liquid.
4, anticoagulant: inject anticoagulant (heparin) from heparin pump (2) to liquid stream, be 2500 ∪ or 20~30 ∪/kg for the first time.
5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (15) is connected venous blood
Pipe, then opens blood pump (2), and blood flow is 100~150ml/min, such as Fig. 1, when arterial blood through arterial blood line pipe (1),
When heparin and blood pump (2) enter blood separator (3), the large volume multinucleated giant cell formed because of HIV is delayed at
In blood separator (3), mononuclear blood cell and blood plasma export (4), blood pump (6) and circulation line (7) through blood successively and flow into
Plasma separator (8), the blood plasma of separation flows into now open depurator (11) through blood plasma pump (9) and blood vessel (10) successively,
Blood plasma to be full of, about 10 minutes, begin paying out blood plasma, through export pipeline (13) flow out, synchronize to depurator (12) irrigate blood plasma,
When blood plasma in depurator (11) has nearly flowed, starting again at perfusion blood plasma, now depurator (12) begins paying out blood plasma, and two
The depurator (11) of individual parallel connection and (12) are alternately.As represented Fig. 2 of the internal structure of the blood separator (3) in Fig. 1, blood
There are a lot of micropore (3), multinucleated giant cell (4) micropore (3) can not be filtered and hindered on the tube wall of the inner chamber (2) of liquid/gas separator (1)
Stay inner chamber (2), thus can be eliminated, can pass through in micropore (3), outside the mononuclear blood cell (5) of small size and blood plasma enters
Chamber (6), then flows out through outlet (7), and then isolates hemocyte and blood plasma through the plasma separator (8) shown in Fig. 1.As represented
Fig. 3 of the internal structure of the plasma separator (8) in Fig. 1, the tube wall of the inner chamber (2) of plasma separator (1) has a lot of micropore
(3), it is impossible to export (8) by the mononuclear blood cell (4) of micropore (3) through the hemocyte with switchable valve and flow out, enter Fig. 1
Shown hemocyte export pipeline (14);Plasma separator exocoel can be entered by the blood plasma of micropore (3) and chemical composition (5) thereof
(6), then depurator is entered through the blood plasma pump (9) shown in blood plasma flow export (7), Fig. 1 and blood vessel (10).As represented in Fig. 1
Depurator (11) and Fig. 4 of (12) internal structure, when the blood plasma containing HIV (1) enters depurator, HIV therein (1) is respectively
It is thin that the HIV antibody (2) that is fixed in agar gel (6), CD4+T cell (4) are combined into antigen antibody complex (3), CD4+T
Born of the same parents' conjugate (5), combined after HIV no longer move down, the most combined large volume of HIV is again by because of concentration more
High and that micropore is less bottom agar gel molecular sieves microporous barrier at (7) place.Purification blood plasma after adsorbed HIV is through Fig. 1 institute
The individual cells that the export pipeline (13) shown separates with plasma separator (8) after export pipeline (14) converges through venous line
(15) fluid confluence circulation.So purifying blood, remove HIV, until the plasma circulation amount being previously set (usually 9L), treatment is
End.Whole therapeutic process is by computer control, and can detect duty, easy to use, automatization and safety at any time.
Six, the checking of acquired immune deficiency syndrome (AIDS) immunization therapy instrument effect
1, blood separator filters the checking of HIV cell effect
The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take Disease Control and Prevention Center and infection
The some parts of anticoagulated whole blood of acquired immune deficiency syndrome (AIDS) (AIDS) patient made a definite diagnosis that sick laboratory biological Sample Storehouse preserves, respectively peek part phase
It is mixed into 5 examples with the anticoagulated whole blood of abo blood group, makes blood flow volume sufficiently large, then entrust HC blood station, Zhejiang Province proportionately
The blood component separation method of part blood transfusion, isolates leukocyte, erythrocyte, blood plasma through blood component piece-rate system, takes leukocyte
Composition centrifugation routinely, inhales and abandons supernatant, with appropriate normal saline suspension leukocyte cell pellet, be subsequently adding proper ratio
Gp120 antibody (Guang Rui bio tech ltd, Shanghai), mix rearmounted 37 DEG C react 5 minutes, then with aperture be 20~
The blood component piece-rate system of 30um isolates the leukocyte (the biggest leukocyte) of large volume, to the leukocyte filtrate filtered again
The leukocyte (leukocyte in referred to as) of medium volume is isolated further with the blood component piece-rate system that aperture is 15~25um,
Leukocyte in filtrate is the leukocyte (referred to as SL) of small size, collects large, medium and small leukocyte separation suspension respectively,
Conventional centrifugal precipitates, and inhales and abandons supernatant, with the large, medium and small leukocyte cell pellet of quantitative liquid shifter draws equal amounts respectively, conventional method
(machinery or cell pyrolysis liquid) cell lysis (as used lysate of the same race, need dosage equal), takes supernatant, then after centrifugation
Operate according to HIV-1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai), with known dense
The p24 antigen conduct of degree 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml
Comparison, lowest detectable limit is less than 5pg/ml, measurement range 0~400pg/ml, range of linearity 0.5pg/ml~80pg/ml, 15min
Interior 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than
0.100,1000pg/ml absorbance is not less than 1.000, is considered as positive as absorbance > 0.12, and testing result (table 1) is said
Bright, the HIV-p24 content in the leukocyte of AIDS patient different volumes size is different, HIV-p24 in large, medium and small leukocyte
Average content be respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, in the most large and small leukocyte, HIV-p24 is average
Content difference 148.6pg/ml, decreases 54.4%;In large, medium and small leukocyte, the total content of HIV-P24 is respectively
1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, HIV-p24 total content difference 742.9pg/ in the most large and small leukocyte
Ml, decreases 54.3%, through statistical test, t=2.43, p < 0.05.Illustrate the large volume leukocyte in AIDS patient body or
The large volume leukocyte formed after gp120 antibody effect contains the HIV of high level, can be by implementing the skill of the present invention
Art scheme is separated removing.
HIV-p24 testing result (p24:pg/ml) in the large, medium and small leukocyte of table 1 AIDS patient peripheral blood
2, blood purification (agent) removes the checking of HIV effect
(1) checking of CD4+T cell strain adsorption removal HIV effect
In order to verify effect of CD4+T cell strain adsorption removal HIV, the present invention devises easy method of testing: takes and goes out
2.5 × 300mm Westergren's blood sedimentation tube of bacterium 5, draws by centrifugation CD4+T cell that (1000r/min, 5min) precipitate extremely respectively
200mm scale, then draws and is incubated after 100 DEG C dissolve at 39~41 DEG C of 0.9% standby agarose C1-4B, reach about
The long scale of 10mm, after putting blood sedimentation stand cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not stop little
The material of the water of molecule and chemical analysis etc passes through.The acquired immune deficiency syndrome (AIDS) that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve
5 example blood plasma, the most about 10mL of patient, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube upper end blank pipe, erythrocyte sedimentation rate to be flowed through in batches
The CD4+T cellular layer of pipe lower floor after flowing out in blood sedimentation tube, collects effluent, blood plasma after referred to as AIDS filter.Before taking AIDS filter
Blood plasma after blood plasma and filter, according to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), the limited public affairs of biotechnology are inspired in Shanghai
Department) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml,
The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0~400pg/ml, the range of linearity as comparison, lowest detectable limit
In 0.5pg/ml~80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than
0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance > 0.12
For being positive, testing result (table 1) illustrates, after AIDS blood plasma filters the simple purifier containing CD4+T cell, part HIV is
Being adsorbed by CD4+T cell, after the 1st time filters, HIV total body clearance is 22.84%, and after the 2nd time filters, total body clearance is
35.31%, after the 3rd time filters, total body clearance is 41.9%.Illustrate that HIV can be by the most clear along with the increase filtering number of times
Remove, thus reach to treat AIDS purpose.
Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after the simple purifier containing CD4+T cell
(2) HIV-1gp120 antibody, the checking of gp41 antibody adsorption removal HIV effect
The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take HIV-1gp120 antibody,
Gp41 antibody (Rui Qi bio tech ltd, Shanghai), joins and is incubated after 100 DEG C dissolve at 50 DEG C of 1.0% standby fine jades
In lipolysaccharide C1-4B, after mixing, titre is 1: 300~500, takes 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draws respectively
1.0% agarose C1-4B solution is to 200mm scale, and after cooling, agarose becomes semi-solid.Ling Qu Disease Control and Prevention Center and infectious disease are real
Test the specimen of the 5 example HIV sufferers that room Sample Storehouse preserves, remove each about 10mL of the blood plasma after cell, respectively take blood before 9mLAIDS filter
Slurry injects blood sedimentation tube upper end blank pipe in batches, the 1.0% agarose C1-4B containing antibody of blood sedimentation tube lower floor to be flowed through from blood
After flowing out in immersed tube, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, according to HIV-
1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operate, with concentration known 0pg/ml,
The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, minimum
Detection limit is less than 5pg/ml, measurement range 0~400pg/ml, range of linearity 0.5pg/ml~80pg/ml, and in 15min, 450nm surveys
Determining absorbance (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/
Ml absorbance is not less than 1.000, is considered as positive as absorbance > 0.12, and testing result (table 1) illustrates, AIDS blood plasma is filtered
After crossing simple purifier, part HIV is adsorbed by corresponding antibodies, and after the 1st time filters, HIV total body clearance is 20.01%,
After the 2nd time filters, total body clearance is 27.99%, and after the 3rd time filters, total body clearance is 37.36%.Illustrate along with filtration
The increase HIV of number of times can constantly be removed, thus reaches to treat AIDS purpose.
Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after simple purifier
In a word, above-mentioned simple confirmatory experiment shows, is easily fused into large volume by the peripheral blood leucocyte of HIV many
Core giant cell or many cells polymer, can be separated by the blood separator of the present invention and remove;And HIV free in blood plasma, can quilt
Blood purification agent (HIVgp120 antibody, HIVgp41 antibody, CD4+T cell strain, the agar gel micropore) absorption of the present invention is clear
Remove.Show by above-mentioned be that the acquired immune deficiency syndrome (AIDS) immunization therapy instrument that critical component is constituted has with blood separator and blood purification (agent)
Significantly remove the therapeutic efficiency of the inside and outside inhibition of HIV of blood cell.