CN106166313A

CN106166313A - A kind of acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator

Info

Publication number: CN106166313A
Application number: CN201610526046.6A
Authority: CN
Inventors: 翁炳焕; 李兰娟
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2016-11-30
Anticipated expiration: 2036-07-01
Also published as: CN106166313B

Abstract

The invention discloses a kind of acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator, the critical piece of depurator is decontaminating column, decontaminating column multilamellar by can in conjunction with the CD4+T cell strain of HIV, hybridoma macrophage strain, free HIV antibody, be incorporated into the HIV antibody of goat-anti Ig, purifying layer that agarose forms forms, multilamellar purifying layer constitutes decontaminating column；Blood to be clean after blood and plasma separator process, HIV therein is cleaned agent adsorption removal, the mononuclear blood cell that blood plasma after purification is separated with separator feeds back internal after converging, thus reaches to remove the purpose of the acquired immune deficiency syndrome (AIDS) blood purification treatment of the inside and outside HIV of hemocyte.

Description

A kind of acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator

Technical field

The present invention relates to a kind of acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator, be mainly used in the inside and outside AIDS of HIV sufferers hemocyte The removing of virus, thus reach to prevent, control and treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

Body can be stimulated after HIV to produce envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody.? Measuring low-level antiviral neutralizing antibody in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers are the highest, and the most protected effect of this antibody is described.But antibody can not be with the virus that retains in mononuclear phagocyte Contacting, and HIV envelope protein easily occurs antigenic variation, original antibody is ineffective, makes neutralizing antibody not play due Effect.In the latent infection stage, HIV provirus is integrated in host cell gene group, and therefore HIV will not be known by immune system Not, so only relying on autoimmune function and cannot being removed.The critically important reason of another one should be to kill according to antibody Go out, remove antigen mechanism speculate, after immune antibody is combined with antigen, immunological effect to be produced, or pass through activating complement, Mediation ADCC effect dissolves cellular antigen, but HIV is not cellular antigen；Phagocyte is attracted to gulp down by chemotaxis Bite removing antigen, but HIV is protected in phagocyte on the contrary, breeds；Antibody has been combined neutralization with antigen, is allowed to Lose appeal, but HIV antigenic structure is changeable, often make antibody be difficult to.

In terms of the treating AIDS method having been used for clinic at present, effect is less preferable: (1) hiv reverse transcriptase suppresses Agent: be only capable of preventing the permissive cell of not yet infected by HIV from infecting, the cell infected do not had therapeutical effect, and toxic and side effects is relatively Many, including gland plastochondria toxicity, bone marrow depression, globular anemia, granulocyte and thrombocytopenia, pancreatitis, intersect resistance in addition The generation of the property of medicine, this kind of medicine is not used alone, and mainly does drug combination, and is easily generated drug resistance mutant, causes under clinical efficacy Fall or inefficacy.(2) hiv protease inhibitor: be easily generated the toxic and side effects such as drug induced hepatic injury, lipid metabolic disorder and drug resistance Property.(3) hiv integrase inhibitor: integrated with host cell DNA by suppression inhibition of HIV DNA and play a role, reverse with HIV Transcriptase inhibitors and protease inhibitor associating use simultaneously and antiviral are had synergism.(4) suppression inhibition of HIV enters suppression Agent: include block gp120 with CD4 be combined, block HIV be combined with accessory receptor, act on gp41 film subunit and act on T pouring Bar cell surface CC-chemokine receptor 5 (CCR5) blocks HIV and enters host cell, but liver and heart are had side effect.(5) Cytokine therapy: be mainly used in regulatory T-cell dynamic equilibrium.(6) HIV vaccine treatment: due to the particularity of HIV, as intrinsic Immunity is not enough to resist HIV and targeting destroys immune system, and virus mutation is rapid, causes the most not yet developing real safety Effective vaccine.(7) gene therapy: the research of HIV gene therapy is never interrupted, does including antisense technology, RNA bait, RNA Disturb, intrabody, dominant negative mutant, suicide gene etc., but enter II clinical trial phase stage gene therapy almost No.(8) monoclonal antibody passive immunization therapy: reduce the susceptibility of HIV by lowering CD4+T cell surface CCR5, prolong The progress of slow acquired immune deficiency syndrome (AIDS) and the diffusion of minimizing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10 are applied to HIV person to be had Good toleration and safety, can delay but can not stop virus bounce-back (9) adoptive immunity cell therapy: external in a large number Virus a large amount of amplification can be caused when cultivating CD4+T cell autologous for HIV, increase the CD4+T cell quantity that virus infects, and feed back CD4+T cell may increase the place of internal virus replication, causes virus load to rebound, on the whole adoptive immunity cell Treatment, without obvious toxic and side effects, does not obtains satisfied therapeutic effect yet.

In gel, Ag-Ab precipitation was initially applied to study Liesegang's phenomenon, application in 1932 in 1905 In identifying bacterial isolates, nineteen forty-six Oudin carries out immunodiffusion in test tube, is used for analyzing antigen mixture, 1948 years Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, compares two or more antigens or anti- Body.The media gel agar of Ag-Ab precipitation in gel or agarose are a kind of polysaccharide bodies containing sulfate, high temperature Time can be dissolved in water, cold after congeal into gel, be internally formed the network structure of a kind of porous, and aperture be very big, can allow macromole Material (molecular weight is up to more than million) pass freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To less, agar concentration is little, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, owing to agar or agarose have Well chemical stability, after gel, water content is big, and transparency is good, and convenient sources is disposable, is therefore a kind of well diffusion Medium.The molecular weight of antigen and antibody is general all below 200,000, spreads to low concentration region from area with high mercury in gel Time suffered resistance the least, be substantially in the form of free diffusing form.When antigen meets after diffusion with corresponding antibodies in gel, shape Become antigen antibody complex, if both are suitable in place's ratio of meeting, then form the complex of maximum.Molecular weight due to complex Increasing, granule increases, thus does not continues to diffusion and produce precipitation, presents wire or banding, and this precipitation is the formation of one Individual " specific barrier ", all antigen same in immunology or antibody molecule can not pass through, and different those of character Molecule can continue diffusion by this barrier, until forming the complex of themselves.This kind of reaction is referred to as agar Gel or immunodiffusion, be the normal experiment checkup item at present with known antibodies detection unknown quantity corresponding antigens, is also " China Pharmacopeia " in 2010 editions regulation for the standard method of influenza virus vaccine hemagglutinin content detection.Generally by a certain amount of goat-anti People's Ig antiserum composition is mixed in agar gel, makes containing the specificity goat-anti sero-fast agar plate of people Ig, to be solidified after Punching, and in respective aperture, add human serum to be checked (IgG, IgA, IgM etc.), make serum to be checked expand to surrounding in agar plate Dissipate, properly locate to combine at antigen and antibody concentration ratio, form macroscopic white precipitate ring and no longer spread.Thus Visible, when a kind of solution is by semi-solid gel, macromole solute therein is just existed by the gel pore detention of molecular sieve effect In gel, antibodies that antigen the most therein can be fixed in gel in advance and be attracted in gel.

CD4 molecule is the receptor of HIV, and HIV is susceptible in CD4+T cell, and CD4+T cell is the one of T lymphocyte, averagely Life-span is generally about 7 days, but some T cell particularly after immortality chemical conversion cell line (strain) can long-term surviving, infinitely expand. Foreign literature is reported, simian virus 40 (SV40) can make some human cell that immortalization occurs.Poulin DL, Kung AL and The research such as Sullivan CS shows, the importing of SV40T antigen gene can accelerate to convert the growth rate of cell, immortalized cells After the most repeatedly passing on, still there is metastable multiplication characteristic and functional status, also can retain its germinal cell simultaneously Many phenotypic differentiations.Vascular smooth muscle cell strain is set up in Reilly simian virus large T antigen gene transformation, builds cell Model is with the inhibitory action mechanism of research heparin for vascular smooth muscle.Su etc. utilize the superficial cell strain converted through SV40, Build cell model and analyze the regulating and controlling effect of epithelial cell internal protein synthesis.The cuticulated epithelium that Miquel etc. convert with SV40 Cell strain, as the cell adhesion of cell model research laminin，LN 5 mediation.Webber etc. are with before SV40 converts Row glandular epithelium strain studies physiological function and the secretory function of prostate epithelial cell as cell model.Racusen etc. Damage and the disease of proximal convoluted tubule is studied with converting renal cells model through Ad12-SV40.The SV40 such as Hougton Conversion set up Bone marrow Stromal cell as cell model with research under certain condition of culture, cell have to adipose cell and The potential of osteoblastic two-way differentiation, studies osteoporotic mechanism further.Foreign study is it is also shown that import exogenous human Reverse transcriptase of telomere (hTERT) can make cell keep normal phenotype and differentiating characteristic.HTERT has the most been utilized successfully to build Found the immortalized cell line of some cell, substantially keep that chromosome stabilityX, differentiation be normal, contact inhibition, without phases such as oncogenicity To normal growth characteristics.In stomatology field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes forever Biochemical people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number reach 150 times with On, cell all shows original biological characteristics, can express the associated protein of derived cell after inducing culture.Kitagawa Establishing people cementoblast system Deng transfection hTERT, cell multiplication reaches more than 200 times, cell differentiation mark such as alkalescence phosphorus Acid enzyme, type i collagen etc. are expressed stable.Because of the needs of research work, almost every kind of disease has respective cell model.Such as glycosuria Sick cell model, cancer cell model, transgenic cell model, climacteric syndrome cell model, endometrial cell mould Type, epilepsy cell model, E-Cell models, alcoholic dementia cell model, cerebral edema cell model etc..So, can prepare CD4+T cell strain, after mass propgation, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS), with the absorption of CD4+T cell, removes blood plasma In HIV, simultaneously by be defeated by CD4+T cell produce cytokine treat acquired immune deficiency syndrome (AIDS).

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophilic granulocyte in peripheral blood, macrophagocyte Being the mononuclear cell in blood and the macrophage in multiple organ, tissue, both constitute mononuclear phagocyte system.Mononuclear cell Being formed by the monocyte precursor Development And Differentiation in bone marrow, account for 3% one the 8% of blood middle leukocytes sum, its volume relatively drenches Bar cell is bigger, and mononuclear cell the most only stops 12 24 hours, subsequently into connective tissue or organ, reach maturity into Macrophage, macrophage is the differentiation of mononuclear phagocyte system camber, ripe cell type, has stronger phagocytosis merit Can, wandering macrophage is more than mononuclear cell several times, lasts a long time, some months of can surviving in the tissue, the macrophage settled down Having different titles, be Kupffer Cell, be microglia in brain, be osteoclast etc. in bone in liver, it expresses Fc Receptor, C3b receptor and CD14, play defense function in inherent immunity, is also that the professional antigen participating in adaptive immunity is offered Cell.The CD4 molecule of Expression of Macrophages, is the receptor of HIV (human immunodeficiency virus) (HIV), after HIV enters human body, first suffers huge biting carefully The phagocytosis of born of the same parents, but HIV quickly changes the sour environment at macrophage some position interior, creates condition of its existence applicable, The most it is not killed amount reproduction the most within it to assemble, then HIV is passed to CD4+T cell.So, can be with conventional hybridization Oncocyte technology of preparing, prepares macrophage hybridoma, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS) after a large amount of amplifications, The HIV in blood plasma is removed with the phagocytic function of macrophage.

In a word, various medicines and biological product cannot effectively kill internal HIV (human immunodeficiency virus), and price, and side effect is big, So far there is no the effective ways for the treatment of acquired immune deficiency syndrome (AIDS), it has also become attack the global problem being unable to for a long time.

Summary of the invention

In order to solve to attack for a long time the global problem in the treating AIDS field being unable to, it is pregnant that the present invention proposes a kind of acquired immune deficiency syndrome (AIDS) Woman's blood purification.

The object of the present invention is achieved like this: a kind of acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator, is provided with multilamellar in depurator The purifying layer being made up of agar gel and daf molecule, multilamellar purifying layer constitutes decontaminating column；Described daf molecule is by can be in conjunction with HIV CD4+T cell strain, hybridoma macrophage strain according to quantity than 1:0.5～3 composition；In each layer, cell is of the total volume 4/5；Described agar gel comprises free HIV antibody, is incorporated into the HIV antibody of goat-anti Ig, agarose；From the import of depurator Place to exit, the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody titer the most from high to low Successively decreasing successively, agarose concentration is incremented by the most successively.

Further, the import and export of described depurator is provided with screen cloth, and the mesh number of import department's screen cloth is 800 mesh, goes out At Kou, the mesh number of screen cloth is 2.0～5.0 mesh.

Further, the number of plies of described purifying layer is 5 layers, the antibody titer of free HIV antibody, is incorporated into goat-anti Ig's The antibody titer of HIV antibody the most from high to low, is followed successively by 1:100,1:200,1:300,1:500,1:700.

Further, the number of plies of described purifying layer is 5 layers, and from low to high, content is followed successively by 0.7g/ to agarose concentration 100ml、0.8g/100ml、0.9g/100ml、1.0g/100ml、1.1g/100ml。

Further, described can in conjunction with the CD4+T cell strain of HIV, hybridoma macrophage strain quantity than for 1:0.5～ 3。

Further, described HIV antibody by HIV-1gp120 antibody, HIV-1gp41 antibody one or both according to Arbitrarily proportioning composition.

Further, the strain of hybridoma macrophage is by hybridoma technology oncocyte and macrophage merged with cell Prepare.

Further, described decontaminating column is prepared by the following method and obtains:

(1) cleaning hybridoma macrophage strain and CD4+T cell strain with physiological saline solution, 1000r/min is centrifuged, and cleans Rear recentrifuge precipitates, and takes sedimentation cell in the ratio that ratio is 1:0.5～3 of hybridoma macrophage strain and CD4+T cell strain, In the hydrostatic column that assembling high-biocompatibility material is made, make blood purification cell column；

(2) by goat-anti Ig with excess HIV antibody mix, make the goat-anti in mixed liquor be fully tied, but remain have free HIV antibody, and free HIV antibody titre is equal with the HIV antibody titre being incorporated into goat-anti Ig；

(3) being mixed with a certain amount of normal saline after 100 DEG C dissolve by agarose, insulation, at 39～41 DEG C, adds step Rapid 2 mixtures of antibodies obtained, wherein, the antibody titer of free HIV antibody, are incorporated into the antibody of the HIV antibody of goat-anti Ig Titre is 1:700, and the content of agarose is 1.1g/100ml.

(4) product step 3 prepared joins in lymphocyte depletion post prepared by step 1 with volume ratio 1:4, is cooled to After semi-solid gel, complete the preparation of bottom purifying layer.

(5) repeat step 1～4, be sequentially completed the preparation of each layer purifying layer from top to bottom, be purified post.And free The antibody titer of HIV antibody, the antibody titer of the HIV antibody being incorporated into goat-anti Ig are 1:500,1:300,1:200,1:100； The content of agarose is followed successively by 1.0g/100ml, 0.9g/100ml, 0.8g/100ml, 0.7g/100ml.

The beneficial effects of the present invention is: the present invention is thin by filtering the large volume containing HIV that many cells are combined into The blood separator of born of the same parents, mononuclear blood cell and the plasma separator of blood plasma can be separated and inhibition of HIV in blood plasma can be carried out height The depurator that effect purifies combines, it is achieved the high purification to blood.

HIV antibody that cleanser in depurator is combined with goat-anti Ig by the HIV antibody being fixed on agar gel, CD4+T Cell strain and macrophage strain are made, the unconjugated HIV of molecular weight ratio of its conjugate of HIV antibody being wherein combined with goat-anti Ig Antibody molecule amount is big, not easily passs through gel molecular sieve, and contained goat-anti Ig is easy and the feature of agar gel secure bond, HIV Antibody is more easy to be fixed in agar gel the most therewith, the meeting when HIV in blood plasma and the HIV antibody being fixed in agar gel are met Occur association reaction to form antigen antibody complex, thus be fixed in agar gel by HIV antibody, because of CD4+T cell The CD4 molecule on surface is the receptor of HIV and becomes the permissive cell of HIV, can adsorb HIV, adsorbed HIV with HIV when meeting It is fixed in agar gel with CD4+T cell, because of the natural phagocytosis characteristic of hybridoma macrophage, can quilt when HIV meets therewith Swallowing and be fixed in agar gel therewith, these compositions are respectively provided with phagocytosis and/or combine the function of absorption HIV, and agar The filter opening that gel is formed reduces along with increasing of agarose concentration, and depurator import department agarose concentration is low, and filter opening is the biggest, Be conducive to the association reaction of plasma perfusion and high titer antibody or cell and HIV；And exit concentration is high, filter opening is the least, it is easy to Detention HIV or Large molecular conjugates, depurator is combined with multiple special and non-specific HIV and removes composition, in order to avoid extraordinary strain Because of the futile treatment caused by immunity difference, so in the extracorporeal circulation of acquired immune deficiency syndrome (AIDS) blood purification treatment, big containing a large amount of HIV First volume cells is filtered by blood separator, and HIV free in blood plasma is by blood purification adsorption removal, the blood after purification Slurry converges in rear reflux with the mononuclear blood cell of blood separator isolated, thus reaches removing and be combined in cell surface And/or intracellular HIV and be free on the purpose of HIV of blood plasma.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of the blood purifying therapeutical instrument that the depurator according to the present invention is assembled.

Fig. 2 is 5 Rotating fields for decontaminating column and the schematic diagram of screen cloth.

Fig. 3 is the purification utensil process schematic proposed according to the present invention.

In Fig. 1, arterial blood line pipe 1, heparin and blood pump 2, blood separator 3, blood export 4 waste liquid outlets 5, blood pump 6, circulation line 7, plasma separator 8, blood plasma pump 9, blood vessel 10, depurator 11 are connected with 12, and export pipeline 13, hemocyte go out Mouth pipeline 14, venous line 15.

In Fig. 3,102,104,106 are respectively HIV antibody, macrophage, CD4+T cell；101 is free HIV；103、 105, it is delayed at the knot in agar gel 108 after 107 respectively HIV and HIV antibody, macrophage, CD4+T Cell binding Compound, 109 is by the large volume of HIV of agar gel 108 molecular sieve detention.

Detailed description of the invention

Below in conjunction with Fig. 1, Fig. 2 and Fig. 3, the acquired immune deficiency syndrome (AIDS) blood purification proposing the present invention is explained in detail.

One, the preparation of acquired immune deficiency syndrome (AIDS) pregnant woman blood cleanser

(1) preparation of hybridoma macrophage strain

1, primary cell source

(1) single core blood cell: refer to the lymphocyte separated from blood with density－gradient centrifuga－tion method and monokaryon is huge bites Cell.Concrete grammar is: the White Blood Cells Concentrate buying Blood Center or the Cord blood preserved for scientific research, takes 2mL specimen, PBS 6mL anticoagulation dropper, by hemodilution 2～3 times, is fully slowly superimposed on along tube wall after mixing that to have added 4mL lymph thin by liquid Born of the same parents separate in the 10mL centrifuge tube of liquid horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided in pipe after Li Xin 3 layers, upper strata is blood plasma and PBS liquid, and lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level Interface has the white cloud and mist layer narrow band based on mononuclearcell to be PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC inserts in another 50mL centrifuge tube, adds 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new life Sanguis Bovis seu Bubali + 2mmol/LEDTA clearly, pH7.2) 2mL re-suspended cell, take 15uL cell suspension and add counted under microscope 4 on blood counting chamber Cell (PBMC) sum in individual block plaid.

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, its preparation method is: the 1. acquisition of spleen tissue and transhipment: in the approval of reason committee and patient's informed consent Under, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Machine Tool grinds spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.By 200 mesh stainless steel filtering net mistakes Filter is outstanding is mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing erythrocyte, lymphocyte, Macrophage etc.).3. erythrocytic cracking in spleen tissue cell suspension: then centrifugal with the washing of RPMI-1640 liquid (1000r/min, 3min), to remove cell debris, adds Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 washing from The heart 1 time, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, mainly contains in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as cultivation Cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) × 10⁶/ L, To adjust the cell suspension inoculation of concentration in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, Cultivate 2～3h respectively, under phase contrast microscope, observe form.The digestion of adherent spleen tissue macrophage: adherent spleen tissue is huge Phagocytal digestion: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, gained cell suspension washing from The heart (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or the discarded specimen of Post operation Extract preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..

(3) amniotic fluid, villus cell: attached hospital for obstetrics and gynaecology of Zhejiang University reproduction genetic laboratory is standby.Reason committee member Can ratify with under patient's informed consent, treating excess syndrome tests the residue amniotic fluid after diagnosis report, villus cell, selects exponential phase cell Continue to employ.

2, cell is cultivated and the adherent preliminary sorting of macrophage

Cell is cultivated routinely, but according to the difference of cellularity, suitably adjusts incubation time, condition of culture etc., typically Single core blood cell (PBMC) or single core histiocyte (macrophage) are placed in and cultivate containing RPMI 1640 by adherent method In the culture dish of base, in 37 DEG C, 5%CO₂Cell culture incubator (Themo electro corporation CLASS 100, beautiful State) in hatch 2h, after mononuclearcell is adherent, inhale abandon upper strata suspension cell (cell beyond macrophage be difficult to adherent and Remove with upper liquid), PBs buffer washs 3 times gently, adds a small amount of RPMI 1 culture medium, scrapes patch with cell scraper Parietal cell (predominantly macrophage, but also have other attached cells a small amount of).1 000r/min is centrifuged 5min, abandons supernatant.Amniotic fluid Cell, villus cell are cultivated 1～7 day, occur that cell growth clone, cell growth converge rate and reach the logarithmic growth of 60～80% Phase cell, with trypsinization, PBS, obtains cell suspension, is made into proper cell concentration.

3, cd4 cell sorting

Employing immunomagnetic beads method sorting cd4 cell: 1. main agents and instrument: (Germany U.S. sky Ni is biological for CD4 immunomagnetic beads Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnology service company)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany).2. cd4 cell immunity Magnetic bead sorting method: cell suspension is divided equally to two 1.5mLEppendorf pipes, centrifugal (300r/min, 20 DEG C) 10min, discards Supernatant, the every 80uLBuffer of re-suspended cell contains cell number 10⁷Individual, every 10⁷Individual cell add 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, and hatches 15min at 4～8 DEG C, uses 1mLBuffer washed cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed in the magnetic field of MACS separator, with 500uLBuffer rinses, and by 500uL cell suspension by detached dowel, rinses detached dowel repetitive operation 3 times with 500uLBuffer, Collect effluent, containing non-cd4 cell in effluent, in separator, take out detached dowel, divide with 1000uLBuffer pressure flush From post, collecting effluent, this is that (cell viability detects cd4 cell: takes 15uL cell suspension before and after cell purification respectively and waits body Long-pending trypan blue solution mixing, the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates 200 The percentage ratio of living cells in individual cell).The cell now sorted is mainly macrophage.

4, CD14 cell (macrophage) sorting

CD14 is mononuclear cell and the distinctive surface marker of macrophage, if in theory from single core histiocyte, sheep Sort in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, then institute Obtain cell and include mononuclear cell and macrophage；But because the mononuclear cell life-span is short, only survives 1 day in peripheral blood and can not show a candle to huge Phagocyte is prone to adherent growth, so the most substantially removing in the cell attachment of the present invention is cultivated, the cell sorted out is basic For macrophage.

Basic skills is analogous to cd4 cell, uses immunomagnetic beads method.1. reagent: people's CDl4 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead with Specificity target cell one mononuclear cell combines: every 1 × 10⁸Magnetic bead and the 800uL of individual PBMC addition 200uL coupling CDl4 antibody delay Rush liquid (containing the bovine serum albumin 2.5mL and 2mol/L EDTA0.5mL of 10%, 4 DEG C of refrigerator pre-coolings), at 15mL centrifuge tube In fully mix, hatch 15min for 4 DEG C, centre can slightly shake 1 time.Taking-up centrifuge tube after 15min, every 1 × 10⁷Individual cell adds Enter 1～2mL pre-cooling buffer, 1 000r/min, centrifugal 8min, abandon supernatant, add O.5mL buffer blow and beat into unicellular outstanding Liquid.(B) collect the mononuclear cell of marked by magnetic bead: be placed on MACS magnetic frame by cell separation post, add lmL buffer balance thin Born of the same parents' detached dowel, treats that no liquid is dripped, and is added by above-mentioned cell suspension in people's cell separation post immediately, thin with 0.5mL wash buffer Born of the same parents' detached dowel 3 times.After to be rinsed, add 1mL buffer, emigrated cells detached dowel from magnetic frame, quickly push away with pin post Dynamic, go out the cell combined with CDl4 antibody one magnetic bead in detached dowel, be the macrophage of CDl4+.

Additionally, following 2 kinds of methods sorting also can be used, including 1. adherent method: be placed in by PBMC and train containing RPMI 1640 Support base culture dish in, in 37 DEG C, containing 5%C0: cell culture incubator (Themo electro corporation CLASS 100, U.S.) in hatch 2h.After adherent mononuclear cells, inhaling and abandon upper strata suspension cell, PBs buffer washs 3 times gently, adds A small amount of RPMI 1 culture medium, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant.2. streaming Cell art method: CDl4 labelling: take PBMC, with buffer (containing bovine serum albumin 2.5mL and 2mol/LEDTA of 10% 0.5mL) adjusting cell density is 1 × 10⁸/ mL, addition CDl4+-FITC antibody 100uL in every milliliter of cell suspension, 4 DEG C Lucifuge labelling 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, with containing 2% blue or green chain It is 2 × 107/mL that the PBS of mycin adjusts cell density.Selected by flow cytometry apoptosis: by the cell of preparation at flow cytometer (BD FAcsAria II, U.S.) upper sorting, according to the fluorescence intensity of CDl4 antibody, the relative size of cell and relative of cell Graininess and the complexity of internal structure, collect the cell of CDl4+.

5, prepared by CD14 hybridoma cell strain (hybridoma macrophage strain)

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire cattle Jinshi City Hao on daytime ocean biological product science and technology responsibility company limited purchased by serum (FBS)；DMSO (--methyl sulfoxide) it is domestic analytical pure Reagent.

(2) myeloma cell prepares: merges and takes out the myeloma cell (SP2/ that a pipe is frozen the last week in liquid nitrogen container 0), it is immediately placed in hot water and thaws that (applying most is Sp2/0 cell strain, and this cell strain growth and fusion efficiencies are the best, and itself is not Secreting any heavy chain immunoglobulin or light chain, the Seedling height scale of cell is 9 × 10⁵/ ml, the doubling time be usually 10～ 15h；The actual application relevant to human body considers select homologous cell strain, if Fu Xiang bio tech ltd, Shanghai is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Adding appropriate complete culture solution after thawing, 1000r/m is centrifuged 3min；It is repeated 1 times.Precipitate is moved in Tissue Culture Flask, add DMEM culture fluid, put CO2 incubator and cultivate, within 3-4 days, carry out Once pass on or amplification culture, in merging first 24 hours, adjust cell state, it is ensured that before merging, cellular morphology is good, it is prosperous to grow Contain.Adding appropriate basal medium in centrifuge tube, after beaing mixing gently, 1000r/m is centrifuged 5-10min, repeated washing cell 2 times.

(3) CD14 cell (macrophage) to be hybridized prepares: the mononuclear phagocyte of present invention sorting is with basal medium Adjust total cell and count to 1 × 10⁸～2 × 10⁸Merge for cell.Blue dyeing phase-contrast microscopy, viable count is expected with platform Should be higher than that 80% for qualified.

(4) cell merges: added with the ratio of 10:1-5:1 with myeloma cell by CD14 cell (mononuclear phagocyte) In centrifuge tube, being mixed evenly, 1000r/m is centrifuged 5min, supernatant discarded, beats gently at the bottom of pipe to cell without granular precipitate, weight Multiple 2 times.Preheating centrifuge tube is rotated gently, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up in 37 DEG C of water-baths PEG3000 is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, afterwards by the basis training of the 25mL of preheating Support base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, rotating centrifugal pipe lightly during adding, then stand In 37 DEG C of water-bath 10min, 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T culture medium.Suitably it is inoculated into after mixing In 96 well culture plates, it is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) there is the mononuclear phagocyte strain screening of phagocytic function: observe cell growth status in 96 well culture plates, 7-10 Only have hybridoma after it and can grow division, now discard HAT culture medium, change complete medium.Cell clone grows When area reaches 1/10 cell hole, culture supernatant, selection is gone to have the culture hole of the hybridoma cell strain that growth conditions is good, aobvious The position of labelling cell strain growth, size under micro mirror, using aseptic rifle head to draw cell clone in the position of mark has to new In the culture hole of complete medium, doubling dilution counts hole to below the most successively, 37 DEG C, cultivates one week left side in 5%CO2 incubator The right side, basis of microscopic observation cell growth status, when cell clone covers with to hole floor space more than 1/10, take in cell or cultivation Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain phagocytosis antibacterial Function detection: macrophage is hanged with staphylococcus or Candida albicans Liquid mixing incubation, smear, fixing, serge blue liquid dyes, and in oil Microscopic observation phagocytosis situation, counts phagocytosis antibacterial and does not gulps down Bite the macrophage number ratio of antibacterial, to swallow antibacterial function strong macrophage alternately positive clone strain.

2. hybridoma macrophage strain phagocytosis HIV Function detection: take acquired immune deficiency syndrome (AIDS) (AIDS) patient's of Disease Control and Prevention Center's preservation After blood plasma is mixed with hybridoma macrophage strain, separates cell strain, PBS 3 times, measure the phagocyte strain through cracking The function of phagocytosis HIV, with specific reference to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai inspires biotechnology limited Company) 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 the positive.Concrete test kit description of pressing operates.

3. the strain of hybridoma macrophage produces macrophage cytokines detection: with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) by specification operates, and detection range is 0～800pg/ml, Sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, and the positive is the strongest, negative Reaction for colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: at ELISA detector On, in 450nm (if developing the color with ABTS, then 410nm) place, survey each hole OD value after returning to zero with blank control wells, if more than regulation 2.1 times of negative control OD value, are the positive, specifically press the operation of test kit description.MIF be collection cytokine, somatomedin, Hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays central Effect, in various infection and active chronic inflammation disease play panimmunity function.

According to testing result, the cell clone selected in the culture hole with stronger macrophage function repeats next Wheel dilution is cultivated, and repeats 2-3 wheel, takes out, proceed to culture bottle mass propgation after detection function-stable.

(6) preservation of hybridoma macrophage strain and recovery: preserve first 12 hours and adjust cell growth state, take one bottle of life Making cell suspension after long vigorous, that form is good cell, suitably digestion, 1000r/min is centrifuged 5min, removes supernatant, flicks Making cell loose at the bottom of pipe, add 4 DEG C of 9 parts of complete culture solutions preserved and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and-70 DEG C refrigerator overnight, puts into cryopreservation tube in liquid nitrogen container after taking-up and saves backup.The hot water of about 40 DEG C is got out before recovery, will Cryopreservation tube carefully takes out from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, is centrifuged at 1000r/min after defrosting 5min, opens cryopreservation tube under aseptic condition in superclean bench, and the cell complete culture solution after thawing washed once, then It is centrifuged 5min, supernatant discarded, in case making amplification culture at 1000r/min.

6, prepared by hybridoma macrophage strain treatment cell

The i.e. amplification cultivation of hybridoma macrophage strain.Move after using complete culture solution the most resuspended above-mentioned cell precipitation Enter in culture bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Repeatedly pass on amplification cultivation, until required hybridoma cell strain Quantity, every Secondary Culture positive hybridoma cell strain 10 generation, the function of detection macrophage hybridoma cell strain, see whether steady Fixed.Continue in several bottles, carry out extensive industrialization to prepare, save backup.

(2) 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)；(perseverance letter in Shanghai is raw for lymphocyte separation medium Change reagent company limited)；Ethylenediaminetetraacetic acid (EDTA), (the Shanghai raw work biotechnology service of 0.2% Trypan Blue liquid Company)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (U.S. of Germany sky limited public affairs of Ni biotechnology Department)；EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. the separation of mononuclearcell (PBMC): by close Degree gradient centrifugation separates.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell suspension is divided equally to two 1.5mLEppendorf manages, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, and the every 80uLBuffer of re-suspended cell contains cell Several 10⁷Individual, every 10⁷Individual cell adds 20uLCD4MicroBeads or CD8MicroBeads, fully mixes, 4～8 DEG C of hatchings 15min, uses 1mLBuffer washed cell, and centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer is resuspended carefully Born of the same parents, are placed on MS detached dowel in the magnetic field of MACS separator, rinse with 500uLBuffer, by 500uL cell suspension by dividing From post, rinse detached dowel repetitive operation 3 times with 500uLBuffer, collect effluent, containing non-CD4+T lymphocyte in effluent Or non-CD8+T lymphocyte, in separator, take out detached dowel, with 1000uLBuffer pressure flush detached dowel, collect and flow out Liquid, this is CD4+T lymphocyte or (the cell viability detection: take 15uL cell before and after cell purification respectively and hang of CD8+T lymphocyte Liquid mixes with equal-volume trypan blue solution, and the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, Calculate the percentage ratio of living cells in 200 cells).

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 with amplification, purification picking resistant to ammonia benzyl Penicillin bacterium colony extracting plasmid, with lipofection import pass on the T lymphocyte in logarithmic growth in vitro, make recon with The DNA of cell integrates, and the clone of positive recombinant that amplification culture screen through G418, screening cellular morphology, growth curve, dye The test of colour solid caryogram, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, Cell generation cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT forever Biochemical CD4+T cell.

With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4DNA 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.(ii) hTERT electrophoresis: With 10% agarose gel electrophoresis, separate hTERT fragment.(iii) hTERT purification and recovery: by purpose hTERT fragment from gel Middle separation, it is purified.(II) connection of hTERT Yu pLXSNneo carrier: build pLXSNneo-hTERT recon.(III) Purification, expand, identify pLXSNneo-hTERT recon.(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 RPMI 1640 liquid in, or be inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin In, typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% hyclone (FBS)；1% is blue or green Mycin and streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, Centrifugal, remove supernatant, standby.(VI) pLXSNneo-hTERT recon imports T lymphocyte and amplification culture: micro-at 1.5ml Measure and centrifuge tube is prepared following solutions: pipe A, pLXSNneo-hTERT recon is dissolved in 100 μ l serum-free mediums；Pipe B, 20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums, pipe A and pipe B are mixed, left at room temperature 45min, use Serum-free medium washs above-mentioned T lymphocyte 2 times.Lipofectamine-pLXSNneo-hTERT mixture adds 1ml serum-free medium, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone is dense to add 1ml serum-free medium Degree is 20ml/L), at CO₂Incubator cultivates 10h, and sucking-off transfection liquid, (hyclone concentration is to add 4ml complete culture solution 20%), continuing to cultivate 20h, discard culture fluid, replacing concentration is 400mg^。L^-1G418 culture fluid continue cultivate, after 8 days select Select after living cells makees amplification culture, then strengthen G418 concentration to 800mg^。L^-1, raw by stablizing in the G418 environment of high concentration Long cell proceeds amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, occur assembling now As.If cell increases slow, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent and changes Liquid.Transfer in 75ml culture bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh culture.Cell is cultivated extremely 9-10 week (the about the 75th generation), still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, dead cell (the increase situation of cell quantity is judged by reading the scale of culture vessel less than 10%；Differentiated dead by trypan blue staining Cell and living cells.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, the permeability of after birth increases, can be dyed blueness by trypan blue, can determine whether as cell the most dead.Method It is to draw weekly a certain amount of suspension culture, mixes rearmounted room temperature 5～10 minutes with Trypan Blue agent, then make thin Born of the same parents' thin slice, under the microscope 1000 total cellular score of counting, calculate dead cell and the percentage of non-staining living cells of coloring Than).Hereafter along with cultivating the increase of algebraically and the prolongation of incubation time, the increase of cell quantity is slack-off, dead cell increasingly Many, until cell is not further added by, even dissolve, reduce, all dead.When total amount reaches 45ml, put in 50ml centrifuge tube, from 1500 turns of the heart, 10 minutes, after abandoning supernatant, addition 3ml freezing media (5% DMSO (dimethyl sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/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 significantly increases, and goes out Existing clustering phenomena, has lymphocyte blastogenesis feature.(ii) observation of cell growth curve: with incubation time as transverse axis, cell number Amount for the longitudinal axis (logarithm), be depicted on semi-logarithmic scale make after curve the growth curve of this cell, immortalized cell line Formed in typical " S " feature or " arched roof "；(iii) chromosome is checked: by analyzing karyotype, if karyotype For diploid " 46, XX " or " 46, XY ", then illustrate that this cell line does not occur vicious transformation.(iv) Flow cytometry: inspection Survey the cell proportion synthesizing, dividing in the 19th continuous cell line, 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 sequence Row.HTERT detection in (v) transfectional cell DNA: as with Immunohistochemical detection, in the nucleus of hTERT transfection, dyeing is visible A large amount of brown particles, show that hTERT has been integrated into intracellular；(vi) mrna expression product measures: the PCR taking 100 μ l systems expands Volume increase thing, reclaims test kit (Takara, Japan) with gel and reclaims product, take 2 μ l DNA solutions and dilute 100 times, survey concentration, remaining Under DNA and each 10 μ l of upstream and downstream primer check order.(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, Amplification culture meets immortalized cells characteristic the cell same or like with primary cell after above-mentioned qualification, takes growth conditions Well, it is in the cell of the different generations of exponential phase, is performing centrifugal separation on (1 200r/min, 6min), with containing dimethyl sulfoxide Frozen stock solution 0.5～1ml re-suspended cell, 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 cryopreservations, build the immortalization CD4+T cell bank that biological characteristics is stable in this way 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, is dissolved in appropriate TE buffer, adds 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, add 5uL electrophoresis sample loading buffer and terminate Reaction is in case electrophoresis.(ii) SV40DNA electrophoresis: power taking swimming level agarose is made into 10% agarose gel with electrophoretic buffer, Electrophoresis under the voltage of 1-10V/cm gel, isolation of DNA fragments.(iii) from agarose, separate about 2600bp SV40 large T antigen DNA.(II) connection of SV40LT Yu pcDNA3.1 carrier: build SV40T/pcDNA3.1 recon.(III) expand, separate with Identify SV40T/pcDNA3.1 recon: (i) prepares competence escherichia coli.(ii) screen, expand and extract recon.(i Ii) qualification of recon: the above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competence escherichia coli, ibid Method restricted enzyme BamH I carries out enzyme action, and 10g/L agarose gel electrophoresis is identified, it is thus achieved that size about 2600bp and 2 bands of 5600bp, the former meets the size of SV40T fragment in GenBank.(IV) CD4+T cell: from the present invention " (2) CD4+ The preparation of T cell " sampling preparation.(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, In RPMI 1640 liquid of 20% hyclone, or it is inoculated in containing 20% hyclone, 5～the low sugar of 10nmol/L insulin In DMEM cell culture medium, typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% tire Sanguis Bovis seu Bubali Clearly (FBS)；1% penicillin and streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, volume fraction 5%CO2 incubator In, cultivate 1-2 days, centrifugal, remove supernatant, standby.(VI) importing of SV40T/pcDNA3.1 and amplification culture: micro-at 1.5ml Measure and centrifuge tube is prepared following solutions: pipe A, SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free medium (hyclone concentration For 20ml/L) in；20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums by pipe B, are mixed by pipe A and pipe B, room The underlying 45min of temperature.Above-mentioned T lymphocyte is washed 2 times with serum-free medium.At Lipofectamine-SV40T/pcDNA3.1 Mixture adds 1ml serum-free medium, mixes gently, then drop to, in above-mentioned T lymphocyte, be subsequently adding 1ml depletion of blood Clear culture fluid (hyclone concentration is 20ml/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 replacing concentration is 400mg^。L^-1G418 culture fluid continue Continuous cultivate, after selecting living cells to make amplification culture after 8 days, then strengthen G418 concentration to 800mg^。L^-1, by can be in high concentration In G418 environment, the cell of stable growth proceeds amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte is obvious Increase, clustering phenomena occurs.If cell increases slow, or cell density is low, or medium pH value is acidity, and cultivation is partly measured in sucking-off Liquid, carries out equivalent oil changing.Transfer in 75ml culture bottle when total amount reaches 14ml, every 2-3 week adds 5-10ml fresh cultured Base.Cell cultivates about 6-8 week (the about the 55th generation), and still in logarithmic growth, i.e. incubation time and cell is accelerated and closed in multiplication System, dead cell is less than 10% (differentiating dead cell and living cells by trypan blue staining).Hereafter along with the increasing cultivating algebraically Adding the prolongation with incubation time, the increase of cell quantity is slack-off, dead cell gets more and more, until cell is not further added by, even Dissolving, minimizing, all death.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 suspending liquid (cell concentration is about 10⁵/ml).Cryopreservation tube subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen at-196 DEG C In liquid nitrogen (or under zero setting immediately 80 degree, move in liquid nitrogen container after 1-2 hour).(VII) immortalization CD4+T characteristics of cell biology Qualification: authentication method is carried out routinely.(VIII) SV40LT gene mediated CD4+T cell bank: screen and continue to pass on, expand Cultivate after above-mentioned qualification, meet immortalized cells characteristic the cell same or like with primary cell, take growth conditions good Get well, be in the cell of the different generations of exponential phase, be performing centrifugal separation on (1 200r/min, 6min), with containing dimethyl sulfoxide Frozen stock solution 0.5～1ml re-suspended cell, cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；20 DEG C, 2h；70 DEG C, overnight, entering 196 DEG C of liquid nitrogen cryopreservations, 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.

(3) 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 2x 10⁶Epstein-Barr virus (EBV) stock solution that rear addition is appropriate, is placed in 370C, 5%C02 overnight incubation, Preparing B cell to be hybridized, screen the positive hole of AntiHIV1 RT activity-l outer membrane protein (gpl20) by ELISA method, transfer cell is to 24 orifice plates Continue to cultivate 2 weeks, repeat to measure anti-gpl20 by ELISA method and confirm positive, continuously clone's secondary a large amount of amplification cultivation.Will After positive cell strain myeloma cell heterogeneous with people Mus (being buied by Zhejiang University's siberian crabapple) mixes (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-gpl20 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, T4DNA 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 base Because of fragment, recombinant plasmid transformed enters E.colistrain XL1 blue, checks order.The abduction delivering of recombiant protein and qualification: restructuring 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: express and produce Thing centrifugal collecting precipitation hangs through PBS, after 30W Ultrasonic Pulverization instrument cracking antibacterial, is centrifuged collection supernatant and filters, and filtrate is used AKTA PURIFYER100 protein purification instrument, GST column purification, obtain fusion protein GST-HIV, concentrates centrifuge tube and concentrates, 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.

(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 adds in centrifuge tube with the ratio of 10:1-5:1, 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, and whether observe this positive hybridoma cell strain can stably excreting 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, take ascites with method.The ascites collected, 10000r/m is centrifuged 5min, takes supernatant subpackage ,-20 DEG C of preservations.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out with reference to " molecular cloning " method, with Semidry method transfers.

(9) HIV-1gp120 monoclonal antibody applies (specialty businessman can be entrusted to complete) after the most refined.

(4) preparation (with the preparation of HIV-1gp120 antibody) of HIV-1gp41 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 uses ELISA method to measure, and specifically enters by test kit description OK.

Two, the preparation of acquired immune deficiency syndrome (AIDS) blood purification

(1) gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, make the goat-anti in mixed liquor Gp120 and gp41 antibody is all fully tied, but surplus free gp120 and the gp41 antibody having sufficiently high titre, then will contain There are conjunction type and the mixed antibody of sequestered gp120 antibody and the mixed antibody containing conjunction type and sequestered gp41 antibody again Secondary with etc. titer mixing, be made into final mixed antibody.

(2) cleaning hybridoma macrophage strain and CD4+T cell strain with physiological saline solution, 1000r/min is centrifuged, and cleans Rear recentrifuge precipitates, and takes sedimentation cell in the ratio that ratio is 1:0.5～3 of hybridoma macrophage strain and CD4+T cell strain, It is assemblied in the hydrostatic column that high-biocompatibility material is made, makes blood purification cell column；

(3) being mixed with a certain amount of normal saline after 100 DEG C dissolve by agarose, insulation, at 39～41 DEG C, adds step Rapid 1 mixtures of antibodies obtained, wherein, the antibody titer of free HIV antibody, is incorporated into the antibody of the HIV antibody of goat-anti Ig Titre is 1:700, and the mass fraction of agarose is 1.1%.

(5) on bottom gel, sedimentation cell is added, the agar mixtures of antibodies being subsequently adding at 39～41 DEG C, wherein, In agar mixtures of antibodies, the titre of antibody is 1:500, and the mass fraction of agarose is 1.0%, and agar mixtures of antibodies is with heavy The volume ratio of shallow lake cell is 1:4；After being cooled to semi-solid gel, complete the preparation of second layer purifying layer.

(6) on second layer gel, sedimentation cell is added, the agar mixtures of antibodies being subsequently adding at 39～41 DEG C, its In, in agar mixtures of antibodies, the titre of antibody is 1:300, and the mass fraction of agarose is 0.9%, agar mixtures of antibodies It is 1:4 with the volume ratio of sedimentation cell；After being cooled to semi-solid gel, complete the preparation of third layer purifying layer.

(7) on third layer gel, sedimentation cell is added, the agar mixtures of antibodies being subsequently adding at 39～41 DEG C, its In, in agar mixtures of antibodies, the titre of antibody is 1:200, and the mass fraction of agarose is 0.8%, agar mixtures of antibodies It is 1:4 with the volume ratio of sedimentation cell；After being cooled to semi-solid gel, complete the preparation of the 4th layer of purifying layer.

(8) on the 4th layer of gel, sedimentation cell is added, the agar mixtures of antibodies being subsequently adding at 39～41 DEG C, its In, in agar mixtures of antibodies, the titre of antibody is 1:100, and the mass fraction of agarose is 0.7%, agar mixtures of antibodies It is 1:4 with the volume ratio of sedimentation cell；After being cooled to semi-solid gel, complete layer 5 purifying layer, i.e. top layer purifying layer Preparation, is purified post.

The specification of blood purification and material

The shell of blood purification typically selects the cylinder that footpath, the end is little, footpath, top is big, or square, infundibulate, and volume is 200 ～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh；At the bottom of exit, footpath sieve 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, 2.5 mesh, 7 kinds of different sizes of 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop the inhibition of HIV of 120 nanometers or bigger Antibacterial；Liquid outlet arranges the cell strainer that mesh number is 100 mesh (being equivalent to 4 microns), in order to stop the cell that may leach； 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 adsorber 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 adsorber inner surface, can suppress blood coagulation, improve biocompatibility, also can reduce Heparin consumption, and likely realize no-rod tractor.Covalent immobilisation linoleic acid film on cellulose acetate film, maybe will be covalently bound to gather Acrylic acid linoleic acid is grafted onto polysulfone membrane surface, can have more preferable histocompatibility and anticoagulant effect.

The blood purification of the present invention generally coordinates blood separator and plasma separator to use, blood separator Screen out the HIV cell existed with multinucleated giant cell or many cells polymer state for by volume size, be i.e. used for removing Endoglobar HIV；Plasma separator is used for separating mononuclear blood cell and blood plasma；Blood purification is in adsorbed plasma HIV.The blood purified, after blood separator separates, filters off multinucleated giant cell, and small volume hemocyte and blood plasma enters blood Slurry separator；Small volume hemocyte and blood plasma are separated by plasma separator, isolated blood plasma through two or two with After upper purification utensil in parallel, after small volume isolated with plasma separator mixes, complete to purify.

Three, the checking of acquired immune deficiency syndrome (AIDS) blood purification effect

As it is shown in figure 1, the acquired immune deficiency syndrome (AIDS) blood purification of the present invention is applied to acquired immune deficiency syndrome (AIDS) blood purification, by it with existing Blood separator and plasma separator connect, and for convenience of operation, are provided with additional member, including blood pump, liver on connecting line Element pump, sound pulse pressure and air monitering, temperature control system, off gas system, monitored conductivity system, ultrafiltration monitoring and leakage blood prison The part compositions such as survey.Wherein, (1) blood pump (Blood Pump): be used for promoting blood circulation to maintain the suitable of blood purification treatment Profit is carried out, and usual blood pump part often has rotary test speed function, and to monitor the blood circumstance of patient, therefore blood pump runner is with recessed Separation sets to be wanted accurately and it needs to often adjust, according to the situation of bloody path pump line, typically spacing is set as 3.2～ 3.3mm, can not be the most loose, and blood flow detection otherwise can be caused to be forbidden；Also can not be too tight, otherwise can cause pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, in order to continue to sieving pipeline (Blood of Patients Liquid) in injecting heparin, owing to the blood of patient circulates and air contact in vitro, be susceptible to blood coagulation phenomenon, use heparin pump It is possible to prevent the generation of blood coagulation.(3) sound pulse pressure monitoring: arterial blood pressure monitoring is mainly in order to dynamic monitoring blood separator micropore Stopping state, additionally in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When blood flow deficiency, arterial pressure will drop Low；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 is not enough and venous return syringe needle When coming off, venous pressure will decline, if the distortion blocking of bloody path return duct or backflow syringe needle occur blocking, venous pressure will rise High.(4) air monitering (Air Detector): be used for monitoring the air bubble of blood pathway, general ultrasonic listening former Reason, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble, detecting system can drive artery and vein Bloody path folder carrys out blocking blood flow, prevents the generation of danger.

Come in accordance with the following methods blood is purified, to verify the clean-up effect of blood purification of the present invention:

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 most again goes through aerofluxus The most complete, liquid stream is the most unobstructed, and avoids managing the pollution of interior flow liquid.

4, anticoagulant: from heparin pump 2 to injecting anticoagulant (heparin) liquid stream, be for the first time 2500 ∪ or 20～30 ∪/.

5, start: one end of arterial blood line pipe 1 is connected with arteries, venous line 15 is connected vein blood vessel, Then opening heparin pump 2, blood flow is 100～150ml/min, such as Fig. 1, when arterial blood is through arterial blood line pipe 1, heparin regulating liver-QI When element pump 2 enters blood separator 3, the large volume multinucleated giant cell formed because of HIV is delayed at blood separator 3 In, 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 of separation Slurry 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, begins paying out Blood plasma, flows out through export pipeline 13, synchronizes to irrigate blood plasma to depurator 12, when the blood plasma in depurator 11 has nearly flowed, then Secondary beginning irrigates blood plasma, and now depurator 12 begins paying out blood plasma, and two depurators 11 and 12 in parallel are alternately.

As it is shown on figure 3, when the blood plasma containing HIV101 enters depurator, HIV101 therein is fixed on HIV respectively Antibody 102, macrophage 104, CD4+T cell 106 are combined into antigen antibody complex 103, macrophage phagocytic body 105, CD4 + T cell conjugate 107, combined after HIV no longer move down, the most combined large volume of HIV is again by because of dense Spend higher and that micropore is less bottom agar gel molecular and sieve microporous barrier at 109.Purification blood plasma after adsorbed HIV is through figure The individual cells that export pipeline 13 shown in 1 separates with plasma separator 8 converges through venous line 15 after export pipeline 14 converges Fluid circulates.So purify blood, remove HIV, until the plasma circulation amount being previously set (usually 9L), treatment just declaration Terminate.Whole therapeutic process is by computer control, and can detect duty, easy to use, automatization and safety at any time.

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-in large, medium and small leukocyte The average content of p24 is respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, HIV-p24 in the most large and small leukocyte 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 2) 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 2 AIDS blood plasma filters p24 testing result (pg/ml) before and after the simple purifier containing CD4+T cell

(2) checking of hybridoma macrophage strain adsorption removal HIV effect

For effect of check cross tumor macrophage strain adsorption removal HIV, the present invention devises easy method of testing: Take 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draw by centrifugation the macrophage that (1000r/min, 5min) precipitates respectively Hybridoma cell strain, to 200mm scale, is then drawn and is incubated after 100 DEG C dissolve at 39～41 DEG C of 0.9% standby agaroses C1-4B, reaches the long scale of about 10mm, and after putting blood sedimentation tube cooling, agarose becomes semi-solid, can stop blood sedimentation tube inner cell stream Go out but the material of water and the chemical analysis that will not stop little molecule etc passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient that storehouse preserves, before respectively taking 9mLAIDS filter, blood plasma injects erythrocyte sedimentation rate in batches Pipe (simple purifier) upper end blank pipe, the hybridoma macrophage strain layer of blood sedimentation tube lower floor to be flowed through outflow in blood sedimentation tube After, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, move suppression with human macrophage The factor (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operates, detection Scope is 0～800pg/ml, and sensitivity is 1.0pg/ml, directly can detect by an unaided eye: color in reacting hole under white background The deepest, the positive is the strongest, and negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also can survey OD value: on ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, surveys each hole after returning to zero with blank control wells OD value, if 2.1 times of the negative control OD value more than regulation, it is the positive.Result such as table 3, MIF testing result in blood plasma before filter It is feminine gender (or preserving cause degraded etc. for a long time because content is less than detection sensitivity, blood plasma), and after filtering, in blood plasma, testing result is equal For the positive.Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF is collection cytokine, growth The factor, hormone and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays The effect of central, plays panimmunity function in various infection and active chronic inflammation disease.Filter making AIDS blood plasma Before and after simple depurator while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, 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 result (table 4) illustrates that AIDS blood plasma filters letter Easily after purifier, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, and the blood plasma HIV after filtration significantly reduces, After the 1st time filters, HIV clearance rate is 20.55%, and after the 2nd time filters, HIV clearance rate is 42.83%, p ＜ 0.01, tool There is obvious effect, illustrate that the increase HIV along with filtering number of times can constantly be removed, thus reach to treat AIDS purpose.

Table 3 AIDS blood plasma filter MIF testing result before and after the simple purifier containing hybridoma macrophage (quantitatively: pg/ml)

Table 4 AIDS blood plasma filters p24 testing result (p24:pg/ before and after the simple purifier containing hybridoma macrophage ml)

(3) 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 The specimen of the 5 example HIV sufferers that laboratory Sample Storehouse preserves, removes each about 10mL of the blood plasma after cell, before respectively taking 9mLAIDS filter Blood plasma injects blood sedimentation tube upper end blank pipe in batches, and the 1.0% agarose C1-4B containing antibody of blood sedimentation tube lower floor to be flowed through is also After flowing out in blood sedimentation 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) operates, with concentration known The p24 antigen of 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as right According to, 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 5) is said Bright, after AIDS blood plasma filters simple purifier, part HIV is adsorbed by corresponding antibodies, and after the 1st time filters, HIV always removes Rate is 20.01%, and after the 2nd time filters, total body clearance is 27.99%, and after the 3rd time filters, total body clearance is 37.36%. Illustrate that the increase HIV along with filtering number of times can constantly be removed, thus reach to treat AIDS purpose.

Table 5 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 Cleanser (HIVgp120 antibody, HIVgp41 antibody, CD4+T cell strain, hybridoma macrophage strain, the agar gel of the present invention Micropore) adsorption removal.Show had by the above-mentioned acquired immune deficiency syndrome (AIDS) blood purifying therapeutical instrument constituted for critical component with blood purification Significantly remove the therapeutic efficiency of the inside and outside inhibition of HIV of blood cell.

Claims

1. an acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator, it is characterised in that be provided with multilamellar in depurator by agar gel and purification The purifying layer of cell composition, multilamellar purifying layer constitutes decontaminating column；Described daf molecule is by can be in conjunction with the CD4+T cell strain of HIV, miscellaneous Hand over tumor macrophage strain according to quantity than 1:0.5～3 composition；In each layer, cell of the total volume 4/5；Described agar gel Comprise free HIV antibody, be incorporated into the HIV antibody of goat-anti Ig, agarose；From the import department of depurator to exit, free The antibody titer of HIV antibody, the antibody titer of HIV antibody that is incorporated into goat-anti Ig successively decrease the most successively, agarose Concentration is incremented by the most successively.

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that the import and export of described depurator Being provided with screen cloth, the mesh number of import department's screen cloth is 800 mesh, and the mesh number of exit screen cloth is 2.0～5.0 mesh.

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that the number of plies of described purifying layer is 5 Layer, the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody titer the most from high to low, be followed successively by 1: 100,1:200,1:300,1:500,1:700.

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that the number of plies of described purifying layer is 5 Layer, from low to high, content can be 0.7g/100ml, 0.8g/100ml, 0.9g/100ml, 1.0g/ to agarose concentration successively 100ml、1.1g/100ml。

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that described HIV antibody is by HIV- One or both in 1gp120 antibody, HIV-1gp41 antibody form according to any proportioning.

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that hybridoma macrophage strain is passed through The hybridoma technology that oncocyte and macrophage merge with cell is prepared.

Acquired immune deficiency syndrome (AIDS) pregnant woman blood depurator the most according to claim 1, it is characterised in that described decontaminating column is by with lower section Method prepares:

(1) cleaning hybridoma macrophage strain and CD4+T cell strain with physiological saline solution, 1000r/min is centrifuged, after cleaning again Secondary centrifugation, takes sedimentation cell in the ratio that ratio is 1:0.5～3 of hybridoma macrophage strain and CD4+T cell strain, assembling In the hydrostatic column that high-biocompatibility material is made, make blood purification cell column；

(2) HIV antibody of goat-anti Ig with excess is mixed, make the goat-anti in mixed liquor be fully tied, but remain and have free HIV Antibody, and the HIV antibody titre dissociated is equal with the HIV antibody titre being incorporated into goat-anti Ig；

(3) being mixed with a certain amount of normal saline after 100 DEG C dissolve by agarose, insulation, at 39～41 DEG C, adds step 2 and obtains Mixtures of antibodies, wherein, the antibody titer of free HIV antibody, the HIV antibody being incorporated into goat-anti Ig antibody titer equal For 1:700, the content of agarose is 1.1g/100ml.

(4) product step 3 prepared joins in lymphocyte depletion post prepared by step 1 with volume ratio 1:4, is cooled to half solid After body gel, complete the preparation of bottom purifying layer.

(5) repeat step 1～4, be sequentially completed the preparation of each layer purifying layer from top to bottom, be purified post.And free HIV resists The antibody titer of body, the antibody titer of the HIV antibody being incorporated into goat-anti Ig are 1:500,1:300,1:200,1:100；Agar The content of sugar is followed successively by 1.0g/100ml, 0.9g/100ml, 0.8g/100ml, 0.7g/100ml.