CN106166313B - A kind of AIDS pregnant woman blood clarifier - Google Patents

Abstract

The invention discloses a kind of AIDS pregnant woman blood clarifiers, the critical piece of clarifier is decontaminating column, for decontaminating column multilayer by can be formed in conjunction with the purifying layer that the CD4+T cell strains of HIV, the strain of hybridoma macrophage, free HIV antibody, the HIV antibody for being incorporated into goat-anti Ig, agarose form, multilayer purifying layer constitutes decontaminating column；Blood and plasma separator treated blood to be clean, HIV therein is cleaned agent absorption and removes, purified blood plasma is fed back in vivo after converging with the separated mononuclear blood cell of separator, to achieve the purpose that remove the AIDS blood purification treatment of the inside and outside HIV of haemocyte.

Description

A kind of AIDS pregnant woman blood clarifier

Technical field

The present invention relates to a kind of AIDS pregnant woman blood clarifiers, are mainly used for the inside and outside AIDS of AIDS patient's haemocyte The removing of virus, to achieve the purpose that prevention, control and treatment AIDS.

Background technology

Body production envelope protein (Gp120, Gp41) antibody and core protein (P24) antibody can be stimulated after HIV infection. Low-level antiviral neutralizing antibody is measured in HIV carriers, AIDS patients serum, wherein AIDS patients level is minimum, HIV carriers' highest illustrates that the antibody has protective effect in vivo.But antibody cannot be with the virus that is retained in mononuclear macrophage Contact, and antigenic variation easily occurs for HIV envelope proteins, original antibody is ineffective, keeps neutralizing antibody due from playing Effect.In the latent infection stage, HIV provirus is integrated into host cell gene group, therefore HIV will not be known by immune system Not, so relying solely on itself immune function can not be removed.Another critically important reason should be killed according to antibody Go out, remove the mechanism speculate of antigen, immune antibody with after antigen binding, to generate immunological effect or by activating complement, ADCC effects are mediated to dissolve cellular antigen, but HIV is not cellular antigen；Phagocyte is attracted to gulp down by chemotaxis Removing antigen is bitten, but HIV is protected in phagocyte, is proliferated instead；Antibody plays neutralization with antigen binding, is allowed to Appeal is lost, but HIV antigenic structures are changeable, often antibody made to be difficult to.

In terms of the treating AIDS method for having been used for clinic at present, effect is less ideal：(1) hiv reverse transcriptase inhibits Agent：Be only capable of preventing the permissive cell of not yet infected by HIV from infecting, there is no a therapeutic effect to the cell infected, and toxic side effect compared with It is more, including gland plastochondria toxicity, bone marrow suppression, globular anemia, granulocyte and decrease of platelet, pancreatitis, intersect in addition resistance to The generation of pharmacological property, this kind of medicine are not used alone, mainly do drug combination, and are also easy to produce drug resistance mutant, cause under clinical efficacy Drop or failure.(2) hiv protease inhibitor：It is also easy to produce the toxic side effects such as drug induced hepatic injury, disorders of lipid metabolism and drug resistance Property.(3) hiv integrase inhibitor：It is played a role by inhibiting inhibition of HIV DNA to be integrated with host cell DNA, is reversed with HIV Transcriptase inhibitors and protease inhibitors are combined while use has synergistic effect to antiviral.(4) inhibition of HIV is inhibited to enter inhibition Agent：Including blocking gp120 that T leaching is combined, HIV is blocked to be combined with accessory receptor, gp41 films subunit is acted on and acted on CD4 Bar cell surface CC-chemokine receptor 5 (CCR5) has side effect to block HIV to enter host cell to liver and heart.(5) Cytokine therapy：It is mainly used for regulatory T-cell dynamic equilibrium.(6) HIV vaccine is treated：It is such as intrinsic due to the particularity of HIV Immune to be not enough to resist HIV and its targeting destruction immune system, virus mutation is rapid, causes not yet to develop real safety so far Effective vaccine.(7) gene therapy：The research of HIV gene therapies is never interrupted, including antisense technology, RNA baits, RNA dry It disturbs, intrabody, dominant negative mutant, suicide gene etc., but enters the gene therapy in II clinical trial phase stage almost No.(8) monoclonal antibody passive immunization therapy：It reduces the neurological susceptibility of HIV by lowering CD4+T cell surfaces CCR5, prolong The progress of slow AIDS and the diffusion for reducing HIV, neutralizing monoclonal antibody 2G12,2F5 and 4E10, which are applied to HIV infection person, to be had Good tolerance and safety can delay but cannot prevent rebound (9) adoptive immunity cell therapy of virus：It is external a large amount of It can lead to viral large amplification when the culture self CD4+T cells of HIV, increase the CD4+T cell quantities of virus infection, and feed back CD4+T cells may increase the place of internal virus replication, lead to virus load bounce-back, on the whole adoptive immunity cell Treatment does not obtain satisfied therapeutic effect without apparent toxic side effect yet.

Ag-Ab precipitation reaction was initially applied to research Liesegang's phenomenon in 1905 in gel, applied within 1932 In identification bacterium bacterial strain, nineteen forty-six Oudin carries out immunodiffusion in test tube, for analyzing antigen mixture, 1948 Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, more two or more antigens or anti- Body.The media gel agar or agarose of Ag-Ab precipitation reaction in gel are a kind of polysaccharide body containing sulfate, high temperature When can be dissolved in water, congeal into gel after cold, inside forms a kind of porous reticular structure, and aperture is very big, allows macromolecular Substance (molecular weight is up to million or more) passes freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To smaller, agar concentration is small, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, since agar or agarose have Good chemical stability, water content is big after gel, and transparency is good, and convenient sources are disposable, therefore is a kind of diffusion well Medium.The molecular weight of antigen and antibody in gel from high concentration region to low concentration region generally all 200,000 hereinafter, spread When suffered resistance very little, be substantially in free diffusing form.When antigen and corresponding antibodies meet after diffusion in gel, shape At antigen antibody complex maximum compound is formed if the two is appropriate in place's ratio of meeting.Due to the molecular weight of compound Increase, particle increase, thus do not continue to spread and generate precipitation, show threadiness or band-like, this precipitation is formed one A " specific barrier ", all antigen or antibody molecule same in immunology cannot pass through, and those of property difference Molecule can continue to spread by this barrier, until forming the compound of themselves.Such reaction is known as agar Gel or Immune proliferation, be at present with known antibodies detect unknown quantity corresponding antigens routine experiment checkup item, and《China Pharmacopeia》Standard method of the regulation for the detection of influenza virus vaccine hemagglutinin content in 2010 editions.Usually by a certain amount of goat-anti People's Ig antiserum ingredients are mixed in agar gel, are made containing the specificity sero-fast agar plates of goat-anti people Ig, after to be solidified Punching, and human serum to be checked (IgG, IgA, IgM etc.) is added in corresponding aperture, so that serum to be checked is expanded around in agar plate It dissipates, properly locates to combine in antigen and antibody concentration ratio, form macroscopic white precipitate ring and no longer spread.Thus As it can be seen that when a kind of solution passes through semi-solid gel, macromolecular solute therein is just existed by the gel pore detention that molecular sieve acts on In gel, antibody that antigen especially therein can be in advance fixed in gel in conjunction with and be attracted in gel.

CD4 molecules are the receptors of HIV, and HIV is susceptible in CD4+T cells, and CD4+T cells are one kind of T lymphocytes, average Service life is generally 7 days or so, but certain T cells can long-term surviving, unlimited amplification especially after immortality chemical conversion cell line (strain). Foreign literature reports that simian virus 40 (SV40) can be such that certain human cells immortalize.Poulin DL, Kung AL and Sullivan CS etc. are studies have shown that the importing of SV40T antigen genes can accelerate the growth rate of transformed cells, immortalized cells Repeatedly still there is metastable multiplication characteristic and functional status, while can also retain its initial cell after passage in vitro Many phenotypic differentiations.Reilly establishes vascular smooth muscle cells strain with the genetic transformation of simian virus large T antigen, builds cell membrane Type is to study the inhibiting effect mechanism of heparin for vascular smooth muscle.Su etc. utilizes the superficial cell strain converted through SV40, structure Cell model is built to analyze the regulating and controlling effect of epithelial cell internal protein synthesis.Miquel etc. is thin with the cuticulated epithelium that SV40 is converted Born of the same parents' strain, the cell adhesion mediated as cell model research laminin 5.The forefront converted through SV40 such as Webber The physiological function and secreting function of prostate epithelial cell are studied in glandular epithelium strain as cell model.The use such as Racusen Renal cells model is converted through Ad12-SV40 to study damage and the disease of proximal convoluted tubule.Hougton etc. is turned with SV40 Change establishes Bone marrow Stromal cell as cell model to study under certain condition of culture, cell with to adipocyte and at The potential of the two-way differentiation of osteocyte further studies the mechanism of osteoporosis.Foreign study, which is also shown that, imports exogenous human end Human telomerase reverse transcriptase (hTERT) can make cell keep normal phenotype and differentiating characteristic.It is successfully established using hTERT in recent years The immortalized cell lines of certain cells keeps normal chromosome stabilityX, differentiation, contact inhibition, opposite without oncogenicity etc. substantially Normal growth characteristics.In dentistry field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes immortality Change people Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number up to 150 times or more, Cell shows original biological characteristics, and the GAP-associated protein GAP of derived cell can be expressed after Fiber differentiation.Kitagawa etc. Transfection hTERT establishes people cementoblast system, and cell multiplication is up to 200 times or more, cell differentiation marker such as alkaline phosphatase The expression such as enzyme, type i collagen are stablized.Because of the needs of research work, almost each disease has respective cell model.Such as diabetes Cell model, cancer cell model, transgenic cell model, climacteric syndrome cell model, endometrial cell model, Epilepsy cell model, E-Cell models, alcoholic dementia cell model, brain edema cell model etc..So CD4+ can be prepared T cell strain is used to prepare the clarifier for the treatment of AIDS after mass propgation, with the absorption of CD4+T cells, removes in blood plasma HIV, while treating AIDS by being defeated by the cell factor of CD4+T cells generation.

The phagocyte of the mankind has large and small two kinds, and microphage is the neutrophil leucocyte in peripheral blood, macrophagocyte It is the macrophage in the monocyte and a variety of organs, tissue in blood, the two constitutes mononuclear phagocyte system.Monocyte It is formed by the monocyte precursor Development And Differentiation in marrow, accounts for about 3% one the 8% of blood middle leukocytes sum, volume is relatively drenched Bar cell is bigger, and monocyte only stops 12-24 hours in blood, subsequently into connective tissue or organ, reach maturity for Macrophage, macrophage are highly differentiation, ripe cell type in mononuclear phagocyte system, have stronger phagocytosis work( Can, wandering macrophage is more than monocyte several times, lasts a long time, can in the tissue survive some months, the macrophage of colonization There is different titles, be Kupffer Cell in liver, be microglia in brain, in bone be osteoclast etc., expresses Fc Receptor, C3b receptor and CD14 play defense function in inherent immunity, and the professional antigen of participation adaptive immunity is offered Cell.The CD4 molecules of Expression of Macrophages, are the receptors of AIDS virus (HIV), thin by macrophage first after HIV enters human body The phagocytosis of born of the same parents, but HIV changes the acidic environment at certain positions in macrophage quickly, creates the condition for being suitble to it to survive, It is not killed not only and assembles instead in its interior mass propagation, then HIV is passed into CD4+T cells.So can be with conventional hybridization Oncocyte technology of preparing, prepares macrophage hybridoma, and the clarifier for the treatment of AIDS is used to prepare after large amplification, The HIV in blood plasma is removed with the phagocytic function of macrophage.

In short, various drugs and biological products can not effectively kill internal AIDS virus, and price, side effect is big, So far the effective ways for the treatment of AIDS be there is no, it has also become attack the global problem being unable to long.

Invention content

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

The object of the present invention is achieved like this：A kind of AIDS pregnant woman blood clarifier is provided with multilayer in clarifier The purifying layer being made of agar gel and daf molecule, multilayer purifying layer constitute decontaminating column；The daf molecule is by that can combine HIV CD4+T cell strains, hybridoma macrophage strain according to quantity than 1：0.5~3 composition；In each layer, cell is of the total volume 4/5；The agar gel includes the HIV antibody dissociated, the HIV antibody for being incorporated into goat-anti Ig, agarose；From the import of clarifier Place to exit, the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody titer from high to low Successively decrease successively, agarose concentration is incremented by successively from low to high.

Further, the import and export of the clarifier is both provided with sieve, and the mesh number of entrance sieve is 800 mesh, is gone out The mesh number of sieve is 2.0~5.0 mesh at mouthful.

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

Further, the number of plies of the 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 be 1 in conjunction with the CD4+T cell strains of HIV, the quantity ratio of hybridoma macrophage strain：0.5~ 3。

Further, the HIV antibody by one or both of HIV-1gp120 antibody, HIV-1gp41 antibody according to Arbitrary proportioning composition.

Further, the strain of hybridoma macrophage by by oncocyte and macrophage with the hybridoma technology of cell fusion It prepares.

Further, the decontaminating column is prepared by the following method to obtain：

(1) strain of hybridoma macrophage and CD4+T cell strains are cleaned with sterile saline, 1000r/min centrifugations are cleaned Centrifugation again afterwards is 1 by the ratio between the strain of hybridoma macrophage and CD4+T cell strains：0.5~3 ratio takes sedimentation cell, It assembles in hydrostatic column made of high-biocompatibility material, blood purification cell column is made；

(2) goat-anti Ig is mixed with excessive HIV antibody, the goat-anti in mixed liquor is made to be fully tied, but it is surplus have it is free HIV antibody, and free HIV antibody titre is equal with the HIV antibody titre of goat-anti Ig is incorporated into；

(3) agarose is mixed after 100 DEG C dissolve with a certain amount of physiological saline, keeps the temperature at 39~41 DEG C, step is added It is rapid 2 obtain antibody mixtures, wherein the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody Titre is 1：700, the content of agarose is 1.1g/100ml.

(4) product for preparing step 3 is with volume ratio 1:4 are added in the lymphocyte depletion column of step 1 preparation, are cooled to After semi-solid gel, the preparation of bottom purifying layer is completed.

(5) step 1~4 are repeated, the preparation of each layer purifying layer is sequentially completed from top to bottom, is purified column.And free The antibody titer of HIV antibody, the HIV antibody for being incorporated into goat-anti Ig antibody titer be 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 are：It is thin that the present invention will filter out the large volume containing HIV that many cells are combined into The blood separator of born of the same parents, the plasma separator that mononuclear blood cell and blood plasma can be detached and inhibition of HIV in blood plasma can be carried out high The clarifier of effect purification combines, and realizes the high purification to blood.

Cleanser in clarifier is by being fixed on the HIV antibody of agar gel, the HIV antibody combined with goat-anti Ig, CD4+T Cell strain and macrophage strain are made, wherein the molecular weight of HIV antibody its conjugate combined with goat-anti Ig is than unbonded HIV Antibody molecule amount is big, not easily pass through gel molecular sieve and contained goat-anti Ig easily with agar gel secure bond the characteristics of, HIV Antibody is also more easy to be fixed in agar gel therewith, the meeting when HIV in blood plasma meets with the HIV antibody for being fixed in agar gel Association reaction occurs and forms antigen antibody complex, to be fixed in agar gel by HIV antibody, because of CD4+T cells The CD4 molecules on surface are the receptor of HIV as the permissive cell of HIV, HIV can be adsorbed when meeting with HIV, the HIV adsorbed It is fixed in agar gel with CD4+T cells, it, can quilt when HIV meets therewith because of the natural phagocytosis characteristic of hybridoma macrophage It swallows and is fixed in agar gel therewith, these compositions all have phagocytosis and/or combine the function of absorption HIV, and agar Gel is formed by filter opening and is reduced with increasing for agarose concentration, and clarifier entrance agarose concentration is low, filter opening with regard to big, Be conducive to the association reaction of plasma perfusion and high titre antibody or cell and HIV；And exit concentration is high, filter opening is just small, is easy to Detention HIV or Large molecular conjugates, clarifier are combined with a variety of special and non-specific HIV and remove composition, in order to avoid extraordinary strain Futile treatment caused by immunity difference contains the big of a large amount of HIV so in the extracorporal circulatory system of AIDS blood purification treatment Volume cells are filtered out by blood separator first, and the HIV to dissociate in blood plasma is adsorbed by blood purification and removed, purified blood Slurry converges with the isolated mononuclear blood cell of blood separator in rear reflux, and cell surface is incorporated in reach removing And/or the purpose of intracellular HIV and the HIV for being free on blood plasma.

Description of the drawings

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

Fig. 2 is for 5 layers of structure of decontaminating column and the schematic diagram of sieve.

Fig. 3 is according to clarifier purification process schematic diagram proposed by 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, clarifier 11 are connected with 12, and export pipeline 13, haemocyte go out Mouth pipeline 14, venous line 15.

In Fig. 3,102,104,106 be respectively HIV antibody, macrophage, CD4+T cells；101 be free HIV；103、 105,107 be respectively the knot being delayed at after HIV and HIV antibody, macrophage, CD4+T cell combinations in agar gel 108 Object is closed, 109 is by the large volume of HIV of 108 molecular sieve detention of agar gel.

Specific implementation mode

With reference to Fig. 1, Fig. 2 and Fig. 3, AIDS blood purification proposed by the present invention is explained in detail.

One, the preparation of AIDS pregnant woman blood cleanser

(1) preparation of hybridoma macrophage strain

1, primary cell source

(1) single core blood cell：Refer to the lymphocyte detached from blood with density-gradient centrifugation method and monokaryon macrophage Cell.Specific method is：The White Blood Cells Concentrate of purchase Blood Center is the Cord blood that scientific research preserves, and takes 2mL samples, PBS 6mL anticoagulations are slowly superimposed on dropper that 4mL lymphs have been added is thin by 2~3 times of hemodilution, after mixing well by liquid along tube wall Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge in the 10mL centrifuge tubes of born of the same parents' separating liquid；It is divided into pipe after centrifugation 3 layers, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle level is lymphocyte separation medium, in upper, middle level It is PBMC that, which there be the white cloud and mist layer narrow band based on mononuclearcell in interface, and cloud and mist layer is inserted into capillary syring, is drawn PBMC is placed in another 50mL centrifuge tubes, is added 5 times and is centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandons supernatant Cell is resuspended in 50mLPBS, centrifuges (350r/min, 20 DEG C) 15min, abandons supernatant, and Buffer (PBS+0.5% new life ox bloods are added + 2mmol/LEDTA clearly, pH7.2) 2mL resuspension cells, it takes 15uL cell suspensions to be added on blood counting chamber and counts 4 under microscope Cell (PBMC) sum in a block plaid.

(2) single core histocyte：It is provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, preparation method are：1. the acquisition and transhipment of spleen tissue：In the approval of the reason committee and patient's informed consent Under, the spleen sample tissue for taking operation to cut off shreds into volume about 1mm immediately³Small tissue blocks, move into equipped with precooling 4 DEG C In sterile sealing bottle, it is transported to cell culture chamber rapidly.2. the preparation of spleen tissue cell suspension：Spleen tissue block is moved to Aseptic operating platform, PBS are washed 3 times, and RPMI-1640 is washed 2 times, to remove the blood in tissue and ensure the sterile of tissue.Machine Tool grinds spleen tissue, at this moment just has a large amount of histocyte is outstanding to be mixed in RPMI-1640 liquid.With 200 mesh stainless steel filtering net mistakes Filter is outstanding to be mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly contain red blood cell, lymphocyte, Macrophage etc.).3. the cracking of red blood cell in spleen tissue cell suspension：Then centrifugation is washed with RPMI-1640 liquid (1000r/min, 3min) is added Tris-NH4Cl and acts on 5min, splitting erythrocyte, Quick spin to remove cell debris (1000r/min, 3min), remove supernatant in splitting erythrocyte fragment, PBS washing centrifugation 3 times, RPMI-1640 wash from The heart 1 time avoids it from influencing the survival of cell, at this point, mainly containing in suspension to remove Tris-NH4Cl remaining in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage：Using aforementioned suspension as culture Cell stoste, Trypan Blue judgement vigor simultaneously count, and are (3~5) × 10 with RPMI-1640 liquid adjustment cell concentration⁶/ L, will The cell suspension inoculation of concentration is adjusted in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2~3h, observe form under phase contrast microscope.The digestion of adherent spleen tissue macrophage：Adherent spleen tissue macrophage The digestion of cell：Culture supernatant is sucked, macrophage is adherent, and PBS blows and beats, digests repeatedly, the washing centrifugation of gained cell suspension (1000r/min, 3min), the macrophage isolated and purified.Further, it is also possible to which the sample discarded after treatment or operation is taken to carry Take preparation, such as cavum peritoneale liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa.

(3) amniotic fluid, villus cell：Zhejiang University's attached hospital for obstetrics and gynaecology's reproduction heredity laboratory is spare.In reason committee member Can ratify under patient's informed consent, take laboratory diagnosis report after remaining amniotic fluid, villus cell, select exponential phase cell Continue to employ.

2, cell culture and the adherent preliminary sorting of macrophage

Routinely cell culture, but according to the difference of cellularity, incubation time, condition of culture etc. are suitably adjusted, generally Single core blood cell (PBMC) or single core histocyte (macrophage) are placed in and are cultivated containing RPMI -1640 by adherent method In the culture dish of base, in 37 DEG C, 5%CO₂Cell incubator (Themo electro corporation CLASS 100, it is beautiful State) in be incubated 2h, after mononuclearcell is adherent, suction abandon upper layer suspension cell (cell other than macrophage be not easy it is adherent and Removed with upper liquid), PBs buffer solutions gently wash 3 times, and mono- 1640 culture mediums of a small amount of RPMI are added, and patch is scraped with cell scraper Parietal cell (predominantly macrophage, but also have other a small amount of attached cells).1 000r/min centrifuges 5min, abandons supernatant.Amniotic fluid There is cell growth clone in cell, villus cell culture 1~7 day, cell growth converges the logarithmic growth that rate reaches 60~80% Phase cell, is digested with pancreatin, and PBS cleanings obtain cell suspension, are made into proper cell concentration.

3, cd4 cell sorts

Cd4 cell is sorted using immunomagnetic beads method：1. main agents and instrument：CD4 immunomagnetic beads (Germany U.S. day Ni biologies Technology Co., Ltd.)；0.2% Trypan Blue liquid (Shanghai Sheng Gong biotechnologies service company)；Newborn bovine serum (Hyclone companies)；MiniMACS magnetic separations system (German Mei Tian Ni Bioisystech Co., Ltd).2. cd4 cell is immune Magnetic bead sorting method：Cell suspension, which is divided equally to two 1.5mLEppendorf, manages, and centrifuges (300r/min, 20 DEG C) 10min, discards Supernatant is resuspended cell and contains cell number 10 per 80uLBuffer⁷It is a, every 10⁷A cell add 20uLCD4MicroBeads or CD8MicroBeads is mixed well, and hatches 15min at 4~8 DEG C, and cell is washed with 1mLBuffer, centrifuge (300r/min, 20 DEG C) 10min, it discards supernatant 500uLBuffer and cell is resuspended, MS splitters are placed in the magnetic field of MACS separators, with 500uLBuffer is rinsed, and by 500uL cell suspensions by splitter, splitter repetitive operation 3 times is rinsed with 500uLBuffer, Efflux is collected, contains non-cd4 cell in efflux, splitter is taken out from separator, with 1000uLBuffer pressure flush point From column, efflux is collected, (cell viability detects for cd4 cell for this：15uL cell suspensions and equal bodies are taken before and after cell purification respectively Product trypan blue solution mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, calculates 200 The percentage of living cells in a cell).The cell sorted at this time is mainly macrophage.

4, CD14 cells (macrophage) sort

CD14 is monocyte and the distinctive surface marker of macrophage, theoretically if from single core histocyte, sheep It is sorted in water cell and villus cell, then gained cell is macrophage；If sorted from single core blood cell, gained Cell includes monocyte and macrophage；But it survives 1 day because of monocyte short life, only in peripheral blood and can not show a candle to macrophage Cell is easy to adherent growth, so being removed substantially in the cell adhere-wall culture of the present invention, the cell sorted out is essentially Macrophage.

Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent：People's CDl4 immunomagnetic beads kits (Miltenyi Biotec, Germany), RPMI-1640 culture mediums (Hyclone, the U.S.)；2. immunomagnetic beads method：(A) magnetic bead with One monocyte of specificity target cell combines：Every 1 × 10⁸The magnetic bead of 200uL coupling CDl4 antibody is added in a PBMC and 800uL delays Fliud flushing (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator precoolings), in 15mL centrifuge tubes In mix well, 4 DEG C incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 10⁷A cell adds Enter 1~2mL precooling buffer solutions, 1 000r/min centrifuges 8min, abandons supernatant, and O.5mL buffer solution is added and blows and beats at unicellular outstanding Liquid.(B) monocyte of marked by magnetic bead is collected：Cell splitter is placed on MACS magnetic frames, it is thin that lmL buffer solutions balance is added Born of the same parents' splitter waits for that no liquid is dripped, and immediately adds above-mentioned cell suspension in people's cell splitter, thin with 0.5mL wash buffers Born of the same parents' splitter 3 times.After to be rinsed, 1mL buffer solutions are added, the emigrated cells splitter from magnetic frame is quickly pushed away with needle column It is dynamic, go out the cell being combined with one magnetic bead of CDl4 antibody in splitter, the as macrophage of CDl4+.

In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used：PBMC is placed in and is trained containing RPMI -1640 In the culture dish for supporting base, in 37 DEG C, contain 5%C0：Cell incubator (Themo electro corporation CLASS 100, U.S.) in be incubated 2h.After adherent mononuclear cells, upper layer suspension cell is abandoned in suction, and PBs buffer solutions gently wash 3 times, is added Mono- 1640 culture mediums of a small amount of RPMI, attached cell is scraped with cell scraper.1 000r/min centrifuges 5min, abandons supernatant.2. streaming Cell art method：CDl4 is marked：PBMC is taken, (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/LEDTA with buffer solution 0.5mL) adjustment cell density is 1 × 10⁸/ mL, the addition CDl4+-FITC antibody 100uL in every milliliter of cell suspension, 4 DEG C It is protected from light label 18min, then 1mL streamings buffer solution is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with containing 2% green chain The PBS adjustment cell densities of mycin are 2 × 107/mL.Selected by flow cytometry apoptosis：By the cell of preparation in flow cytometer (BD FAcsAria II, the U.S.) on sort, according to opposite of the fluorescence intensity of CDl4 antibody, the relative size of cell and cell The complexity of graininess and internal structure collects the cell of CDl4+.

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

(1) culture medium and main agents:DMEM culture mediums, HAT, HT Selective agar medium are purchased from Sigma companies, top grade tire ox Serum (FBS) purchases the oceans Jinshi City Hao on daytime biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is that domestic analysis is pure Reagent.

(2) myeloma cell prepares：Fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container 0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strains, the cell strain growth and fusion efficiencies are good, itself is not Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 10⁵/ ml, the doubling time is usually 10~ 15h；With selection homologous cell strain is considered in the relevant practical application of human body, if the Shanghai bio tech ltd Fu Xiang is to ATCC The NCI-H929 human myeloma cells strain that cell bank is introduced).Appropriate complete culture solution, 1000r/m centrifugations are added after thawing 3min；It is repeated 1 times.Sediment is moved into Tissue Culture Flask, DMEM culture solutions are added, sets CO2 incubator cultures, is carried out within 3-4 days Primary passage expands culture, merges and adjusts cell state in first 24 hours, ensures that cellular morphology is good, it is prosperous to grow before fusion It contains.It is added in appropriate basal medium to centrifuge tube, gently beats 1000r/m centrifugation 5-10min after mixing, wash repeatedly cell 2 times.

(3) CD14 cells (macrophage) to be hybridized prepare：The mononuclear macrophage that the present invention sorts is with basal medium Total cell number is adjusted to 1 × 10⁸~2 × 10⁸For cell fusion.Expect blue dyeing phase-contrast microscopy, viable count with platform 80% be should be higher than that as qualification.

(4) cell fusion：By CD14 cells (mononuclear macrophage) and myeloma cell with 10:1-5:1 ratio is added It in centrifuge tube, is mixed evenly, 1000r/m centrifuges 5min, discards supernatant, and gently beats tube bottom to cell grainless and precipitates, weight It is 2 times multiple.Gently rotation preheats centrifuge tube in 37 DEG C of water-baths, by the 50% of 1000 μ L of preheating under aseptic condition after taking-up PEG3000 is added drop-wise in fusion pipe along tube wall in 60s while gently rotating centrifugal pipe, later trains the basis of the 25mL of preheating It supports base to be also added drop-wise in centrifuge tube along tube wall in 3-5min, lightly rotating centrifugal pipe during addition is then allowed to stand In 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T culture mediums are added.It is inoculated into after appropriate mixing In 96 well culture plates, 37 DEG C are placed in, is cultivated in 5% CO2 incubators.

(5) the mononuclear macrophage strain with phagocytic function is screened:Observe cell growth status in 96 well culture plates, 7-10 Division can be grown by only having hybridoma after it, discarded HAT culture mediums at this time, replaced complete medium.Cell clone is grown When area reaches 1/10 cell hole, goes culture supernatant, selection to have the culture hole of the good hybridoma cell strain of growth conditions, show Position, the size that cell strain growth is marked under micro mirror, draw cell clone in the position of mark using sterile pipette tips has to new In the culture hole of complete medium, then doubling dilution to hole is counted below successively, and 37 DEG C, 5%CO2 incubators are interior to cultivate one week left side The right side, microscopically observation cell growth status take when cell clone is covered with to 1/10 or more hole floor space in cell or culture Clear detection hybridoma macrophage (M φ) strain function.

1. hybridoma macrophage strain swallows bacterium Function detection：Macrophage and staphylococcus or Candida albicans are hanged Liquid mixing incubates, and smear is fixed, the dyeing of serge blue liquid, in oily phagocytosis situation under the microscope, counts phagocytosis bacterium and does not gulp down The number of macrophages ratio for biting bacterium, to swallow the strong macrophage of bacterium function alternately positive clone strain.

2. hybridoma macrophage strain swallows HIV Function detections：Take AIDS (AIDS) patient's of Disease Control and Prevention Center's preservation Blood plasma detaches cell strain, PBS is cleaned 3 times, measures the phagocyte strain through cracking with after hybridoma macrophage strain mixed culture The function of swallowing HIV, with specific reference to HIV-1p24 antigen detection kits, (enzyme-linked immunization, Shanghai inspire biotechnology limited Company) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, As a contrast, minimum detection limit is less than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity to the p24 antigens of 80pg/ml 450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbances, is recognized as absorbance ＞ 0.12 To be positive.Specifically operated by kit specification.

3. hybridoma macrophage strain generates macrophage cytokines detection：With human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen bio tech ltd of Shanghai) by specification operates, and detection range is 0~800pg/ml, Susceptibility is 1.0pg/ml, can directly be detected by an unaided eye under white background：Color is deeper in reacting hole, positive stronger, negative Reaction is colourless or extremely shallow, according to the depth of be in color, is indicated with "+", "-" number.Also OD values can be surveyed：In ELISA detectors On, at 450nm (if developing the color with ABTS, 410nm), each hole OD values are surveyed after returning to zero with blank control wells, if more than defined 2.1 times of negative control OD value, it is as positive, specifically operated by kit specification.MIF be collection cell factor, growth factor, The multi-effect protein molecular of hormone and enzyme characteristic plays central as inherent immunity and the regulatory factor of inflammatory reaction Effect, it is various infection and active chronic inflammation disease in play panimmunity function.

According to testing result, select the cell clone in the culture hole with stronger macrophage function repeat it is next Wheel dilution culture, repeats 2-3 wheels, and detection function is taken out after stablizing, and is transferred to culture bottle mass propgation.

(6) preservation and recovery of hybridoma macrophage strain:Preceding 12 hour adjustment cell growth state is preserved, one bottle of life is taken Long vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min centrifuges 5min, removes supernatant, flick Tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, and 1mL/ is managed, and -70 Cryopreservation tube, is put into liquid nitrogen container after taking-up and saves backup by DEG C refrigerator overnight.40 DEG C or so of hot water is got out before recovery, it will Cryopreservation tube carefully takes out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and is centrifuged in 1000r/min after defrosting 5min opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting washed once with complete culture solution, then 5min is centrifuged in 1000r/min, is discarded supernatant, in case making to expand culture.

6, prepared by hybridoma macrophage strain treatment cell

That is the amplification cultivation of hybridoma macrophage strain.It is moved after above-mentioned cell precipitation is gently resuspended using complete culture solution Enter in culture bottle, sets 37 DEG C, cultivated in 5%CO2 incubators.Amplification cultivation is passed on repeatedly, until required hybridoma cell strain Quantity detects the function of macrophage hybridoma cell strain per 10 generation of secondary culture positive hybridoma cell strain, sees whether steady It is fixed.Continuation carries out extensive industrialization preparation in several bottles, saves backup.

(2) preparation of CD4+T cell strains

1, the source of primary lymphocyte

Have with sow by way of：1. lymphocyte strain (the warp frozen in the Infectious Diseases Lab sample database preserved for scientific research Inactivation HIV totivirus is immune but is uninfected by the lymphocyte of HIV)；2. buying the fresh White Blood Cells Concentrate in blood station, then went out The immune lymphocyte of HIV infection strain living；3. the T lymphocytic series (strain) directly bought from businessman；4. being preserved for scientific research Cord blood lymphocytes cell (immune through inactivating HIV)；5. being directly derived from the peripheral blood lymphocytes of HIV-1 the infected (for certainly Body), using Histopaque lymphocyte separation mediums separation mononuclearcell (PBMC).

2, the preparation of CD4+T cells

1. main agents and instrument：CD4, CD8 immunomagnetic beads (German Mei Tian Ni Bioisystech Co., Ltd)；Isothiocyanic acid Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech companies)；(perseverance letter in Shanghai is biochemical for lymphocyte separation medium Reagent Co., Ltd)；(it is public that the service of work biotechnology is given birth in Shanghai for ethylenediamine tetra-acetic acid (EDTA), 0.2% Trypan Blue liquid Department)；Newborn bovine serum (Hyclone companies)；MiniMACS magnetic separations system (German Mei Tian Ni Bioisystech Co., Ltd)； EpicsXL types flow cytometer (BeckmanCoulter companies of the U.S.).2. the separation of mononuclearcell (PBMC)：By density level bands Spend centrifugal process separation.3. CD4+T cells and CD8+T cells isolate and purify：PBMC cell suspensions are divided equally to two 1.5mLEppendorf is managed, and is centrifuged (300r/min, 20 DEG C) 10min, is discarded supernatant, and cell is resuspended and contains cell per 80uLBuffer Number 10⁷It is a, every 10⁷A cell adds 20uLCD4MicroBeads or CD8MicroBeads, mixes well, and hatches at 4~8 DEG C 15min washs cell with 1mLBuffer, centrifuges (300r/min, 20 DEG C) 10min, discards supernatant 500uLBuffer and is resuspended carefully MS splitters are placed in the magnetic field of MACS separators, are rinsed with 500uLBuffer by born of the same parents, by 500uL cell suspensions by dividing From column, splitter repetitive operation 3 times is rinsed with 500uLBuffer, efflux is collected, contains non-CD4+T lymphocytes in efflux Or non-CD8+T lymphocytes, splitter is taken out from separator, with 1000uLBuffer pressure flush splitters, collects outflow Liquid, this is CD4+T lymphocytes or (the cell viability detection of CD8+T lymphocytes：Take 15uL cells outstanding before and after cell purification respectively Liquid is mixed with isometric trypan blue solution, and the not colored shinny person of microscopically observation is living cells, and the coloring person of swelling is dead cell, Calculate the percentage of living cells in 200 cells).

3, amplification in vitro CD4+T cells

There is document report using the monoclonal antibody of T cell surface C D3 molecules as the stimulant of cell growth, great Liang Pei After the T cell for supporting AIDS patient's separation, fed back as itself therapeutic cells.But HIV is also with the culture of HIV infection cell It breeds in endogenous multiplication, the feedback of increment T cell also results in the feedback of increment HIV.The present invention is with SV40 and/or hTERT CD4+T cells are immortalized, and using CD3 monoclonal antibodies as cell growth stimulant, large amplification CD4+T cells.

It is by the method for cell growth stimulant of CD3 monoclonal antibodies：By anti-CD49d McAb, (CD4+T cells contain simultaneously CD3 molecules) it is coated with to stimulation mononuclearcell (lymphocyte) growth on culture plate, referred to as anti-cd 3 antibodies are coated with method, available Good expanding effect, the lymphocyte that should be expanded in this way have been used for the second stage of clinical treatment of tumour and achieve certain The effect of.Foreign literature reports [Shimizu etc.] also with the lymphocyte of 5 full-blown AIDS patients of the method culture, training Support the amplification for being achieved with 1000 times in 4 weeks, and expand cell mass in CD4+/CD8+T can large amplification (CD4+T cells are more Obviously).Another kind is the dual anti-cross-linking methods of AntiCD3 McAb/CD28, i.e., grows AntiCD3 McAb/CD28 dual anti-are crosslinking on pearl as stimulant training HIV infection person's peripheral blood mononuclear cells (lymphocyte) is supported, a large amount of CD4+T cells, and the CD4+T expanded can be expanded Cell has the ability of confrontation HIV infection, and virus finds that this may be with CD28 later also below detection level in incubation Second signal is provided, a large amount of Th1 cell factors of selective induction secretion are related with chemotactic factor (CF), expanded with the method The clinical treatment that CD4+T cells have been used for HIV infection person is fed back, devoid of risk but effect is general.

It is in the hTERT methods for immortalizing CD4+T cells：With I double digestion plasmid pCIneo- of restriction endonuclease EcoR I and Xho HTERT and carrier pLXSNneo, the hTERT and pLXSNneo detached through PCR amplification, gel electrophoresis with Ligation Mix connections Digestion products, build pLXSNneo-hTERT recons, and conversion DH5a competent cells are green to expand, purify simultaneously picking resistant to ammonia benzyl Mycin bacterium colony extracts plasmid, and T lymphocyte of the in vitro passage in logarithmic growth is imported with lipofection, make recon with it is thin The DNA of born of the same parents is integrated, and expands the clone for the positive recombinant that culture is screened through G418, screening cellular morphology, growth curve, dyeing It is body caryogram, the experiment of nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mRNA expression products, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic and with the same or similar person of primary cell as hTERT immortality The CD4+T cells of change.

It is in the SV40 methods for immortalizing CD4+T cells：It is connected simultaneously through BamHI digestions with T4DNA ligases The SV40LTag DNA of pcDNA3.1 (-) DNA and PCR amplification, agarose gel electrophoresis separation, build SV40LTag- PcDNA3.1 (-) recombinant plasmid, conversion DH5a competent escherichia coli cells are amp-R to expand, purify simultaneously picking Bacterium colony extracts plasmid, and the T lymphocytes of in vitro culture are imported with lipofection, and the DNA of recon and cell is made to integrate, with The cell containing positive recombinant of G418 screenings passes on, expands culture, screening cellular morphology, cell growth curve, chromosome The big T genetic tests of SV40, mRNA expression products measurement and determined dna sequence in caryogram, the experiment of nude mice tumorigenesis, transfectional cell DNA As a result the CD4+T cells for meeting immortalized cells characteristic and being immortalized as SV40 with the same or similar person of primary cell.

1. immortalizing the specific method of CD4+T cells with hTERT

(I) extraction of hTERT：(i) digestion pClneo-hTERT：HTERT be located at the EcoRI of plasmid pClneo-hTERT with Between the sites SalI, pLXSNneo vector multiple cloning sites (MCS) are containing EcoRI and XhoI restriction enzyme sites.(ii) hTERT electrophoresis： With 10% agarose gel electrophoresis, hTERT segments are detached.(iii) hTERT purifying and recycling：By purpose hTERT segments from gel Middle separation is purified.(II) connection of hTERT and pLXSNneo carriers：Build pLXSNneo-hTERT recons.(III) Purifying, amplification, identification pLXSNneo-hTERT recons.(IV) CD4+T cells：From " preparation of (2) CD4+T cells " of the invention It is prepared by sampling.(V) CD4+T cells preculture：By above-mentioned cell inoculation in containing 5~10nmol/L insulin, 20% fetal calf serum 1640 liquid of RPMI in, or be inoculated in the low sugar DMEM cell culture mediums containing 20% fetal calf serum, 5~10nmol/L insulin In, generally it is inoculated in culture medium (the 1.6%1M HEPES buffer solutions of 3ml Fresh；15% fetal calf serum (FBS)；1% is green Mycin and streptomysin；PRMI 1640 is supplied to 100%), being placed in 37 DEG C, in volume fraction 5%CO2 incubators, is cultivated 1-2 days, Centrifugation, removes supernatant, spare.(VI) pLXSNneo-hTERT recons import T lymphocytes and expand culture：It is micro- in 1.5ml Following solutions are prepared in amount centrifuge tube：PLXSNneo-hTERT recons are dissolved in 100 μ l serum-free mediums by pipe A；Pipe B, 20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums, by pipe A and pipe B mixings, stand 45min at room temperature, are used Serum-free medium washs above-mentioned T lymphocytes 2 times.It is added in Lipofectamine-pLXSNneo-hTERT mixtures 1ml serum-free mediums, gently mixing, is added dropwise in above-mentioned T lymphocytes, and 1ml serum-free mediums are added, and (fetal calf serum is dense Degree is 20ml/L), in CO₂Transfection liquid is sucked out in incubator culture 10h, and adding 4ml complete culture solutions, (fetal calf serum is a concentration of 20%), continue to cultivate 20h, discard culture solution, replace a concentration of 400mg^。L^-1G418 culture solutions continue to cultivate, selected after 8 days Living cells is made after expanding culture, then increases G418 concentration to 800mg^。L^-1, growth will be stablized in the G418 environment of high concentration Cell continue amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte significantly increases, and clustering phenomena occurs. If cell increases, slow or cell density is low or medium pH value is in acidity, and half amount culture solution is sucked out, carries out equivalent oil changing.When Total amount is transferred to when reaching 14ml in 75ml culture bottles, and 5-10ml fresh cultures were added per 2-3 weeks.Cell culture was to 9-10 weeks (the about the 75th generation), still in exponential phase, i.e., it is in multiplication relation that cell, which is accelerated with incubation time, and dead cell is less than 10% (judges the increase situation of cell quantity by the scale of reading culture vessel；Differentiate dead cell by trypan blue staining And living cells.Because of normal living cells, after birth structural integrity can be repelled, and make trypan blue that can not enter intracellular；And it loses Active cell, the permeability of after birth increase, and can dye blue by trypan blue, can determine whether for cell it is dead.Method is every Week draws a certain amount of suspension culture, mixes postposition room temperature 5~10 minutes with Trypan Blue agent, it is thin that cell is then made Piece counts 1000 total number of cells, calculates the percentage of the dead cell and non-staining living cells of coloring under the microscope).This Afterwards with the extension of the increase and incubation time of culture algebraically, the increase of cell quantity is slack-off, dead cell is more and more, until Cell is not further added by, or even dissolving, reduction, all death.It when total amount reaches 45ml, sets in 50ml centrifuge tubes, centrifugation 1500 Turn, 10 minutes, after abandoning supernatant, addition 3ml freezing medias (5% dimethyl sub-maple (dimethyl sufoxide), 95% FBS) mixing, at cell suspending liquid, (cell concentration is about 10⁵/ml).Cryopreservation tube dispenses, and 1ml/ pipes set -20 DEG C of 2h, then set -70 Then DEG C 2h freezes in -196 DEG C of liquid nitrogen (or immediately under zero setting moved into liquid nitrogen container after 80 degree, 1-2 hours).(VII) immortal Change the identification of CD4+T characteristics of cell biology：(i) cellular morphology is observed：It can be seen that lymphocyte significantly increases, it is existing to there is aggregation As having lymphocyte blastogenesis feature.(ii) cell growth curve is observed：Using incubation time as horizontal axis, cell quantity is the longitudinal axis (logarithm), be depicted in after curve is made on semi-logarithmic scale the cell growth curve, immortalized cell line is in typical " S " feature or " arched roof " are formed；(iii) chromosome is checked：By analyzing karyotype, if karyotype is diploid " 46, XX " or " 46, XY " then illustrate that vicious transformation does not occur for the cell line.(iv) Flow cytometry：Detected for the 19th generation The cell proportion for synthesizing, dividing in cell line, if its proliferative capacity is not obviously than building the normal cell for being enhancing, explanation is The result that hTERT is integrated, expressed.(vii) determined dna sequence：Routinely sequenator detects, and shows hTERT gene orders.(v) HTERT is detected in transfectional cell DNA：Such as with Immunohistochemical detection, the visible a large amount of palm fibres of the interior dyeing of nucleus of hTERT transfections Coloured particles show that hTERT has been integrated into the cell；(vi) mRNA expression products measure：The pcr amplification product of 100 μ l systems is taken, With gel reclaims kit (Takara, Japan) recovery product, takes 2 μ l DNA solutions to dilute 100 times, survey concentration, remaining DNA And primer each 10 μ l in upstream and downstream are sequenced.(VIII) hTERT mediates CD4+T cell banks：It screens and continues passage, expand training Support and meet immortalized cells characteristic after above-mentioned identification and the cell same or similar with primary cell, take growth conditions it is good, The cell of different generations in exponential phase is centrifuged (1 200r/min, 6min), with freezing containing dimethyl sulfoxide Cell is resuspended in 0.5~1ml of liquid, and cell density is 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；—70 DEG C, overnight, enter -196 DEG C of liquid nitrogen cryopreservations, it is spare to build the immortalization CD4+T cell banks that biological characteristics are stablized in this way.

2. immortalizing the specific method of CD4+T cells with SV40

(I) extraction of SV40 large T antigens DNA：(i) SV40DNA digestions：Contain large T antigen gene from commercially available purchase SV40 freeze dried powders are dissolved in appropriate TE buffer solutions, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O adds restriction enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C are heated 15min, and inactivator is added 5uL electrophoresis sample loading buffers and terminates Reaction is in case electrophoresis.(ii) SV40DNA electrophoresis：Electrophoresis grade agarose is taken to be made into 10% Ago-Gel with electrophoretic buffer, Electrophoresis under the voltage of 1-10V/cm gels, isolation of DNA fragments.(iii) about 2600bp SV40 large T antigens are detached from agarose DNA.(II) connection of SV40LT and pcDNA3.1 carriers：Build SV40T/pcDNA3.1 recons.(III) expand, detach with Identify SV40T/pcDNA3.1 recons：(i) competent E.coli is prepared.(ii) it screens, expand and extraction recon.(i Ii) the identification of recon：The above-mentioned DNA (containing recon SV40T/pcDNA3.1) extracted from competent E.coli, ibid Method carries out digestion with restriction enzyme BamH I, the identification of 10g/L agarose gel electrophoresis, obtain size about 2600bp and 2 bands of 5600bp, the former meets the size of SV40T segments in GenBank.(IV) CD4+T cells：From the present invention " (2) CD4+ It is prepared by the preparation of T cell " sampling.(V) CD4+T cells preculture：By above-mentioned cell inoculation in containing 5~10nmol/L insulin, In 1640 liquid of RPMI of 20% fetal calf serum, or it is inoculated in the low sugar containing 20% fetal calf serum, 5~10nmol/L insulin In DMEM cell culture mediums, it is generally inoculated in culture medium (the 1.6%1M HEPES buffer solutions of 3ml Fresh；15% tire ox blood (FBS) clearly；1% penicillin and streptomysin；PRMI 1640 is supplied to 100%), being placed in 37 DEG C, volume fraction 5%CO2 incubators It is interior, it cultivates 1-2 days, supernatant is removed in centrifugation, spare.(VI) importing and expansion culture of SV40T/pcDNA3.1：It is micro- in 1.5ml Following solutions are prepared in amount centrifuge tube：SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free medium (fetal calf serum concentration by pipe A For 20ml/L) in；20 μ l Lipofectamine are dissolved in 80 μ l serum-free mediums by pipe B, by pipe A and pipe B mixings, room The underlying 45min of temperature.Above-mentioned T lymphocytes are washed with serum-free medium 2 times.In Lipofectamine-SV40T/pcDNA3.1 1ml serum-free mediums are added in mixture, gently mixing, then are added dropwise in above-mentioned T lymphocytes, 1ml is then added without blood Clear culture solution (a concentration of 20ml/L of fetal calf serum), in CO₂Incubator culture 10h is sucked out transfection liquid, adds 4ml complete culture solutions (a concentration of 20%) of fetal calf serum continues to cultivate 20h, discards culture solution, replace a concentration of 400mg^。L^-1G418 culture solutions after Continuous culture selects living cells to make after expanding culture, then increases G418 concentration to 800mg after 8 days^。L^-1, will be in high concentration The cell for stablizing growth in G418 environment continues amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte is apparent Increase, clustering phenomena occurs.If cell increases, slow or cell density is low or medium pH value is in acidity, and half amount culture is sucked out Liquid carries out equivalent oil changing.It is transferred to when total amount reaches 14ml in 75ml culture bottles, 5-10ml fresh cultureds was added per 2-3 weeks Base.In about 6-8 weeks (the about the 55th generation) of cell culture, still in logarithmic growth, i.e. incubation time is accelerated with cell and is closed in multiplication System, dead cell by trypan blue staining less than 10% (differentiating dead cell and living cells).Hereafter with the increasing of culture algebraically The extension of incubation time is summed it up, the increase of cell quantity is slack-off, dead cell is more and more, until cell is not further added by, even Dissolving reduces, is all dead.It when total amount reaches 45ml, sets in 50ml centrifuge tubes, centrifuges 1500 turns, 10 minutes, abandon supernatant, 3ml freezing medias (5% dimethyl sub-maple (dimethyl sufoxide), 95%FBS) mixing is added, at cell suspending liquid (cell concentration is about 10⁵/ml).Cryopreservation tube dispenses, and 1ml/ pipes are set -20 DEG C of 2h, then set -70 DEG C of 2h, then frozen at -196 DEG C In liquid nitrogen (or immediately under zero setting after 80 degree, 1-2 hours move into liquid nitrogen container in).(VII) CD4+T characteristics of cell biology is immortalized Identification：Routinely identification method carries out.(VIII) SV40LT gene mediateds CD4+T cell banks：It screens and continues passage, expand Culture meets immortalized cells characteristic and the cell same or similar with primary cell after above-mentioned identification, takes growth conditions good The cell of different generations good, in exponential phase, is centrifuged (1 200r/min, 6min), with containing dimethyl sulfoxide Cell is resuspended in 0.5~1ml of frozen stock solution, and cell density is 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enter -196 DEG C of liquid nitrogen cryopreservations, it is spare to build the CD4+T cell banks that biological characteristics are stablized in this way.

(4) with the preparation of CD4+T cell identity function particles：It can be by CD4 molecules, the CD4 molecules of genetic recombination and similar The molecule of function, which is fixed on by conventional chemical coupling, crosslinking, affine absorption etc. on carrier, is made for being coated with CD4 molecules Grain, or directly take intimate particle and substitute CD4+ cell applications.It is thin that the CD4+T cells of the present invention represent other CD4+ Born of the same parents, including prepared with other methods and immortalize CD4+T cells.

(3) preparation of HIV-1gp120 antibody

Antibody according to the present invention can entrust professional businessman to prepare, or directly be bought from professional businessman, as Shanghai is auspicious The neat units such as bio tech ltd and Shanghai Linc-Bio Science Co., Ltd. all specialize in HIV-1gp120 antibody, The preparation and sale of the various antibody such as gp41 antibody and goat anti-human igg.Method includes that hybridoma technology prepares monoclonal antibody, EB Virus Transformation technology prepares monoclonal antibody, hybridoma technology is combined with Epstein-Barr virus transformation technology and prepares monoclonal antibody and base Because of engineered antibody, specifically it is listed below.

1, it converts to be combined with hybridoma technology using lymphocyte Epstein-Barr virus and prepares HIV-1gp120 monoclonal antibodies

Specimen origin have it is following it is several by way of：Take the lymphocyte strain frozen in Infectious Diseases Lab sample database (through inactivation The immune lymphocytes with EBV transfections of HIV)；The fresh White Blood Cells Concentrate in blood station is bought, inactivation HIV infection strain was then carried out and exempts from The lymphocyte of epidemic disease；It is derived from the cord blood lymphocytes cell (immune through inactivating HIV) preserved as scientific research；Directly it is derived from HIV-1 The peripheral blood lymphocytes (being used for itself) of the infected itself, it is thin to detach single core using Histopaque lymphocyte separation mediums Born of the same parents (PBMC), adjust a concentration of 2x 10⁶After suitable Epstein-Barr virus (EBV) stoste is added, be placed in 370C, 5%C02 overnight incubations, B cell to be hybridized is prepared, with the positive hole of ELISA method screening AntiHIV1 RT activity-l outer membrane proteins (gpl20), metastatic cells to 24 orifice plates Continue culture 2 weeks, repeats to measure anti-gpl20 confirmations positive with ELISA method, continuously clone secondary and large amplification culture.It will Positive cell strain mixes (3 with the heterogeneous myeloma cell of people mouse (being bought by Zhejiang University's siberian crabapple)：1) after, 1ml50% is added PEG makes the two merge, and cell is then resuspended and cultivated liquid in IMDM culture solutions, Peritoneal Cells of Mice is added within second day (by Zhejiang Jiang great Xue siberian crabapples are bought) it is used as trophocyte, continue to screen anti-gpl20 antibody with ELISA after cultivating 3 weeks, selects strong positive Hole hybrid tumor cell amplification culture, and repeatedly clone is carried out until obtaining stable cell line, with this cell line culture, prepare HIV-1 antibody, using ELISA detection kit, by specification operation is measured the Ig subclass of antibody, and is surveyed with conventional ELISA method The potency and specificity for determining antibody select the high antibody of high specificity, potency.

2, antibody is prepared using genetic recombination HIV-1gp120 combination hybridoma technologies

(1) reagent and recombinant antigen：It is related to reagent：HIV-1gp120 genetic fragments, HIV-1gp120 antigens, HIV antigens Nitrocellulose membrane item provides I restriction endonuclease Xho of BamH, I restriction endonucleases, nucleic acid coprecipitator, T4DNA by Beijing Wan Tai Pharma Inc.s Ligase is purchased from precious biological Co., Ltd；Liagen plastic recovery kits are purchased from QIAquick companies；1640 dry powder cultures of RPMI Base is purchased from Gibco companies；Top grade newborn bovine serum is purchased from bright marine growth Engineering Co., Ltd；SupersignalWest Pico Trial Kit, TMB Substrate Kit are purchased from Pierce companies；Mouse Ig subclass detection kit, freund adjuvant and PEG, Hy-poxanthine, Thymidine and Aminoptem are purchased from Sigma companies.HIV- antibody diagnosing reagent kits are purchased from Shanghai section Magnificent biology Co., Ltd, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) label are purchased from doctor's moral Co., Ltd. Construction of recombinant plasmid and identification：Vector plasmid PEGX-4T-2 BamH I, I digestions of Xho, T4DNA Ligase connection gp120 bases Because of segment, recombinant plasmid transformed enters E.colistrain XL1 blue, is sequenced.The induced expression of recombinant protein and identification：Recombination Plasmid is transformed into XL1-Blue Escherichia coli, 25 DEG C under the effect of IPTG derivants, 190r/min concussions, overnight, 4 000r/ Min centrifuges 10min, collects bacterium, SDS-PAGE testing goal protein expression situations.Fusion protein purification and identification：Expression production Object is collected by centrifugation precipitation and is hanged through PBS, after cracking bacterium with 30W Ultrasonic Pulverization instrument, supernatant filtering is collected by centrifugation, filtrate is used AKTA PURIFYER100 protein purifications instrument, GST column purifications obtain fusion protein GST-HIV, and concentration centrifuge tube is concentrated, S21 type biology spectrophotometer measurement concentration, SDS-PAGE identify purifying protein.

(2) animal immune：6 week old of BALB/c mouse, female, take mouse vein blood by 4 before immune, separation serum, which stays, to be done Negative serum.The GST-HIV fusion proteins of 50-100 μ g are mixed with isometric Freund's complete adjuvant, pneumoretroperitoneum is emulsified and injects and exempt from Epidemic disease mouse.After initial immunity, every 2 weeks using incomplete Freund's adjuvant and booster immunization mouse after fusion protein emulsification, it is immunized Dosage and approach are the same, repeat to be immunized 2-3 times, the GST-HIV that 50-100 μ g are directly injected intraperitoneally in last time booster immunization melts Hop protein.

(3) foundation of Detection of Monoclonal Antibody：The immune latter all tail vein bloods of third time, positive serum is determined with square formation method Best effort concentration and GST-HIV fusion proteins best peridium concentration.

(4) cell fusion:Myeloma cell prepares：It is thin that fusion the last week takes out the myeloma that a pipe freezes out of liquid nitrogen container Born of the same parents are immediately placed in hot water defrosting.Appropriate complete culture solution is added after thawing, 1000r/min centrifuges 3min；It is repeated 1 times.It will precipitation Object moves into Tissue Culture Flask, adds DMEM culture solutions, sets CO2 incubator cultures, carries out within 3-4 days once passing on or expanding culture, Fusion adjusts cell state in first 24 hours, ensure that the preceding cellular morphology of fusion is good, growth is vigorous.Appropriate pancreatin is used before fusion It is collected, is added in appropriate basal medium to centrifuge tube using centrifuge tube after digestion, 1000r/min is centrifuged after gently beaing mixing 5-10min, repeated washing cell 2 times.Splenocyte prepares：Before fusion, a Balb/c mouse is taken, eyeball is won and takes blood, bloodletting Complete post-tensioning neck is put to death, and is soaked in 75% ethyl alcohol.It takes out layback and puts and be fixed on dissection plate, spleen is taken under gnotobasis, it will Spleen moves into plate.Then 1640 basal mediums of 10mL RPMI are added in plate, are repeatedly extruded with flat mouth tweezers broken Afterwards, it aspirates piping and druming repeatedly using asepsis injector, single cell suspension is made.1000r/min centrifuges 10min after counting viable count, Basal medium is added and adjusts total cell number to 1 × 10⁸~2 × 10⁸For cell fusion.Cell fusion：By splenocyte and marrow Oncocyte is with 10:1-5:1 ratio is added in centrifuge tube, is mixed evenly, and 1000r/min centrifuges 5min, discards supernatant, gently strikes It beats tube bottom to cell grainless to precipitate, be repeated 2 times.Gently rotation preheats centrifuge tube, sterile item after taking-up in 37 DEG C of water-baths The 50%PEG3000 of 1000 μ L of preheating is added drop-wise to along tube wall in fusion pipe in 60s under part while gently rotating centrifugal pipe, The basal medium of the 25mL of preheating is also added drop-wise to along tube wall in centrifuge tube in 3-5min later, it is light during addition Lightly rotating centrifugal pipe is then allowed to stand in 37 DEG C of water-bath 10min, 1000r/m centrifugation 5min, discards supernatant, 50mL HA T are added Culture medium.It is inoculated into 96 well culture plates after appropriate mixing, is placed in 37 DEG C, is cultivated in 5% CO2 incubators.

(5) screening of positive clone strain:Cell growth status in 96 well culture plates is observed, it is thin to only have hybridoma after 7-10 days Born of the same parents can grow division, discard HAT culture mediums at this time, replace complete medium.Cell clone growth area reaches 1/10 thin When hilum, culture supernatant is gone, positive hybridoma cell clone is screened by the monoclonal antibody screening technique established before.Using improved Limiting dilution assay --- gradient limiting dilution assay continuously carries out 3-4 wheels to the positive cell hole that indirect ELISA preliminary screening goes out Subclone.Selection has the culture hole of the good positive hybridoma cell strain of growth conditions, and cell strain growth is marked under microscope Position, size, drawing cell clone in the position of mark using sterile pipette tips has to new in the culture hole of complete medium, so Doubling dilution to hole is counted below successively afterwards, and 37 DEG C, 5%CO2 incubators are interior to be cultivated one week or so, microscopically observation cell growth Situation takes cells and supernatant to carry out antibody inspection side when cell clone is covered with to 1/10 or more hole floor space.To testing result Repeat next round dilution culture for the cell clone in positive culture hole, 2-3 wheels is repeated, after detecting supernatant titer plateaus It takes out, is transferred to culture bottle mass propgation.

(6) preservation of hybridoma cell strain and secondary culture:It preserves and recovers:Preserve preceding 12 hour adjustment cell growth shape State.Take one bottle of growth vigorous, cell suspension is made in the good cell of form after appropriate digestion, 1000r/m centrifuges 5min, goes Clear liquid, flicking tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, dispense cell cryopreservation tube, 1mL/ is managed, and cryopreservation tube is put into liquid nitrogen container after taking-up and saves backup by -70 DEG C of refrigerator overnights.40 DEG C of left sides are got out before recovery Right hot water, cryopreservation tube is carefully taken out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, after defrosting 1000r/m centrifuges 5min, opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting is washed with complete culture solution It washs once, then centrifuges 5min in 1000r/m, discard supernatant, cell precipitation moves into training after being gently resuspended using complete culture solution It supports in bottle, sets 37 DEG C, cultivated in 5%CO2 incubators.Secondary culture positive hybridoma cell continuously passed for 10 generations, using indirect 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 culture, dilute by basal medium It is 1 × 10 to release cell number⁷/mL.Mouse peritoneal injects 0.2mL/ only, and mouse ascites production is observed after injection, waits for that abdomen is bright Aobvious distension rises, and punctures abdominal cavity with syringe needle, centrifuge tube collects ascites.Acquisition finishes, and appropriate basal medium is injected to mouse peritoneal, Every 2-3 days, ascites is taken with method.The ascites being collected into, 10000r/m centrifuge 5min, supernatant are taken to dispense, -20 DEG C of preservations.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody:Specificity:Weston-Blotting tests reference《Molecular cloning》Method carries out, with Semidry method transfers.

(9) HIV-1gp120 monoclonal antibodies are applied after further refining and (professional businessman can be entrusted to complete).

(4) preparation of HIV-1gp41 antibody (with the preparation of HIV-1gp120 antibody)

(5) preparation of goat-anti people-Ig

It is antigen by made HIV-1gp120 antibody and gp41 antibody, immune sheep prepares antibody.

1, experimental animal：It is immune that is selected with experimental animal is the white goat of animal institute of Zhejiang University hybridization, 30 jin of weight The between twenty and fifty ewe two of health of left and right, is smeared at the back of animal with dyestuff before immune, makes specific label.Using doing as everybody else does The feeding manner of stable breeding ensures that sheep has to suitable movement daily, and it is left to move general half an hour at dusk every time for run duration The right side, is conducive to healthy and strong in this way, avoids sheep overfertilization, increases the immunity of body, and drinking-water is sufficient, and give suitable fine fodder, Green grass, corn, wheat bran, wheat, vitamin etc. keep its nutrition balanced.Often check experiment sheep health status.

2, HIV-1gp120 antibody and gp41 antibody are antigen：HIV-1gp120 antibody and gp41 antibody prepared by the present invention (IgG) concentration is respectively 2.5mg/mL (can be provided by businessman), and 0.1mL antigens, 1.9mL sterile PBS, 2mL are mixed not before being inoculated with It is spare that immunogen emulsion is made in family name's (or incomplete) adjuvant completely.

3, goat is immune：Choose two goats, be labeled as goat A, goat B, antigen inoculation (HIV-1gp120 antibody and HIV-1gp41 antibody).Immune position is front and back groin, often locates 2 point injections of groin point, a total of 8 injection points.Note It is to be subcutaneously injected to penetrate mode, and every goat per injection 4mL immunogene contains 250 μ g antigens；Each injection point injecting immune is former 0.5mL, containing about 32 μ g antigens.Preceding two goats are immunized to draw blood respectively 10mL, are labeled as 0dP1, -20 DEG C of preservations；It is immune for the first time I.e. 1, exempt from immunogene：The sterile PBS+ Freund's complete adjuvants 2mL (CFA) of 0.1mL antigens+1.9mL；Draw blood 10mL after 7 days immune, Serum is detached, 7dP1 is labeled as, ELISA detects serum titer, -20 DEG C of preservations of remaining serum.Start within 3~4 weeks to be immunized for second, Two immunogenes：The sterile PBS+2mL incomplete Freund's adjuvants (IFA) of 0.1mL antigens+1.9mL, draw blood 10mL after 7 days immune, separation Serum, is labeled as 7dP2, and ELISA detects serum titer, -20 DEG C of preservations of remaining serum.Third time immunization time be 6-8 weeks after, Three exempt from immunogene：The sterile PBS+2mL incomplete Freund's adjuvants (IFA) of 0.1mL antigens+1.9mL, draw blood 10mL after 7 days immune, point From serum, it is labeled as 7dP3, ELISA detects serum titer, -20 DEG C of preservations of remaining serum.If serum titer does not reach at this time 1：10⁶More than, then it needs to exempt from again primary；If serum titer is up to 10⁶Do not have to then be immunized again above, draw blood week about later 50mL detaches serum, -20 DEG C of preservations.

4, prepared by serum：It is general that rear can be detected in sheep jugular vein blood collection for 7~10 days is immunized every time.By assistant Baoding Animal makes it keep standing position, and after neck cropping, sterile cotton balls cleaning disinfection, searches out the blood sampling of jugular vein hand syringes, Syringe position is fixed and takes blood 5-10mL.Bioactivity is carried out after isolating serum.7~10 days after the third immunization, warp It once can use blood 30-50mL after bioactivity qualification.Serum is aseptically detached, in plate or triangular flask to be collected in After blood clotting, 37 are put into after clot and bottle wall stripping with sterile dropper in gnotobasis (such as superclean bench) DEG C, 1~2h is put into 4 DEG C overnight after taking out, serum is made fully to be precipitated and (cannot freeze, otherwise generate haemolysis), through centrifugation point Go out serum, puts low temperature refrigerator preservation into.It must again dispense and save backup after signing is qualified before.

5, the measurement of antiserum titre：Antiserum titre using ELISA method measure, specifically by kit specification into Row.

Two, the preparation of AIDS blood purification

(1) gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti in mixed liquor Gp120 and gp41 antibody is fully tied, but the surplus free gp120 and gp41 antibody for having enough high titres, then will be contained There are the mixed antibody of mating type and sequestered gp120 antibody and mixed antibody containing mating type and sequestered gp41 antibody again It is secondary with etc. potency mix, be made into final mixed antibody.

(2) strain of hybridoma macrophage and CD4+T cell strains are cleaned with sterile saline, 1000r/min centrifugations are cleaned Centrifugation again afterwards is 1 by the ratio between the strain of hybridoma macrophage and CD4+T cell strains：0.5~3 ratio takes sedimentation cell, It is assemblied in hydrostatic column made of high-biocompatibility material, blood purification cell column is made；

(3) agarose is mixed after 100 DEG C dissolve with a certain amount of physiological saline, keeps the temperature at 39~41 DEG C, step is added It is rapid 1 obtain antibody mixture, wherein the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody Titre is 1：700, the mass fraction of agarose is 1.1%.

(4) product for preparing step 3 is with volume ratio 1:4 are added in the lymphocyte depletion column of step 1 preparation, are cooled to After semi-solid gel, the preparation of bottom purifying layer is completed.

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

(6) sedimentation cell is added on second layer gel, the agar antibody mixture at 39~41 DEG C is then added, In, in agar antibody mixture, the titre of antibody is 1：300, the mass fraction of agarose is 0.9%, agar antibody mixture Volume ratio with sedimentation cell is 1:4；After being cooled to semi-solid gel, the preparation of third layer purifying layer is completed.

(7) sedimentation cell is added on third layer gel, the agar antibody mixture at 39~41 DEG C is then added, In, in agar antibody mixture, the titre of antibody is 1：200, the mass fraction of agarose is 0.8%, agar antibody mixture Volume ratio with sedimentation cell is 1:4；After being cooled to semi-solid gel, the preparation of the 4th layer of purifying layer is completed.

(8) sedimentation cell is added on the 4th layer of gel, the agar antibody mixture at 39~41 DEG C is then added, In, in agar antibody mixture, the titre of antibody is 1：100, the mass fraction of agarose is 0.7%, agar antibody mixture Volume ratio with sedimentation cell is 1:4；After being cooled to semi-solid gel, layer 5 purifying layer, i.e. top layer purifying layer are completed It prepares, is purified column.

The specification and material of blood purification

The shell of blood purification generally selects small bottom diameter, the top big cylinder of diameter or rectangular, infundibulate, volume 200 ~300ml, inlet and outlet are equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh 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, to stop 120 nanometers of inhibition of HIV or bigger Bacterium；The cell strainer that mesh number is 100 mesh (being equivalent to 4 microns) is arranged in liquid outlet, to stop the cell that may be filtered out； It is equipped with buffering area between liquid entrance and mesh screen, is conducive to the stability of system circulation.

Blood purification selects the high molecular material of acrylate etc, it is desirable that good biocompatibility, hardly activation are mended Body, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.Can by covalently, The methods of grafting, polymerization improve the structure of material, the inhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidative stress Influence, to improve biocompatibility, reduce complication generation.Add hydrophilic gel in absorber inner surface, by 2 methyl-props Alkene acyloxyethyl phosphocholine-butyl methacrylate is solidificated in cellulose acetate film, by controlling wet-spinning procedure, is produced CA/PMB30, CA/PMB80 and CA/PMB30-80 have higher blood and cell compatibility.There is anticoagulation by certain Substance be solidificated on the material of carrier or absorber inner surface, can inhibit blood clotting, improve biocompatibility, can also reduce Heparin dosage, and be possible to realize no-rod tractor.The covalent immobilisation linoleic acid film on cellulose acetate film, or will be covalently bound to poly- The linoleic acid of acrylic acid is grafted onto polysulfones film surface, can there is better histocompatbility and anticoagulant effect.

The blood purification of the present invention coordinates blood separator and plasma separator to use under normal circumstances, blood separator For screening out with HIV infection cell existing for multinucleate giant cell or many cells condensate state by volume size, that is, it is used to remove Endoglobar HIV；Plasma separator is for detaching mononuclear blood cell and blood plasma；Blood purification is used in adsorbed plasma HIV.The blood of purification filters off multinucleate giant cell after blood separator detaches, and small volume haemocyte and blood plasma enter blood Starch separator；Plasma separator detaches small volume haemocyte and blood plasma, the blood plasma isolated through two or two with After upper parallel connection clarifier purification, after being mixed with the small volume that plasma separator is isolated, purification is completed.

Three, the verification of AIDS blood purification effect

As shown in Figure 1, by the present invention AIDS blood purification be applied to AIDS blood purification, by its with it is existing Blood separator is connected with plasma separator, and for convenience of operating, additional member, including blood pump, liver are provided 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 such as survey forms.Wherein, (1) blood pump (Blood Pump)：For pushing blood circulation to maintain the suitable of blood purification treatment Profit carries out, and usual blood pump part often has rotary test speed function, to monitor the blood circumstance of patient, therefore blood pump runner with it is recessed Separation setting is accurate, and needs often adjustment, the case where according to bloody path pump line, spacing is generally set as 3.2~ 3.3mm, can not be too loose, and no it will cause blood flow detections to be not allowed；Also can not be too tight, it is no that it will cause pipe breakages.(2) heparin pump (Heparin Pump)：Heparin pump is equivalent to the micro-injection pump clinically applied, to continue to sieving pipeline (patient's blood Liquid) in injecting heparin, contacted with air since the blood of patient recycles in vitro, be easy to happen blood coagulation phenomenon, use heparin pump Anticoagulative it can occur.(3) sound pulse pressure monitors：Arterial blood pressure monitoring is mainly to dynamic monitoring blood separator micropore Stopping state, in addition monitoring extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will drop It is low；When having blood coagulation, thrombosis, especially separator blockage of the micro orifice, angiosthenia will increase；Vein pressure monitoring is used for monitoring The pressure of pipeline blood reflux, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow is insufficient and venous return syringe needle When falling off, vein pressure will decline, and when if the distortion of bloody path return duct blocks or flows back, syringe needle blocks, vein pressure will rise It is high.(4) air monitering (Air Detector)：For monitoring the air bubble of blood pathway, the general original for using ultrasonic listening Reason, in order to avoid air embolism occurs for patient and is arranged.When having monitored air bubble, detecting system can drive artery and vein Bloody path folder carrys out blocking blood flow, prevents dangerous generation.

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

1, it installs：Such as Fig. 1, with sterile working connecting components, including blood separator, plasma separator, blood purification Device and each circulation line.

2, it is vented：With sterile saline filling liquid separator, clarifier and each circulation line, separator, clarifier are excluded And its gas, bubble in circulation line, it goes through, confirms without being used after gas, bubble.

3, lead to liquid：The arteries that arterial blood line pipe 1 is connected to AIDS patient, goes through exhaust again in operation Completely whether, whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.

4, anti-freezing：Anti-coagulants (heparin) is injected into liquid stream from heparin pump 2, is 2500 ∪ or 20~30 ∪/㎏ for the first time.

5, start：One end of arterial blood line pipe 1 is connected with arteries, venous line 15 is connected into vein blood vessel, Then heparin pump 2 is opened, blood flow is 100~150ml/min, such as Fig. 1, when arterial blood is through arterial blood line pipe 1, heparin and liver When element pump 2 enters blood separator 3, the large volume multinucleate giant cell formed by HIV infection is delayed at blood separator 3 Interior, mononuclear blood cell and blood plasma flow into plasma separator 8, the blood of separation through blood outlet 4, blood pump 6 and circulation line 7 successively Slurry flows into clarifier 11 open at this time through blood plasma pump 9 and blood vessel 10 successively, blood plasma to be full of, about 10 minutes, begins paying out Blood plasma is flowed out through export pipeline 13, synchronous that blood plasma is perfused to clarifier 12, when the blood plasma in clarifier 11 has nearly flowed, then Secondary to start that blood plasma is perfused, clarifier 12 begins paying out blood plasma at this time, and two clarifiers 11 and 12 in parallel are alternately.

As shown in figure 3, when the blood plasma containing HIV101 enters clarifier, HIV101 therein is fixed on HIV respectively Antibody 102, macrophage 104, CD4+T cells 106 are combined into antigen antibody complex 103, macrophage phagosome 105, CD4 + T cell conjugate 107, the HIV after being combined no longer moves down, and the large volume of HIV not in addition being combined is again by because dense Spend higher and the smaller bottom agar gel molecular of micropore sieves microporous barrier at 109.By the purification blood plasma after absorption HIV through figure The individual cells that export pipeline 13 is detached with plasma separator 8 shown in 1 converge after export pipeline 14 converges through venous line 15 Fluid circulation.So purification blood, removing HIV, until the plasma circulation amount (being usually 9L) being previously set, just declaration is tied for treatment Beam.Entire therapeutic process is controlled by computer, and can detect working condition at any time, easy to use, automation and safety.

1, blood separator filters out the verification of HIV infection cell effect

The present inventor's basic skills according to the invention has done following simple confirmatory experiment：Take Disease Control and Prevention Center and infection The anticoagulated whole blood several pieces for AIDS (AIDS) patient made a definite diagnosis that sick laboratory biological sample database preserves, part phase of fetching respectively Anticoagulated whole blood with abo blood group is mixed into 5, keeps blood volume sufficiently large, then entrusts hospital center of Zhejiang Province blood station proportionately The blood component separation method of part blood transfusion, isolates leucocyte, red blood cell, blood plasma through blood component piece-rate system, takes leucocyte Composition routinely centrifugation, suction abandon supernatant, with suitable physiological saline suspension leukocyte cell pellet, proper ratio are then added Gp120 antibody (the Shanghai bio tech ltd Guang Rui), 37 DEG C of mixing postposition reacts 5 minutes, then with aperture be 20~ The blood component piece-rate system of 30um isolates the leucocyte (be known as big leucocyte) of large volume, again to the leucocyte filtrate of filtration The leucocyte (leucocyte in being known as) of medium volume is further isolated with the blood component piece-rate system that aperture is 15~25um, Leucocyte in filtrate is the leucocyte (being known as small white blood cells) of small size, collects large, medium and small leucocyte separation suspension respectively, Conventional centrifugal precipitates, and supernatant is abandoned in suction, and the large, medium and small leukocyte cell pellet of draws equal amounts, conventional method are distinguished with quantitative liquid shifter (machinery or cell pyrolysis liquid) lytic cell (such as with lysate of the same race, needing dosage equal), takes supernatant, then after centrifugation According to HIV-1p24 antigen detection kits (bio tech ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known dense Spend the p24 antigen conducts of 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml Control, minimum detection limit are less than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity 0.5pg/ml~80pg/ml, 15min Interior 450nm measures absorbance (OD), and blank control calibration object absorbance value is not higher than not higher than 0.050,0pg absorbance values 0.100,1000pg/ml absorbance is not less than 1.000, is considered positive as absorbance ＞ 0.12, testing result (table 1) is said Bright, the HIV-p24 contents in the leucocyte of AIDS patient different volumes size are different, the HIV- in large, medium and small leucocyte The average content of p24 is respectively 275.0pg/ml, 196.0pg/ml, 126.4pg/ml, wherein HIV-p24 in large and small leucocyte Average content differs 148.6pg/ml, reduces 54.4%；The total content of HIV-P24 is respectively in large, medium and small leucocyte 1375.0pg/ml, 979.9pg/ml, 632.1pg/ml, wherein HIV-p24 total contents differ 742.9pg/ in large and small leucocyte Ml reduces 54.3%, through statistical test, t=2.43, p ＜ 0.05.Illustrate large volume leucocyte in AIDS patient body or HIV containing high level in the large volume leucocyte formed after the effect of gp120 antibody, can be by implementing skill of the invention Art scheme is removed by separation.

HIV-p24 testing results (p24 in the 1 large, medium and small leucocyte of AIDS patient peripheral blood of table：pg/ml)

2, blood purification (agent) removes the verification of HIV effects

(1) verification of HIV effects is removed in the absorption of CD4+T cell strains

In order to verify the effect of HIV is removed in the absorption of CD4+T cell strains, the present invention devises easy test method：It takes and goes out 2.5 × 300mm Westergren's blood sedimentation tubes of bacterium 5 draw the CD4+T cells precipitated through centrifugation (1000r/min, 5min) extremely respectively 200mm scales are then drawn the heat preservation after 100 DEG C dissolve and are reached about in 39~41 DEG C of 0.9% spare agarose C1-4B 10mm long scales, after setting blood sedimentation stand cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not prevented small The water of molecule and the substance of chemical analysis etc pass through.The AIDS that Ling Qu Disease Control and Prevention Centers and Infectious Diseases Lab sample database preserve 5 blood plasma of patient, respectively about 10mL, respectively takes the preceding blood plasma of 9mLAIDS filters to inject blood sedimentation tube upper end blank pipe, erythrocyte sedimentation rate to be flowed through in batches The CD4+T cellular layers of pipe lower layer simultaneously after outflow, collect efflux, blood plasma after referred to as AIDS filters out of blood sedimentation tube.Before taking AIDS to filter Blood plasma after blood plasma and filter, according to HIV-1p24 antigen detection kits, (the limited public affairs of biotechnology are inspired in enzyme-linked immunization, Shanghai Department) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, As a contrast, minimum detection limit is less than 5pg/ml, 0~400pg/ml of measurement range, the range of linearity to the p24 antigens of 80pg/ml 450nm measures absorbance (OD) in 0.5pg/ml~80pg/ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value is not less than 1.000 not higher than 0.100,1000pg/ml absorbances, is recognized as absorbance ＞ 0.12 To be positive, testing result (table 2) illustrates, after AIDS blood plasma filters the simple purifier of the cell containing CD4+T, part HIV is It is adsorbed by CD4+T cells, after the 1st filtration, HIV total body clearances are 22.84%, and after the 2nd filtration, total body clearance is 35.31%, after the 3rd filtration, total body clearance 41.9%.Illustrate that the increase with filtration number, HIV can be by constantly clear It removes, to reach treatment AIDS purposes.

P24 testing results (pg/ml) before and after the simple purifier of 2 AIDS blood plasma of table filtration cell containing CD4+T

(2) verification of HIV effects is removed in hybridoma macrophage strain absorption

In order to which the effect of removing HIV is adsorbed in check cross tumor macrophage strain, the present invention devises easy test method： 2.5 × 300mm the Westergren's blood sedimentation tubes 5 for taking sterilizing draw the macrophage precipitated through centrifugation (1000r/min, 5min) respectively Hybridoma cell strain is then drawn and is kept the temperature in 39~41 DEG C of 0.9% spare agaroses after 100 DEG C dissolve to 200mm scales C1-4B reaches about 10mm long scales, and after setting blood sedimentation tube cooling, agarose becomes semisolid, can prevent blood sedimentation tube inner cell stream Go out but not prevent the water of small molecule and the substance of chemical analysis etc to pass through.Ling Qu Disease Control and Prevention Centers and Infectious Diseases Lab sample 5 blood plasma for AIDS (AIDS) patient that library preserves, respectively about 10mL, respectively takes the preceding blood plasma of 9mLAIDS filters to inject erythrocyte sedimentation rate in batches (simple purifier) upper end blank pipe is managed, the hybridoma macrophage strain layer of blood sedimentation tube lower layer to be flowed through simultaneously is flowed out out of blood sedimentation tube Afterwards, efflux, blood plasma after referred to as AIDS filters are collected.Blood plasma after taking the preceding blood plasma of AIDS filters and filtering, is moved with human macrophage and is inhibited The factor (MIF) ELISA detection kit (hundred stamen bio tech ltd of Shanghai) pairing detection, by specification operation, detection Ranging from 0~800pg/ml, susceptibility 1.0pg/ml can directly detect by an unaided eye under white background：Color in reacting hole Deeper, positive stronger, negative reaction is colourless or extremely shallow, according to the depth of be in color, is indicated with "+", "-" number.Also it can survey OD values：On ELISA detectors, at 450nm (if developing the color with ABTS, 410nm), each hole is surveyed after returning to zero with blank control wells OD values, it is as positive if more than 2.1 times of defined negative control OD value.As a result such as table 3, MIF testing results in blood plasma before filtering It is negative (or because content is less than detection sensitivity, the cause degradation of blood plasma long-term preservation etc.), and testing result is equal in blood plasma after filtering For the positive.Illustrate that macrophage hybridoma cell strain produces MIF cell factors in this process.MIF is collection cell factor, growth The multi-effect protein molecular of the factor, hormone and enzyme characteristic is played as inherent immunity and the regulatory factor of inflammatory reaction The effect of central plays panimmunity function in various infection and active chronic inflammation disease.Making the filtration of AIDS blood plasma Before and after simple clarifier while MIF pairings detection, the present invention has also done the pairing detection of HIV-1p24, according to HIV-1p24 Antigen detection kit (enzyme-linked immunization, Shanghai inspire bio tech ltd) operation, with known concentration 0pg/ml, The p24 antigens of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are as a contrast, minimum Detection limit is less than 5pg/ml, 0~400pg/ml of measurement range, and 450nm is surveyed in the range of linearity 0.5pg/ml~80pg/ml, 15min Determine absorbance (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ not higher than 0.050,0pg absorbance values Ml absorbances are not less than 1.000, are considered positive as absorbance ＞ 0.12, as a result (table 4) illustrates the filtration letter of AIDS blood plasma After easy purifier, part HIV is swallowed by macrophage hybridoma cell strain to be adsorbed, and the blood plasma HIV after filtration is significantly reduced, After the 1st filtration, HIV clearance rates are 20.55%, and after the 2nd filtration, HIV clearance rates are 42.83%, p ＜ 0.01, tool It has obvious effects on, illustrates that the increase HIV with filtration number can be removed constantly, to reach treatment AIDS purposes.

MIF testing results are (quantitative before and after the simple purifier of 3 AIDS blood plasma of table filtration macrophage containing hybridoma： pg/ml)

P24 testing results (p24 before and after the simple purifier of 4 AIDS blood plasma of table filtration macrophage containing hybridoma：pg/ ml)

(3) verification of HIV-1gp120 antibody, gp41 antibody absorption removing HIV effects

The present inventor's basic skills according to the invention has done following simple confirmatory experiment：Take HIV-1gp120 antibody, Gp41 antibody (the Shanghai bio tech ltd Rui Qi) is added to and is kept the temperature in 50 DEG C of 1.0% spare fine jades after 100 DEG C dissolve In lipolysaccharide C1-4B, titre is 1 after mixing：300~500,2.5 × 300mm Westergren's blood sedimentation tubes 5 of sterilizing are taken, are drawn respectively 1.0% agarose C1-4B solution is to 200mm scales, and agarose becomes semisolid after cooling.Ling Qu Disease Control and Prevention Centers and infectious disease The sample for 5 AIDS patients that laboratory sample database preserves, removes the respectively about 10mL of the blood plasma after cell, before respectively taking 9mLAIDS to 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 layer to be flowed through is simultaneously After being flowed out out of blood sedimentation tube, efflux, blood plasma after referred to as AIDS filters are collected.Blood plasma after taking the preceding blood plasma of AIDS filters and filtering, according to HIV-1p24 antigen detection kits (bio tech ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known concentration The p24 antigens conduct pair of 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml It is less than 5pg/ml, 0~400pg/ml of measurement range according to, minimum detection limit, in the range of linearity 0.5pg/ml~80pg/ml, 15min 450nm measures absorbance (OD), and blank control calibration object absorbance value is not higher than 0.100 not higher than 0.050,0pg absorbance values, 1000pg/ml absorbances are not less than 1.000, are considered positive as absorbance ＞ 0.12, and testing result (table 5) illustrates, After AIDS blood plasma filters simple purifier, part HIV is adsorbed by corresponding antibodies, after the 1st filtration, HIV total body clearances It is 20.01%, after the 2nd filtration, total body clearance 27.99%, after the 3rd filtration, total body clearance 37.36%.It says The bright increase HIV with filtration number can be removed constantly, to reach treatment AIDS purposes.

5 AIDS blood plasma of table filters p24 testing results (pg/ml) before and after simple purifier

In short, above-mentioned simple confirmatory experiment shows that the peripheral white blood cells by HIV infection are easily fused into the more of large volume Core giant cell or many cells condensate can be detached by the blood separator of the present invention and be removed；And the HIV to dissociate in blood plasma, it can quilt Cleanser (HIVgp120 antibody, HIVgp41 antibody, CD4+T cell strains, the strain of hybridoma macrophage, the agar gel of the present invention Micropore) absorption removing.Show had by the above-mentioned AIDS blood purifying therapeutical instrument constituted as critical component using blood purification The significant therapeutic efficiency for removing the inside and outside inhibition of HIV of blood cell.

Claims (7)

1. a kind of AIDS pregnant woman blood clarifier, which is characterized in that be provided with multilayer in clarifier by agar gel and purification The purifying layer of cell composition, multilayer purifying layer constitute decontaminating column；The daf molecule is by can be in conjunction with the CD4+T cell strains, miscellaneous of HIV Hand over tumor macrophage strain according to quantity than 1：0.5~3 composition；In each layer, cell accounts for the 4/5 of this layer of volume；The agar is solidifying Glue includes the HIV antibody dissociated, the HIV antibody for being incorporated into goat-anti Ig, agarose；From the entrance of clarifier to exit, trip From the antibody titer of HIV antibody, the antibody titer for the HIV antibody for being incorporated into goat-anti Ig successively decrease successively from high to low, agar Sugared concentration is incremented by successively from low to high.

2. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that the import and export of the clarifier It is both provided with sieve, the mesh number of entrance sieve is 800 mesh, and the mesh number of exit sieve is 2.0~5.0 mesh.

3. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that the number of plies of the purifying layer is 5 Layer, the antibody titer of free HIV antibody, be incorporated into goat-anti Ig HIV antibody antibody titer from high to low, be followed successively by 1： 100、1：200、1：300、1：500、1：700.

4. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that the number of plies of the purifying layer is 5 Layer, from low to high, content is followed successively by 0.7g/100ml, 0.8 g/100ml, 0.9 g/100ml, 1.0 g/ to agarose concentration 100ml、1.1 g/100ml。

5. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that the HIV antibody is by HIV- One or both of 1gp120 antibody, HIV-1gp41 antibody are formed according to arbitrary proportioning.

6. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that hybridoma macrophage strain passes through Oncocyte and macrophage are prepared with the hybridoma technology of cell fusion.

7. AIDS pregnant woman blood clarifier according to claim 1, which is characterized in that the decontaminating column passes through with lower section Method is prepared：

（1）The strain of hybridoma macrophage and CD4+T cell strains are cleaned with sterile saline, 1000r/min is centrifuged, after cleaning again Secondary centrifugation is 1 by the ratio between the strain of hybridoma macrophage and CD4+T cell strains：0.5~3 ratio takes sedimentation cell, assembly In hydrostatic column made of high-biocompatibility material, blood purification cell column is made；

（2）Goat-anti Ig is mixed with excessive HIV antibody, the goat-anti in mixed liquor is made to be fully tied, but surplus has free HIV Antibody, and free HIV antibody titre is equal with the HIV antibody titre of goat-anti Ig is incorporated into；

（3）Agarose is mixed after 100 DEG C dissolve with a certain amount of physiological saline, is kept the temperature at 39~41 DEG C, step 2 is added and obtains The antibody mixture obtained, wherein the antibody titer of free HIV antibody, the antibody titer for the HIV antibody for being incorporated into goat-anti Ig are equal It is 1：700, the content of agarose is 1.1 g/100ml；

（4）The product that step 3 is prepared is with volume ratio 1:4 are added in the lymphocyte depletion column of step 1 preparation, and it is solid to be cooled to half After body gel, the preparation of bottom purifying layer is completed；

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