CN106267416A

CN106267416A - AIDS therapeutic equipment

Info

Publication number: CN106267416A
Application number: CN201610539174.4A
Authority: CN
Inventors: 翁炳焕; 李兰娟; 李晓; 陈敏; 李红阁
Original assignee: 翁炳焕
Current assignee: Shu Lan (Hangzhou) Hospital Ltd.; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2017-01-04
Anticipated expiration: 2036-07-01
Also published as: CN106267416B

Abstract

nullA kind of AIDS therapeutic equipment for medical domain，It is characterized in that preparation can washed corpuscles and the separator of blood plasma，Prepare HIVgp120 and gp41 antibody and combine HIVgp120 and the gp41 antibody of goat-anti Ig，Build with exogenous gene transfection and hybridoma technology respectively and not only retained archeocyte characteristic but also can the CD4+T cell strain of indeterminate growth and hybridoma macrophage strain，It is formulated in agar gel after amplifying cells strain and forms depurator with high-biocompatibility material parcel，Gel is made to form antibody titer from high to low from top to bottom and the layer distributed of agarose concentration from low to high，Wherein with goat-anti Ig conjunction type and sequestered gp120 and gp41 antibody、CD4+T cell strain、Hybridoma macrophage strain be all fixed on agar gel rise absorption HIV be used，Made depurator is applied in combination with separator and regulatory process，Extracorporeally circulating blood is divided into blood plasma and hemocyte by separator，The purified device of blood plasma converges feedback with hemocyte after filtering HIV.

Description

AIDS therapeutic equipment

Technical field

The present invention relates to preparation and the application of AIDS therapeutic equipment in medical domain, be mainly used in HIV sufferers blood plasma Chinese mugwort Grow the removing of virus, thus reach to treat the purpose of acquired immune deficiency syndrome (AIDS).

Background technology

After HIV enters human body, first by macrophage phagocytic, but HIV quickly changes macrophage some position interior Sour environment, creates the condition of its existence applicable, is not the most killed and the most within it breeds.Because CD4 is being subject to of HIV Body, thus in macrophage breeding HIV by its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes self hydrophobic interaction mediate retroviral cyst membrane and cell membrane fusion) (cell, monokaryon are huge to be bitten carefully to enter CD4+ cell Born of the same parents, dendritic cell etc.), in intracellular rapid propagation, produce 10 every day⁹～10¹⁰Virion, and it is normal constantly to enter other And regeneration the intracellular duplication of CD4+, manufacture more virus infected cell, make peripheral blood CD4+T cell sustaining breakdown, subtract Few.Gp120 with the CD4 receptor of HIV be combined can directly activate infected apoptosis, even infected by HIV T cell express Envelope antigen also can start normal T-cell, indirectly causes a large amount of broken of CD4+ cell by the crosslinking of cell surface CD4 molecule Bad, result causes the severe immune deficiency centered by CD4+T cell defect, and patient mainly shows: periphery lymphocyte reduces, T4/T8 proportional arrangement, to phytohaemagglutinin and the loss for reaction of some antigen, delayed allergy declines, and NK cell, huge bites Cytoactive weakens, and the synthesis of the cytokine such as IL2, IFN-γ reduces.CD4+T cell is most important immunocyte, infects Person once loses a large amount of CD4+T cell, and whole immune system will suffer deathblow, and the infection to various diseases is all lost Go resistance.HIV can also show as hiding for a long time and not showing clinical symptoms after entering host's CD4+ cell, its genome RNA reverse transcription becomes double-stranded DNA, enters in host cell core with viral integrase enzyme, and under the effect of intergrase, double-stranded DNA is integrated In host cell gene group, integrated viral DNA is referred to as provirus, and the several months of can hiding does not replicates, and causes The AIDS several months is to incubation period for many years.In the incubation period of AIDS, HIV is mainly in the macrophage and dendritic cell of lymph node Breeding, these cells are internal HIV depots, releasably to peripheral blood or transfection peripheral blood in CD4+T cell, have silk to divide Splitting former, antigen, TNF, IL-2 and lymph element (LT) can excite HIV provirus gene multiple at the CD4+T Intracellular transcription infected System.After a large amount of propagation, inhibition of HIV granule constantly discharges from destroyed infection cell and is free on blood, then enters back into Other cells continue course of infection, and inhibition of HIV granule, before the cell release infected to extracellular, is expressed in infection cell table Fusion between gp120 and gp41 energy mediated infection cell and the non-infected cells in face, the CD4 of such as infected by HIV⁺T cell table The gp120 reached is combined with the CD4 of non-infected cells, causes cell to merge and form multinucleated giant cell.

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

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

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

CD4 molecule is the receptor of HIV, and HIV is susceptible in CD4+T cell, and CD4+T cell is the one of T lymphocyte, averagely Life-span is generally about 7 days, but some T cell particularly after immortality chemical conversion cell line (strain) can long-term surviving, infinitely expand. Foreign literature is reported, simian virus 40 (SV40) can make some human cell that immortalization occurs.Poulin DL, Kung AL and The research such as Sullivan CS shows, the importing of SV40T antigen gene can accelerate to convert the growth rate of cell, immortalized cells After the most repeatedly passing on, still there is metastable multiplication characteristic and functional status, also can retain its germinal cell simultaneously Many phenotypic differentiations.Vascular smooth muscle cell strain is set up in Reilly simian virus large T antigen gene transformation, builds cell membrane Type is with the inhibitory action mechanism of research heparin for vascular smooth muscle.Su etc. utilize the superficial cell strain converted through SV40, structure Build cell model to analyze the regulating and controlling effect of epithelial cell internal protein synthesis.The cuticulated epithelium that Miquel etc. convert with SV40 is thin Born of the same parents' strain, as the cell adhesion of cell model research laminin，LN 5 mediation.The prostatitis converted through SV40 such as Webber Glandular epithelium strain studies physiological function and the secretory function of prostate epithelial cell as cell model.Racusen etc. use Convert renal cells model through Ad12-SV40 and study damage and the disease of proximal convoluted tubule.The SV40 such as Hougton turns Change set up Bone marrow Stromal cell as cell model with research under certain condition of culture, cell have to adipose cell with become The potential of the two-way differentiation of osteocyte, studies osteoporotic mechanism further.Foreign study is it is also shown that import exogenous human end Human telomerase reverse transcriptase (hTERT) can make cell keep normal phenotype and differentiating characteristic.HTERT has the most been utilized to be successfully established The immortalized cell line of some cell, keeps that chromosome stabilityX, differentiation be normal, contact inhibition, relative without oncogenicity etc. substantially Normal growth characteristics.In stomatology field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establishes immortality Changing people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number reaches more than 150 times, Cell all shows original biological characteristics, can express the associated protein of derived cell after inducing culture.Kitagawa etc. Transfection hTERT establishes people cementoblast system, and cell multiplication reaches more than 200 times, cell differentiation mark such as alkaline phosphatase Enzyme, type i collagen etc. are expressed stable.Because of the needs of research work, almost every kind of disease has respective cell model.Such as 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, cerebral edema cell model etc..So, CD4+ can be prepared T cell strain, after a large amount of amplifications, for preparing the depurator for the treatment of acquired immune deficiency syndrome (AIDS), with in CD4+T cell adsorption removal blood plasma HIV, the cytokine simultaneously produced by being defeated by CD4+T cell treats acquired immune deficiency syndrome (AIDS).

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

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

Summary of the invention

In order to solve to attack for a long time the global problem in the treating AIDS field being unable to, present inventors have proposed the present invention.

The invention aims to provide AIDS therapeutic equipment；Another object is intended to provide preparation and the application side of therapeutic instrument Method.

The object of the present invention is achieved like this: preparation can washed corpuscles and the separator of blood plasma, preparation gp120 and Gp41 antibody, then prepare goat-anti gp120 and gp41 antibody as corresponding antigens, build CD4+T cell with exogenous gene transfection Strain also expands with its peculiar molecular antibody for cell growth stimulator, not only retains former macrophage feature but also can with hybridoma technology preparation The hybridoma macrophage strain of indeterminate growth amplification cultivation, take cell strain and prepare can prevent with high-biocompatibility material parcel Stop cell and fragment leaches and can provide the depurator in place for removing HIV, take gp120 and gp41 antibody and goat-anti Gp120 and gp41 antibody mixes, and makes goat-anti gp120 and gp41 antibody be fully tied but surplus gp120 and gp41 having high titre Antibody, allocates into mixed antibody with reverse gradient titre and is incubated the agar at the gradient concentration of 39～42 DEG C after 100 DEG C dissolve Sugar, paramount is sequentially added into depurator by antibody titer from low by it, followed by adding next time after being cooled to semi-solid gel, makes Gel in depurator forms antibody titer from high to low and the layer distributed of agarose concentration from low to high from top to bottom, Be conducive to plasma perfusion and molecular sieve and the effect of immune clearance, that be wherein combined and unconjugated gp120 with goat-anti HIV antibody And gp41 antibody, CD4+T cell strain, macrophage strain are all fixed in agarose gel, play an absorption HIV and be used, made only Changing device and separator combination Additional regulatory program and make AIDS therapeutic equipment, the blood in extracorporeal circulation is divided into by separator Blood plasma and hemocyte, then the purified device of blood plasma feeds back after converging with hemocyte after filtering HIV.

The technological core of the present invention is made up of separator and depurator, separator energy washed corpuscles and blood plasma, depurator In cleanser be combined with goat-anti Ig by the HIV antibody being fixed on agar gel HIV antibody, CD4+T cell strain and huge bite thin Born of the same parents' strain is made, and the molecular weight ratio unconjugated HIV antibody molecular weight of its conjugate of HIV antibody being wherein combined with goat-anti Ig is big, Not easily passing through gel molecular sieve, and contained goat-anti Ig is easy and the feature of agar gel secure bond, HIV antibody is more easy to the most therewith Be fixed in agar gel, the HIV in blood plasma and the HIV antibody being fixed in agar gel association reaction can occur when meeting and Form antigen antibody complex, thus be fixed in agar gel by HIV antibody, because the CD4 molecule of CD4+T cell surface is The receptor of HIV and become the permissive cell of HIV, HIV, adsorbed HIV can be adsorbed when meeting with HIV and consolidated with CD4+T cell Due to agar gel, because of the natural phagocytosis characteristic of hybridoma macrophage, can be fixed therewith by phagocytosis when HIV meets therewith In agar gel, and the filter opening that agar gel is formed reduces along with increasing of agarose concentration, depurator import department fine jade Lipolysaccharide concentration is low, and filter opening is just big, beneficially the association reaction of plasma perfusion and high titer antibody or cell and HIV；And exit Concentration is high, and filter opening is the least, it is easy to detention HIV or Large molecular conjugates, and it is clear that depurator is combined with multiple special and non-specific HIV Except composition, in order to avoid extraordinary strain substitutes with the method for external mechanical removal HIV because of the futile treatment caused by immunity difference, the present invention Cannot effectively kill the routine internal anti-reverse transcription drug treatment of HIV for a long time.

Detailed description of the invention

Fig. 1 is the application schematic diagram of the AIDS therapeutic equipment proposed according to the present invention.

Fig. 2 is the internal structure schematic diagram of the separator proposed according to the present invention.

Fig. 3 is the internal structure schematic diagram of the depurator proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) being connected with plasma separator (4), plasma separator (4) is through the purification in parallel with two of blood plasma pump (6) and circulation line (7) Device (8), depurator (9) are connected, and are connected with circulation line (10), venous line (5) the most successively, another of venous line (5) End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 is plasma separator inner chamber, and 3 is the micropore on plasma separator inner chamber tube wall, 4 Being the hemocyte that can not pass through micropore (3), 5 is blood plasma and the chemical analysis that can pass through micropore (3), and 6 is plasma separator exocoel, 7 is blood plasma flow export, and 8 is the hemocyte outlet with switchable valve.

In Fig. 3,1 is free HIV；2,4,6 HIV antibody being respectively fixed in agar gel (8), macrophage, CD4+T cell；3, it is delayed at agar gel after 5,7 respectively HIV and HIV antibody, macrophage, CD4+T Cell binding (8) conjugate in, 9 is by the large volume of HIV of agar gel (8) molecular sieve detention.

Below in conjunction with Fig. 1, Fig. 2 and Fig. 3, preparation and the embodiment of application to the AIDS therapeutic equipment that the present invention proposes It is explained in detail.

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

(1) preparation of hybridoma macrophage strain

1, primary cell source

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

(2) single core histiocyte: provided by Zhejiang University's Tissue Engineering Study platform.Substantially belong to from spleen Macrophage, its preparation method is: the 1. acquisition of spleen tissue and transhipment: in the approval of reason committee and patient's informed consent Under, take the spleen specimen tissue of excision, shred into volume about 1mm immediately³Small tissue blocks, move into equipped with pre-cooling 4 DEG C In sterile sealing bottle, it is transported to rapidly cell culture chamber.2. the preparation of spleen tissue cell suspension: spleen tissue block is moved to Aseptic operating platform, PBS washs 3 times, and RPMI-1640 washs 2 times, to remove in-house blood and to ensure the aseptic of tissue.Machine Tool grinds spleen tissue, the most just has substantial amounts of histiocyte to hang and is mixed in RPMI-1640 liquid.By 200 mesh stainless steel filtering net mistakes Filter is outstanding is mixed with histiocytic RPMI-1640 liquid, filtrate be spleen tissue cell suspension (mainly containing erythrocyte, lymphocyte, Macrophage etc.).3. erythrocytic cracking in spleen tissue cell suspension: then centrifugal with the washing of RPMI-1640 liquid (1000r/min, 3min), to remove cell debris, adds Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), removes the splitting erythrocyte fragment in supernatant, centrifugal 3 times of PBS washing, RPMI-1640 washing from The heart 1 time, to remove the Tris-NH4Cl of remaining in suspension, it is to avoid it affects the survival of cell, now, mainly contains in suspension Spleen tissue macrophage and lymphocyte.4. the adhere-wall culture of spleen tissue macrophage: using aforementioned suspension as cultivation Cell stock solution, Trypan Blue judges vigor and counts, and adjusting cell concentration with RPMI-1640 liquid is (3～5) × 10⁶/ L, will Adjusting the cell suspension inoculation of concentration in glass culture bottle, condition of culture is 37 DEG C, 50mL/LCO₂, 100% humidity, point Not Pei Yang 2～3h, under phase contrast microscope observe form.The digestion of adherent spleen tissue macrophage: adherent spleen tissue is huge to be bitten The digestion of cell: suck culture supernatant, macrophage is adherent, and PBS repeatedly blows and beats, digests, and the washing of gained cell suspension is centrifugal (1000r/min, 3min), obtains isolated and purified macrophage.Further, it is also possible to take treatment or the discarded specimen of Post operation carries Take preparation, such as peritoneal cavity liquid, alveolar, liver, spleen, peritoneal tissues, small intestinal mucosa etc..

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

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

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

3, cd4 cell sorting

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

4, CD14 cell (macrophage) sorting

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

Basic skills is analogous to cd4 cell, uses immunomagnetic beads method.1. reagent: people's CD14 immunomagnetic beads test kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead is with special Opposite sex target cell one mononuclear cell combines: every 1 × 10⁸Individual PBMC adds magnetic bead and the 800uL buffering of 200uL coupling CD14 antibody Liquid (containing the bovine serum albumin 2.5mL and 2mol/L EDTA0.5mL of 10%, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube Fully mixing, hatches 15min for 4 DEG C, and centre can slightly shake 1 time.Taking-up centrifuge tube after 15min, every 1 × 10⁷Individual cell adds 1 ～2mL pre-cooling buffer, 1 000r/min, centrifugal 8min, abandon supernatant, add 0.5mL buffer and blow and beat into single cell suspension. (B) collect the mononuclear cell of marked by magnetic bead: be placed on MACS magnetic frame by cell separation post, add 1mL buffer statocyte Detached dowel, treats that no liquid is dripped, and is added by above-mentioned cell suspension in people's cell separation post immediately, with 0.5mL wash buffer cell Detached dowel 3 times.After to be rinsed, add 1mL buffer, emigrated cells detached dowel from magnetic frame, quickly promote with pin post, Go out the cell combined with CD14 antibody one magnetic bead in detached dowel, be the macrophage of CD14+.

Additionally, following 2 kinds of methods sorting also can be used, including 1. adherent method: be placed in by PBMC and cultivate containing RPMI-1640 In the culture dish of base, in 37 DEG C, containing 5%C0: cell culture incubator (Themo electro corporation CLASS 100, The U.S.) in hatch 2h.After adherent mononuclear cells, inhaling and abandon upper strata suspension cell, PBs buffer washs 3 times gently, adds a small amount of RPMI 1 culture medium, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant.2. fluidic cell Art method: CD14 labelling: take PBMC, with buffer (bovine serum albumin 2.5mL and 2mol/LEDTA 0.5mL containing 10%) Adjusting cell density is 1 × 10⁸/ mL, adds CD14+-FITC antibody 100uL, 4 DEG C of lucifuge labellings in every milliliter of cell suspension 18min, then addition 1mL streaming buffer is to terminate dyeing in centrifuge tube, PBs washs 3 times, with the PBS containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation at flow cytometer (BD FAcsAria II, U.S.) upper sorting, according to the fluorescence intensity of CD14 antibody, the relative size of cell and the relative particle of cell and interior The complexity of portion's structure, collects the cell of CD14+.

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

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

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

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

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

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

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

2. hybridoma macrophage strain phagocytosis HIV Function detection: take acquired immune deficiency syndrome (AIDS) (AIDS) patient's of Disease Control and Prevention Center's preservation After blood plasma is mixed with hybridoma macrophage strain, separates cell strain, PBS 3 times, measure the phagocyte strain through cracking The function of phagocytosis HIV, with specific reference to HIV-1p24 antigen detection kit, (euzymelinked immunosorbent assay (ELISA), Shanghai inspires biotechnology limited Company) operation, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, The p24 antigen of 80pg/ml is less than 5pg/ml, measurement range 0～400pg/ml, the range of linearity as comparison, lowest detectable limit In 0.5pg/ml～80pg/ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050,0pg absorbance is not higher than 0.100, and 1000pg/ml absorbance is not less than 1.000, is recognized as absorbance ＞ 0.12 For being the positive.Concrete test kit description of pressing operates.

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

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

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

6, prepared by hybridoma macrophage strain treatment cell

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

(2) preparation of CD4+T cell strain

1, the source of primary lymphocyte

Have to sow by way of 1. frozen in the Infectious Diseases Lab Sample Storehouse that scientific research preserves lymphocyte strain (warp Inactivation HIV totivirus is immune but is uninfected by the lymphocyte of HIV)；2. buy the fresh White Blood Cells Concentrate in blood station, then went out The lymphocyte that HIV strain of living is immune；3. the T lymphocyte series (strain) directly bought from businessman；4. preserve for scientific research Cord blood lymphocytes cell (through inactivation HIV immunity)；The most directly take from the peripheral blood lymphocyte of HIV-1 the infected (for certainly Body), use Histopaque lymphocyte separation medium separation mononuclearcell (PBMC).

2, the preparation of CD4+T cell

1. main agents and instrument: CD4, CD8 immunomagnetic beads (Mei Tian Ni Bioisystech Co., Ltd of Germany)；Isothiocyanic acid Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；Lymphocyte separation medium (Shanghai perseverance letter biochemistry Reagent company limited)；Ethylenediaminetetraacetic acid (EDTA), 0.2% Trypan Blue liquid (Shanghai raw work biotechnology service public affairs Department)；New-born calf serum (Hyclone company)；MiniMACS magnetic separation system (Mei Tian Ni Bioisystech Co., Ltd of Germany)； EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. separation (the density gradient of mononuclearcell (PBMC) Centrifuging): aseptic take 20mL blood sample (500IU/mL2mL heparin sodium anticoagulant)；PBS liquid, by hemodilution 2～3 times, fully mixes After 6mL anticoagulant venous blood dropper is slowly superimposed on, along tube wall, the 10mL centrifuge tube having added 4mL lymphocyte separation medium Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；Being divided into 3 layers after Li Xin in pipe, upper strata is blood plasma and PBS liquid, Lower floor is mainly erythrocyte and granulocyte, and middle level is lymphocyte separation medium, has one with mononuclearcell in upper, interface, middle level The white cloud and mist layer narrow band being main is PBMC, is inserted into cloud and mist layer with capillary pipette, draws PBMC and inserts another 50mL centrifuge tube In, add 5 times and be centrifuged (300r/min, 20 DEG C) 10min with upper volume PBS, abandon supernatant 50mLPBS re-suspended cell, centrifugal (350r/min, 20 DEG C) 15min, abandons supernatant, adds Buffer (PBS+0.5% new-born calf serum+2mmol/LEDTA, pH7.2) 2mL re-suspended cell, takes the cell (PBMC) that 15uL cell suspension adds on blood counting chamber in 4 block plaid of counted under microscope Sum.3. CD4+T cell and CD8+T cell is isolated and purified: PBMC cell suspension is divided equally to two 1.5mLEppendorf pipes, Centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded, the every 80uLBuffer of re-suspended cell contains cell number 10⁷Individual, every 10⁷Individual carefully Born of the same parents add 20uLCD4MicroBeads or CD8MicroBeads, fully mix, and hatch 15min at 4～8 DEG C, wash with 1mLBuffer Wash cell, centrifugal (300r/min, 20 DEG C) 10min, supernatant discarded 500uLBuffer re-suspended cell, MS detached dowel is placed on In the magnetic field of MACS separator, rinse with 500uLBuffer, by 500uL cell suspension by detached dowel, use 500uLBuffer Rinse detached dowel repetitive operation 3 times, collect effluent, containing non-CD4+T lymphocyte or non-CD8+T lymphocyte in effluent, Taking out detached dowel in separator, with 1000uLBuffer pressure flush detached dowel, collect effluent, this is that CD4+T lymph is thin (cell viability detects: take 15uL cell suspension before and after cell purification respectively molten with equal-volume trypan blue for born of the same parents or CD8+T lymphocyte Liquid mixes, and the not colored shinny person of basis of microscopic observation is living cells, and the coloring person of swelling is dead cell, calculates in 200 cells and lives The percentage ratio of cell).

3, amplification in vitro CD4+T cell

Document is had to report the stimulant utilizing the monoclonal antibody of T cell surface C D3 molecule to grow, great Liang Pei as cell After supporting the T cell that HIV sufferers separates, feed back as self therapeutic cells.But HIV is also with the cultivation of HIV cell Breeding and at endogenous multiplication, the feedback of increment T cell result also in the feedback of increment HIV.The present invention is with SV40 and/or hTERT Immortalization CD4+T cell, and with CD3 monoclonal antibody for cell growth stimulant, a large amount of amplification CD4+T cells.

With the method that CD3 monoclonal antibody is cell growth stimulant it is: by anti-CD49d McAb, (CD4+T cell contains simultaneously CD3 molecule) it is coated on culture plate stimulation mononuclearcell (lymphocyte) growth, referred to as anti-cd 3 antibodies is coated method, can obtain Well expanding effect, the lymphocyte that should expand in this way has been used for the second stage of clinical treatment of tumor and achieves certain Curative effect.Foreign literature report [Shimizu etc.] has also cultivated the lymphocyte of 5 example full-blown AIDS patients, training by the method Support and within 4 weeks, be achieved with the amplification of 1000 times, and in the cell mass of amplification, CD4+/CD8+T all can expand that (CD4+T cell is more in a large number Substantially).Another kind is the dual anti-cross-linking method of AntiCD3 McAb/CD28, will dual anti-being crosslinking on taste pearl of AntiCD3 McAb/CD28 train as stimulant Support HIV person's PERIPHERAL BLOOD MONONUCLEAR CELL (lymphocyte), substantial amounts of CD4+T cell, and the CD4+T expanded can be expanded Cell has the ability of antagonism HIV, and in its incubation, virus is also below detection level, finds that this may be with CD28 afterwards Providing secondary signal, it is relevant with chemotactic factor that selective induction secretes substantial amounts of Th1 cytokine, with the method amplification CD4+T cell have been used for HIV person clinical treatment feed back, devoid of risk but effect is general.

With the method for hTERT immortalization CD4+T cell it is: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo- HTERT and carrier pLXSNneo, connects hTERT and pLXSNneo separated through PCR amplification, gel electrophoresis with Ligation Mix Digestion products, builds pLXSNneo-hTERT recon, converts DH5a competent cell blue or green with amplification, purification picking resistant to ammonia benzyl Mycin bacterium colony extracting plasmid, imports with lipofection and passes on the T lymphocyte in logarithmic growth in vitro, makes recon with thin The DNA of born of the same parents integrates, and the clone of positive recombinant that amplification culture screen through G418, screening cellular morphology, growth curve, dyeing The test of body caryogram, nude mice tumorigenesis, transfectional cell telomerase activation, hTERT mrna expression product, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic person same or like with primary cell as hTERT immortality The CD4+T cell changed.

With the method for SV40 immortalization CD4+T cell it is: connect simultaneously through BamHI enzyme action with T4DNA ligase PcDNA3.1 (-) DNA with PCR amplification, the SV40LTag DNA that separates of agarose gel electrophoresis, structure SV40LTag- PcDNA3.1 (-) recombiant plasmid, convert DH5a competent escherichia coli cell amp-R with amplification, purification picking Bacterium colony extracting plasmid, imports the T lymphocyte of In vitro culture with lipofection, makes the DNA integration of recon and cell, with The cell containing positive recombinant of G418 screening, passes on, amplification culture, screening cellular morphology, cell growth curve, chromosome SV40 big T gene test in the test of caryogram, nude mice tumorigenesis, transfectional cell DNA, mrna expression product measure and determined dna sequence Result meets immortalized cells characteristic person same or like with the primary cell CD4+T cell as SV40 immortalization.

1. with the concrete grammar of hTERT immortalization CD4+T cell

(I) extraction of hTERT: (i) enzyme action pClneo-hTERT:hTERT be positioned at the EcoRI of plasmid pClneo-hTERT with Between SalI site, pLXSNneo vector multiple cloning site (MCS) restriction enzyme site Han EcoRI Yu XhoI.Commercially available purchase pCIneo- HTERT plasmid, is dissolved in appropriate ultra-clean H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restriction Property restriction endonuclease EcoR I and each 0.5ul of Xho I, 37 DEG C of incubation 1h, 75 DEG C heating 15min, inactivator, add 5uL electrophoresis sample-adding Buffer terminates reaction, routinely after PCR method amplification hTERT, collects amplified matter in case electrophoresis.(ii) hTERT electrophoresis: power taking is swum Level agarose is made into 10% agarose gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample comb, After gelling is solid, from glue platform, removes envelope band, extracts comb, put in the electrophoresis tank added with enough electrophoretic buffers, buffering Liquid exceeds gel surface about 1mm, prepares hTERT enzyme action sample with 10 appropriate × sample loading buffer, then will with pipettor Sample adds in sample well, and does suitable standard control thing simultaneously, connects electrode, makes the hTERT Ghandler motion that faces south move, then at 1- Under the voltage of 10V/cm (80V) gel, electrophoresis is to when being sufficiently separated the distance of hTERT fragment (30min), closes power supply.(iii) HTERT purification and recovery: from agarose, separate hTERT band: by coagulating containing target hTERT fragment under long wave ultraviolet light source Adhesive tape band cuts in loading bag filter, adds 2ml electrophoretic buffer, be allowed to submergence gel, and empty steam bubble in bag filter, will Bag filter level puts into electrophoresis tank (length direction is parallel with electrophoresis), adds appropriate amount of buffer solution by bag filter submergence (about 6-7mm), Switching on power, 150 volts of electricity are washed, and observe and treat that hTERT all removes gel under uviol lamp, change direction of an electric field and continue energising 1 point Clock, from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of volume n-butyl alcohol, and mixing extracting is gone EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats secondary, molten from lower floor hTERT Adding equal-volume phenol chloroform (2) in liquid to extract 2 times, supernatant proceeds to add in another Eppendorf pipe 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, it is centrifuged 10 minutes at 4 DEG C, obtains hTERT precipitation, abandon Clearly, after adding 70% washing with alcohol 2 times, abandon dry ethanol, add 50 μ l TE and dissolve hTERT.Additionally, can also be used with low melting-point agarose Purpose hTERT fragment is separated from gel, is purified by gel method, hTERT filter membrane inserted sheet method etc..

(II) connection of hTERT Yu pLXSNneo carrier: take the hTERT composition (0.1-5 μ g) of the 9 above-mentioned purification of μ l, 1 μ l 10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C Incubation 24h, builds pLXSNneo-hTERT recon.

(III) pLXSNneo-hTERT recon purification, expand, identify: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or multiple divalent cation etc. processes antibacterial, feeding them into competence is converted, and uses CaCl₂Preparing fresh or freezing competent escherichia coli cell, be usually used in preparation in quantity competence antibacterial, this law is applicable to greatly Most coli strains, operating process is summarized as follows.(ii) with competence escherichia coli purification, amplification pLXSNneo-hTERT Recon: take 200 μ l from every kind of competent cell suspension with the sterile pipette tip of cooling and transfer to aseptic microcentrifugal tube In, often pipe adds DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng), rotates gently to mix content, at ice Middle placement 30min, is put into centrifuge tube pre-heating to the test tube rack in the circulator bath of 40 DEG C, places 90s～2min, not Shake test tube, quickly transfers in ice bath by pipe, makes cell cooling 1～2min, and every centrifuge tube adds 800 μ lSOC culture medium, uses water Culture medium is warmed to 37 DEG C by bath, is then transferred to by pipe on 37 DEG C of shaking tables, and incubation 45min makes bacteria resuscitation, and expresses matter Antibiotic-resistance marker's gene of grain coding, the competent cell that proper volume (every 90mm flat board is up to 200 μ l) has been converted Transfer in the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, flat board is placed in room temperature and is absorbed to liquid, fall Horizontalization ware, in 37 DEG C of cultivations, may occur in which bacterium colony after 12～16h.(iii) screen, expand recon: connect with sterile toothpick or sterilizing Kind of pin selects single colony inoculation in LB culture medium aseptic for 5mL or rich medium, after overnight incubation, is then added to In the 500mL 2L flask containing LB culture medium, cultivate to saturation (OD then at 37 DEG C₆₀₀≈ 4), what addition 1mL newly joined contains The GTE solution of 25mg/mL lysozyme, resuspended precipitation, place 10min in room temperature, add 10mL and newly join NaOH/SDS solution, Yu Bing Upper placement 10min, adds 7.5mL acetic acid solution, is gently mixed with suction pipe until viscosity declines and formed big precipitation, Yu Bing Upper placement 10min, in 4 DEG C, 20 000g are centrifuged 10min, are poured into gently by supernatant in another clean centrifuge tube, if Have visible drift that the isopropanol of 0.6 times of volume, reverse mixing by several layers of filtered through gauze, can be added, room temperature place 5～ 10min, in room temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration the most centrifugal, inhales Go ethanol, vacuum drying (precipitation can be 4 DEG C of long-term preservations).(iv) qualification of recombiant plasmid and amplification: the list on picking plate Bacterium colony, is inoculated in 3ml and contains in 100ug/ml ampicillin LB culture medium, 37 DEG C, cultivate in 250r/min shaking table, receive after 14h Collection culture, 4 DEG C, 10000r/min be centrifuged 5min, extract in a small amount and purification of Recombinant plasmid by test kit description.

(IV) CD4+T cell: from the present invention " preparation of (2) CD4+T cell " sampling preparation.

(V) CD4+T cell preculture: above-mentioned cell is inoculated in containing 5～10nmol/L insulins, 20% hyclone In RPMI1640 liquid, or it is inoculated in the low sugar DMEM cell culture medium containing 20% hyclone, 5～10nmol/L insulin, Typically it is inoculated in the freshly prepared culture medium of 3ml (1.6%1M HEPES buffer；15% hyclone (FBS)；1% penicillin And streptomycin；PRMI 1640 supplies 100%), it is placed in 37 DEG C, in volume fraction 5%CO2 incubator, cultivates 1-2 days, from The heart, removes supernatant, standby.

(VI) pLXSNneo-hTERT recon imports T lymphocyte and amplification culture: make in 1.5ml microcentrifugal tube Standby following solutions: pipe A, is dissolved in pLXSNneo-hTERT recon in 100 μ l serum-free mediums；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, left at room temperature 45min, trains with serum-free Nutrient solution washs above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-pLXSNneo-hTERT mixture Culture fluid, mixes gently, drops in above-mentioned T lymphocyte, and (hyclone concentration is 20ml/ to add 1ml serum-free medium L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues to cultivate 20h, discards culture fluid, and changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select living cells make expand After cultivation, then strengthen G418 concentration to 800mg L^-1, by can in the G418 environment of high concentration the cell of stable growth continue into Row amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If cell increases Slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches 14ml In time, transferred in 75ml culture bottle, and every 2-3 week adds 5-10ml fresh culture.Cell is cultivated to 9-10 week (the about the 75th generation), Still in exponential phase, i.e. cell is accelerated and incubation time is multiplication relation, and dead cell is less than 10%.When total amount reaches During to 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes, after abandoning supernatant, add 3ml freezing media (5% diformazan Base Asia maple (dimethyl sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Frozen Pipe subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen in-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, Move in liquid nitrogen container after 1-2 hour).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: with incubation time as transverse axis, Cell quantity is the longitudinal axis (logarithm), be depicted on semi-logarithmic scale make after curve the growth curve of this cell, immortalization Cell line is formed in typical " S " feature or " arched roof "；(iii) chromosome is checked: by analyzing karyotype, if dyeing Body caryogram is diploid " 46, XX " or " 46, XY ", then illustrate that this cell line does not occur vicious transformation.(iv) flow cytometry Detection: the cell proportion that synthesizes, divide in detection the 19th continuous cell line, if its multiplication capacity substantially than do not build be normal the most carefully Born of the same parents strengthen, and explanation is the result that hTERT integrates, expresses.(vii) determined dna sequence: sequenator detection routinely, shows hTERT Gene order.HTERT detection in (v) transfectional cell DNA: as with Immunohistochemical detection, dye in the nucleus of hTERT transfection Seen from color, a large amount of brown particles, show that hTERT has been integrated into intracellular；(vi) mrna expression product measures: take 100 μ l systems Pcr amplification product, reclaims test kit (Takara, Japan) with gel and reclaims product, take 2 μ l DNA solutions and dilute 100 times, survey dense Degree, remaining DNA and each 10 μ l of upstream and downstream primer checks order.

(VIII) hTERT mediates CD4+T cell bank: screen and continue to pass on, amplification culture meets forever after above-mentioned qualification OEG cell characteristic the cell same or like with primary cell, take the difference that growth conditions is good, be in exponential phase Cell from generation to generation, is performing centrifugal separation on (1 200r/min, 6min), with the frozen stock solution 0.5～1ml re-suspended cell containing dimethyl sulfoxide, Cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid nitrogen Frozen, the immortalization CD4+T cell bank building biological characteristics stable in this way is standby.

2. with the concrete grammar of SV40 immortalization CD4+T cell

(I) extraction of SV40 large T antigen DNA: (i) SV40DNA enzyme action: contain large T antigen gene from commercially available purchase SV40 freeze dried powder or SV40 plasmid, be dissolved in appropriate H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restricted enzyme BamH I (1-5U/ugDNA), 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, adds 5uL Electrophoresis sample loading buffer terminates reaction in case electrophoresis.(ii) SV40DNA electrophoresis: power taking swimming level agarose is made into electrophoretic buffer 10% agarose gel, pours on the gel casting platform sealed, plugs sample comb, removes after gelling is solid from glue platform Removing envelope band, extract comb, put in the electrophoresis tank added with enough electrophoretic buffers, buffer exceeds gel surface about 1mm, with suitable 10 × sample loading buffer of amount prepares DNA sample, is then added in sample well by sample with pipettor, and does suitably simultaneously DNA molecular amount standard control thing, connect electrode, make DNA face south Ghandler motion move, under the voltage of 1-10V/cm gel, electrophoresis is to enough During the distance of isolation of DNA fragments, close power supply.(iii) from agarose, separate about 2600bp SV40 large T antigen DNA: Under 300-360nm long wave ultraviolet light source, the gel-tape containing target DNA fragments is cut in loading bag filter, add in bag filter Enter 2ml electrophoretic buffer, be allowed to submergence gel, and empty steam bubble, bag filter level is put into electrophoresis tank, add appropriate amount of buffer solution By bag filter submergence (about 6-7mm), switching on power, 150 volts of electricity are washed, and observe and treat that DNA all removes gel, change under uviol lamp Direction of an electric field continues energising 1 minute, and from bag filter, sucking-off buffer is in 1-5ml Eppendorf pipe, adds 1.5 times of volumes N-butyl alcohol, mixing extracting removes EB, on desk centrifuge 2 minutes the most at a high speed, sucks upper strata butanol solution, so repeats two Secondary, in the solution of lower floor speech DNA, add equal-volume phenol chloroform (2) extract 2 times, supernatant proceeds in another Eppendorf pipe Add 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohol, in 20 DEG C overnight, 12000g, be centrifuged 10 minutes at 4 DEG C, DNA precipitation, abandon supernatant, abandon dry ethanol after adding 70% washing with alcohol 2 times, add 50 μ l TE dissolving DNAs.Additionally, can also be used with Target DNA fragment is separated from gel, is purified by low melting-point agarose gel method, DNA filter membrane inserted sheet method etc..

(II) connection of SV40 large T antigen DNA and pcDNA3.1 genophore: take 9 μ l above-mentioned DNA composition (0.1-5 μ g), 10 μ l 2 × connection buffer, 1 μ l 10mmol/L ATP, T4DNA ligase (20～500 sticky end unit) or large intestine bars Bacterium DNA ligase, pcDNA3.1 empty carrier mix, and 15 DEG C of incubation 24h are built into SV40T/pcDNA3.1 recon.

(III) SV40T/pcDNA3.1 recon amplification, separate and identify: the preparation of (i) E. coli competent: from 37 DEG C cultivate 16～20h fresh plate in one single bacterium colony of picking (such as bacillus coli DH 5 2), or 1ml fresh 16～20h Overnight culture, forwards in a 1L or 500ml flask containing 100mlLB culture medium, in 37 DEG C of violent shakings cultivate about 2～ 3h, measures OD600 value ≈ 0.4 every 20～30min, aseptically antibacterial is transferred to one, ice-cold 50ml In polypropylene centrifuge tube, place 10～20min on ice, be centrifuged 10min with SorvallGS2 rotary head with 4000r/min in 4 DEG C, To reclaim cell, culture fluid reciprocal, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, with 10ml ice pre-cooling 0.1mMCaCl₂Resuspended every part of precipitation, is put on ice, is centrifuged 10min with SorvallGS3 rotary head with 4000r/min in 4 DEG C, To reclaim cell, pour out culture fluid, pipe is inverted 1min so that the trace culture fluid of final residual flows to end, every 50ml initial incubation The thing 0.1M CaCl of 2ml ice pre-cooling₂Resuspended every part is sunk calmly, at this point it is possible to cell is distributed into rapidly aliquot, ice in liquid nitrogen Freezing ,-70 DEG C of storages are standby, take 200 μ l with the sterile pipette tip of cooling and transfer to aseptic from every kind of competent cell suspension In microcentrifugal tube, DNA or coupled reaction mixture (volume≤10 μ l, DNA≤50ng) should be added in often pipe, rotate gently with Mixing content, places 30min in ice, centrifuge tube is put into pre-heating to the test tube rack in the circulator bath of 40 DEG C, puts Putting 90s～2min, do not shake test tube, quickly transfer in ice bath by pipe, make cell cooling 1～2min, every centrifuge tube adds 800 μ lSOC culture medium, is warmed to 37 DEG C with water-bath by culture medium, is then transferred to by pipe on 37 DEG C of shaking tables, and incubation 45min makes antibacterial Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, competent cell proper volume converted is transferred to In SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, flat board is placed in room temperature and is absorbed to liquid, be inverted flat Ware, in 37 DEG C of cultivations, may occur in which bacterium colony after 12～16h.(ii) recon screening, expand and extract: with sterile toothpick or go out Bacterium Inoculating needle selects single colony inoculation in LB culture medium aseptic for 5mL or rich medium (such as super broth or the super meat of TB Soup culture medium) in, after overnight incubation, it is then added in the 500mL 2L flask containing LB culture medium (containing suitable antibiotic), then at Cultivate to saturation (OD for 37 DEG C₆₀₀≈ 4), the GTE solution containing 25mg/mL lysozyme that addition 1mL newly joins, resuspended precipitation, in Room temperature places 10min, adds 10mL and newly joins NaOH/SDS solution, and mixes until liquid becomes homogeneous, limpid and viscous gently Thick, place 10min on ice, add 7.5mL acetic acid solution, be gently mixed with suction pipe until viscosity declines and formed big sinking Forming sediment, place 10min on ice, in 4 DEG C, 20 000g are centrifuged 10min, and supernatant is poured into another clean centrifuge tube gently In, if there being visible drift by several layers of filtered through gauze, can add the isopropanol of 0.6 times of volume, reverse mixing, room temperature is placed 5～10min, in room temperature, 1 500g is centrifuged 10min, adds 2mL 70% ethanol and washs precipitation gently, the ofest short duration quickly from The heart, sucks ethanol, and is vacuum dried (precipitation can be 4 DEG C of long-term preservations).(iii) qualification of recon: above-mentioned big from competence The DNA (containing recon SV40T/pcDNA3.1) extracted in enterobacteria, ibid method restricted enzyme BamH I carries out enzyme action, 10g/L agarose gel electrophoresis is identified, it is thus achieved that 2 bands of size about 2600bp and 5600bp, the former meets in GenBank The size of SV40T fragment.

(VI) importing of SV40T/pcDNA3.1 and amplification culture: prepare following solutions in 1.5ml microcentrifugal tube: pipe A, is dissolved in SV40T/pcDNA3.1 in 100 μ l serum-free mediums (hyclone concentration is 20ml/L)；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, is mixed by pipe A and pipe B, the underlying 45min of room temperature.Use serum-free culture Liquid washs above-mentioned T lymphocyte 2 times.The training of 1ml serum-free is added in Lipofectamine-SV40T/pcDNA3.1 mixture Nutrient solution, mixes gently, then drops in above-mentioned T lymphocyte, and (hyclone concentration is to be subsequently adding 1ml serum-free medium 20ml/L), at CO₂Incubator cultivates 10h, sucking-off transfection liquid, adds 4ml complete culture solution (hyclone concentration is 20%), continues Continuous cultivation 20h, discards culture fluid, and changing concentration is 400mg L^-1G418 culture fluid continue cultivate, after 8 days select living cells After making amplification culture, then strengthen G418 concentration to 800mg L^-1, will can stablize the cell of growth in the G418 environment of high concentration Proceed amplification cultivation.Cultivate about 9 days Microscopic observations, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If it is thin Born of the same parents increase slowly, or cell density is low, or medium pH value is acidity, and culture fluid is partly measured in sucking-off, carries out equivalent oil changing.When total amount reaches Transferring in 75ml culture bottle during to 14ml, every 2-3 week adds 5-10ml fresh culture.Cell cultivates about 6-8 week the (the about the 55th Generation), still in logarithmic growth, i.e. incubation time and cell is accelerated in multiplication relation, and dead cell is less than 10%.Work as total amount When reaching 45ml, put in 50ml centrifuge tube, centrifugal 1500 turns, 10 minutes, abandon supernatant, add 3ml freezing media (5% diformazan Base Asia maple (dimethyl sufoxide), 95%FBS) mixing, (cell concentration is about 10 to become cell suspending liquid⁵/ml).Frozen Pipe subpackage, 1ml/ manages, puts-20 DEG C of 2h, then put-70 DEG C of 2h, the most frozen in-196 DEG C of liquid nitrogen (or under zero setting immediately 80 degree, Move in liquid nitrogen container after 1-2 hour).

(VII) qualification of immortalization CD4+T characteristics of cell biology: (i) observation of cell form: visible lymphocyte is obvious Increase, clustering phenomena occurs, there is lymphocyte blastogenesis feature.(ii) observation of cell growth curve: with incubation time as transverse axis, Cell quantity is the longitudinal axis (logarithm), be depicted on semi-logarithmic scale make after curve the growth curve of this cell, immortalization Cell line is formed in typical " S " feature or " arched roof "；(iii) chromosome is checked: by analyzing karyotype, if dyeing Body caryogram is diploid " 46, XX " or " 46, XY ", then illustrate that this cell line does not occur vicious transformation；(iv) flow cytometry Detection: the cell proportion that synthesizes, divide in detection the 19th continuous cell line, if its multiplication capacity substantially than do not build be normal the most carefully Born of the same parents strengthen, and explanation is the result that SV40 large T antigen is integrated, expressed.(viii) determined dna sequence: sequenator detection routinely, aobvious Show SV40 large T antigen DNA sequence.SV40 big T gene test in (v) transfectional cell DNA: as with Immunohistochemical detection, In the nucleus of SV40 transfection, the visible a large amount of brown particles of dyeing, show that SV40T antigen has been integrated into intracellular；(vi) mRNA table Reach product to measure: T antigen mRNA RT-PCR product checks order: takes the amplified production of 100 μ l systems, reclaims test kit with gel (Takara, Japan) reclaims product, takes 2 μ l DNA solutions and dilutes 100 times, surveys concentration, and remaining DNA and upstream and downstream primer are each 10 μ l check order.

(VIII) SV40LT gene mediated CD4+T cell bank: screen and continue to pass on, amplification culture accords with after above-mentioned qualification Close immortalized cells characteristic the cell same or like with primary cell, take that growth conditions is good, be in exponential phase The cell of different generations, is performing centrifugal separation on (1 200r/min, 6min), resuspended carefully with the frozen stock solution 0.5～1ml containing dimethyl sulfoxide Born of the same parents, cell density is 5 × 10⁵Individual/ml, adds cryopreservation tube, through 4 DEG C, 0.5h；-20 DEG C, 2h；-70 DEG C, overnight, enter-196 DEG C of liquid Nitrogen is frozen, and the CD4+T cell bank building biological characteristics stable in this way is standby.

(4) with the preparation of CD4+T cell identity function granule: can be by CD4 molecule, the CD4 molecule of gene recombinaton and similar The molecule of function by conventional chemical coupling, crosslinking, affine absorption etc. be fixed on carrier make be coated with CD4 molecule Grain, or directly take the replacement CD4+ cell application of intimate granule.It is thin that the CD4+T cell of the present invention represents other CD4+ Born of the same parents, prepare immortalization CD4+T cell including with additive method.

(3) preparation of HIV-1gp120 antibody

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

1, use lymphocyte Epstein-Barr virus to convert with hybridoma technology and combine and prepare HIV-1gp120 monoclonal antibody

Specimen origin have following several by way of: take lymphocyte strain frozen in Infectious Diseases Lab Sample Storehouse (through inactivation HIV immunity and the lymphocyte of EBV transfection)；Buy the fresh White Blood Cells Concentrate in blood station, then carried out inactivation HIV strain and exempt from The lymphocyte of epidemic disease；Take from the cord blood lymphocytes cell (through inactivation HIV immunity) preserved as scientific research；Directly take from HIV-1 The peripheral blood lymphocyte (for self) of the infected self, uses Histopaque lymphocyte separation medium separation single core thin Born of the same parents (PBMC), regulation concentration is 2x 10⁶Epstein-Barr virus (EBV) stock solution that rear addition is appropriate, is placed in 370C, 5%C02 overnight incubation, Preparing B cell to be hybridized, with the positive hole of ELISA method screening anti-HIV-1 outer membrane protein (gp120), transfer cell is to 24 orifice plates Continue to cultivate 2 weeks, repeat to measure anti-gp120 by ELISA method and confirm positive, continuously clone's secondary a large amount of amplification cultivation.Will After positive cell strain myeloma cell heterogeneous with people Mus (being buied by Zhejiang University's siberian crabapple) mixes (3: 1), add 1ml50% PEG makes the two merge, and then re-suspended cell cultivated liquid in IMDM culture fluid, within second day, adds Peritoneal Cells of Mice (by Zhejiang Jiang great Xue siberian crabapple is buied) as trophocyte, screen anti-gp120 antibody with ELISA after continuing to cultivate 3 weeks, select strong positive Hole hybrid tumor cell amplification is cultivated, and repeatedly clones until obtaining stable cell line, cultivates with this cell line, prepares HIV-1 antibody, uses ELISA detection kit, and by specification operates, and measures the Ig subclass of antibody, and surveys with conventional ELISA method Determine titer and the specificity of antibody, select high specificity, antibody that titer is high.

2, use gene recombinaton HIV-1gp120 to combine hybridoma technology and prepare antibody

(1) reagent and recombinant antigen: relate to reagent: HIV-1gp120 genetic fragment, HIV-1gp120 antigen, HIV antigen Nitrocellulose membrane bar is provided BamH I restriction endonuclease Xho I restriction endonuclease, nucleic acid coprecipitator, T4DNA by Beijing Wan Tai Pharma Inc. Ligase is purchased from precious biological company limited；Liagen glue reclaims test kit purchased from QIAquick company；RPMI 1640 dry powder is cultivated Base is purchased from Gibco company；Top grade new-born calf serum is purchased from bright marine growth Engineering Co., Ltd；SupersignalWest Pico Trial Kit, TMB Substrate Kit are purchased from Pierce company；Mice Ig subclass detection kit, freund adjuvant and PEG, Hy-poxanthine, Thymidine and Aminoptem are purchased from Sigma company.HIV-antibody diagnosing reagent kit is purchased from Shanghai section Biological company limited of China, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) labelling is purchased from doctor moral company limited. Construction of recombinant plasmid and qualification: vector plasmid PEGX-4T-2 BamH I, Xho I enzyme action, T4DNA Ligase connects gp120 Genetic fragment, recombinant plasmid transformed enters E.colistrain XL1 blue, checks order.The abduction delivering of recombiant protein and qualification: weight Group Plastid transformation enters in XL1-Blue escherichia coli, under IPTG derivant effect 25 DEG C, and 190r/min shakes, overnight, and 4 000r/min is centrifuged 10min, collects antibacterial, SDS-PAGE testing goal protein expression situation.Fusion protein purification and qualification: table Reach product centrifugal collecting precipitation to hang through PBS, after 30W Ultrasonic Pulverization instrument cracking antibacterial, be centrifuged collection supernatant and filter, filter Liquid AKTA PURIFYER100 protein purification instrument, GST column purification, obtain fusion protein GST-HIV, concentrates centrifuge tube and carries out dense Contracting, S21 type biology spectrophotometer measurement concentration, purifying protein is identified by SDS-PAGE.

(2) animal immune: BALB/c mouse 6 week old, female, 4, before immunity, take mouse vein blood, separation serum stays and does Negative serum.Mixed with equal-volume Freund's complete adjuvant by the GST-HIV fusion protein of 50-100 μ g, the injection of emulsifying pneumoretroperitoneum is exempted from Epidemic disease mice.After initial immunity, booster immunization mice after using incomplete Freund's adjuvant and fusion protein emulsifying every 2 weeks, immunity Dosage and approach are the same, repeat immunity 2-3 time, and the GST-HIV of last booster immunization direct lumbar injection 50-100 μ g melts Hop protein.

(3) foundation of Detection of Monoclonal Antibody: third time immunity one week after tail vein blood, determines positive serum by square formation method Best effort concentration and GST-HIV fusion protein be most preferably coated concentration.Operate as follows: according to 1: 1000,1: 500,1: 200,1: 100 four dilution factor, uses and is coated buffer dilution antigen, be longitudinally coated 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of bags By overnight, wash 3 times, every minor tick 3 minutes.Positive serum and negative serum are made doubling dilution respectively by 1: 1000, Laterally it is loaded onto the 10th hole, every hole 100 μ L, is placed in 37 DEG C of incubation 1h in wet box, wash 3 times, every minor tick 3 minutes.Enzyme mark resists Body HRP-sheep anti-mouse igg makees 1: 10000 dilution, every hole 100 μ L, 37 DEG C of incubation 1h to specifications, washs 3 times.Add people now to join OPD substrate solution, every hole 100 μ L, 37 DEG C of lucifuges reaction appropriate times, every hole adds 100 μ L stop buffers and terminates reaction, detects it OD492 value.Positive hybridoma cell screening technique is set up.According to experiment condition and method in square formation method, melt with GST-HIV respectively Hop protein is experimental group, and after abduction delivering, recombinant bacterium (containing plasmid pET-32a) albumen is matched group, screening positive clone.Operation Step is as follows: with the suitableeest concentration envelope antigen that is coated in 96 hole ELISA Plate, and 100 μ l/ holes, 4 DEG C of refrigerators are coated overnight.Take out bag Washed 3 times by adding cleaning mixture after plate, wash 3min every time；Every hole adds cell conditioned medium to be measured (1: 5 dilution) 100 μ L, 37 DEG C of temperature Case hatches 50min, and washing 3 times, wash 3min every time afterwards；Every hole adds two and resists 100 μ l, 37 DEG C of incubation 30min, washes Wash 3 times, wash 3min every time；Every hole adds the OPD substrate solution 100 μ L now joined, room temperature lucifuge reaction 10-15min；In every hole Add 2mol/L H2SO4 stop buffer 100 μ l to be used for terminating reaction；Detection plate is placed in microplate reader survey OD492 value.Matched group Set up: positive controls is the positive serum of suitably dilution, and negative control group is anti-with one to have the most dilution unrelated list Anti-cell conditioned medium.Indirect ELISA the selection result judges.Often group detection OD492 value, with P (sample value)/N (negative value) >=2.0 Person is judged to positive value.Screening positive clone standard: cell conditioned medium reacts in sun with just screening group (fusion protein is coated after purification) Property, react the detection hole being negative with negative screening group (tropina containing pET-32a plasmid after induction) be positive sample simultaneously Product.

(4) cell merges: myeloma cell prepares: merge the myeloma taking out a pipe the last week in liquid nitrogen container frozen thin Born of the same parents, are immediately placed in hot water and thaw.Adding appropriate complete culture solution after thawing, 1000r/min is centrifuged 3min；It is repeated 1 times.Will precipitation Thing moves in Tissue Culture Flask, adds DMEM culture fluid, puts CO2 incubator and cultivates, within 3-4 days, once passes on or amplification culture, Cell state is adjusted, it is ensured that before merging, cellular morphology is good, it is vigorous to grow in merging first 24 hours.Appropriate pancreatin is used before merging Using centrifuge tube to collect after digestion, add appropriate basal medium in centrifuge tube, after beaing mixing gently, 1000r/min is centrifuged 5-10min, repeated washing cell 2 times.Splenocyte prepares: before fusion, takes a Balb/c mice, wins eyeball and take blood, blood-letting Post-tensioning neck completely is put to death, and soaks in 75% ethanol.Taking-up layback is put and is fixed on dissection plate, takes spleen under gnotobasis, will Spleen moves in plate.Then 10mL RPMI 1640 basal medium is added at plate, with flat mouth tweezers attrition crushing repeatedly After, use asepsis injector repeatedly to aspirate piping and druming, make single cell suspension.After meter viable count, 1000r/min is centrifuged 10min, Add the basal medium total cell of adjustment and count to 1 × 10⁸～2 × 10⁸Merge for cell.Cell merges: by splenocyte and bone marrow Oncocyte is with 10: the ratio of 1-5: 1 adds in centrifuge tube, is mixed evenly, and 1000r/min is centrifuged 5min, supernatant discarded, strikes gently Beat at the bottom of pipe to cell without granular precipitate, be repeated 2 times.Preheating centrifuge tube, aseptic bar after taking-up is rotated gently in 37 DEG C of water-baths Under part, the 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe rotating centrifugal pipe the most gently along tube wall in 60s, Afterwards the basal medium of the 25mL of preheating is also added drop-wise in centrifuge tube along tube wall in 3-5min, light during adding Lightly rotating centrifugal pipe, is then statically placed in 37 DEG C of water-bath 10min, and 1000r/m is centrifuged 5min, supernatant discarded, adds 50mL HA T Culture medium.Suitably it is inoculated in 96 well culture plates after mixing, is placed in 37 DEG C, the CO2 incubator of 5% is cultivated.

(5) screening of positive clone strain: observe cell growth status in 96 well culture plates, only has hybridoma thin after 7-10 days Born of the same parents can grow division, now discards HAT culture medium, changes complete medium.Cell clone growth area reaches 1/10 carefully During hilum, go culture supernatant, cloned by the monoclonal antibody screening technique screening positive hybridoma cell set up before.Use improvement The positive cell hole that indirect ELISA Preliminary screening is gone out by limiting dilution assay gradient limiting dilution assay continuously carries out 3-4 wheel Sub-clone.Selection has the culture hole of the positive hybridoma cell strain that growth conditions is good, labelling cell strain growth under microscope Position, size, use aseptic rifle head to draw cell clone in the position of mark in the new culture hole having complete medium, so After successively doubling dilution count hole to below, 37 DEG C, cultivate about one week in 5%CO2 incubator, basis of microscopic observation cell grows Situation, when cell clone covers with to hole floor space more than 1/10, takes cells and supernatant and carries out antibody inspection side.To testing result Repeat next round dilution for the cell clone in positive culture hole to cultivate, repeat 2-3 wheel, after detection supernatant titer plateaus Take out, proceed to culture bottle mass propgation.

(6) preservation of hybridoma cell strain and Secondary Culture: preserve and recover: preserve first 12 hours and adjust cell growth shape State.Taking one bottle of growth vigorous, make cell suspension after the cell that form is good, suitably digestion, 1000r/m is centrifuged 5min, goes Clear liquid, flicks and makes cell loose at the bottom of pipe, add 4 DEG C preserve 9 parts of complete culture solutions and 1 part of DMSO, subpackage cell cryopreservation tube, 1mL/ manages, and cryopreservation tube is put in liquid nitrogen container after taking-up and saved backup by-70 DEG C of refrigerator overnight.40 DEG C of left sides are got out before recovery Right hot water, carefully takes out cryopreservation tube from liquid nitrogen, is immediately placed in hot water uniformly shake and makes cell thawing, after defrosting 1000r/m is centrifuged 5min, opens cryopreservation tube in superclean bench under aseptic condition, and the cell complete culture solution after thawing is washed Washing once, be then centrifuged 5min, supernatant discarded at 1000r/m, cell precipitation moves into training after using complete culture solution the most resuspended Support in bottle, put 37 DEG C, 5%CO2 incubator is cultivated.Secondary Culture positive hybridoma cell passed for 10 generations continuously, used indirectly The method of ELISA measures culture supernatant antibody titer, observes the change of titer, and whether observe this positive hybridoma cell strain can be steady Determine secretory antibody.

(7) preparation of monoclonal antibody mouse ascites: low-speed centrifugal collects the hybridoma after cultivating, dilute through basal medium Releasing cell number is 1 × 10⁷/mL.Mouse peritoneal injection 0.2mL/ only, observes mouse ascites production, treats that abdominal part is bright after injection Aobvious distension rises, and punctures abdominal cavity with syringe needle, and centrifuge tube collects ascites.Gather complete, mouse peritoneal is injected appropriate basal medium, Every 2-3 days, same method took ascites again.The ascites collected, 10000r/m is centrifuged 5min, Aspirate supernatant, subpackage ,-20 DEG C Preserve.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out with reference to " molecular cloning " method.Single Anti-subgroup identification selects mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody i.e. with kit measurement, grasps by test kit description Make.

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

(4) preparation of HIV-1gp41 antibody

Preparation with HIV-1gp120 antibody

(5) preparation of goat-anti people-Ig

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

1, laboratory animal: what immunity laboratory animal was selected is that animal institute of Zhejiang University hybridizes white goat, body weight 30 jin The healthy between twenty and fifty ewe two of left and right, the front dyestuff of immunity smears the back animal, makes clear and definite labelling.Employing is done as everybody else does The feeding manner of stable breeding, ensures that sheep has to appropriate motion every day, and between the lights, general half an hour of every time moving is left for movement time The right side, the most healthy and strong, it is to avoid sheep overfertilization, increase the immunity of body, drinking-water abundance, and give appropriate concentrate, Grass, Semen Maydis, wheat bran, Semen Tritici aestivi, vitamin etc. so that it is balanced in nutrition.Often check experiment sheep health status.

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

3, the immunity of goat: choose two goats, is labeled as goat A, goat B, antigen inoculation (HIV-1gp120 antibody and HIV-1gp41 antibody).Immunity position is front and back's groin, and often place's groin divides 2 some injections, a total of 8 injection points.Note The mode of penetrating is subcutaneous injection, and every goat per injection 4mL immunogen, containing 250 μ g antigens；Each injection point injecting immune is former 0.5mL, containing about 32 μ g antigens.Front two goats of immunity are drawn blood 10mL respectively, are labeled as 0dP1 ,-20 DEG C of preservations；Immunity for the first time I.e. 1, immunogen: 0.1mL antigen+1.9mL aseptic PBS+ Freund's complete adjuvant 2mL (CFA) is exempted from；Draw blood after immune 7 days 10mL, Separate serum, be labeled as 7dP1, ELISA and detect serum titer, remain serum-20 DEG C preservation.Within 3～4 weeks, start second time immunity, Two immunogens: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), draw blood after immune 7 days 10mL, separates Serum, is labeled as 7dP2, ELISA and detects serum titer, remain serum-20 DEG C preservation.After immunization time is 6-8 week for the third time, Three exempt from immunogen: 0.1mL antigen+1.9mL aseptic PBS+2mL incomplete Freund's adjuvant (IFA), and draw blood after immune 7 days 10mL, point From serum, it is labeled as 7dP3, ELISA and detects serum titer, remain serum-20 DEG C preservation.If now serum titer does not reaches 1 ∶10⁶Above, then need to exempt from again once；If serum titer has reached 10⁶Below then need not be immune again, draw blood the most week about 50mL, separates serum ,-20 DEG C of preservations.

4, prepared by serum: within after general each immunity 7～10 days, can detect in sheep jugular vein blood collection.By assistant Baoding Animal so that it is keep stance, after cervical region cropping, aseptic cotton balls cleaning disinfection, searches out the blood sampling of jugular vein hand syringes, Blood 5-10mL is taken by fixing for syringe position.Bioactivity is carried out after isolating serum.7～10 days after the third immunization, warp The most desirable blood 30-50mL after bioactivity is qualified.Aseptically separate serum, in plate to be collected in or triangular flask After blood coagulation, after clot being peeled off with bottle wall in gnotobasis (such as superclean bench) with aseptic dropper, put into 37 DEG C, 1～2h take out after put into 4 DEG C overnight, make serum fully separate out (can not freeze, otherwise produce haemolysis), by centrifugation precipitation point Go out serum, put cryogenic refrigerator into and preserve.Before using must through signing qualified after again subpackage save backup.

5, the mensuration of antiserum titre: antiserum titre is to use ELISA assay method: be with being coated when being coated by sample After liquid is diluted to the concentration of 1: 1000, in ELISA Plate, every hole adds 100 μ L, is then placed in aluminum box and puts in 4 DEG C of refrigerators overnight. Take out the next morning to pat dry and be coated liquid, wash three times with PBST, every minor tick five minutes, pat dry enzyme with gauze for the last time Target, adds the confining liquid of 10% serum, and every hole adds 100UL, puts into 37 DEG C, water-bath 1～2h.Take out the most again and pat dry closing Liquid, with the every minor tick of PBST detersive enzyme target 3 five minutes, pats dry with gauze for the last time, adds 100 μ L/ holes one anti-(1: 2000 Dilution, dilutes with the PBST of 4% Ox blood serum, puts into 37 DEG C, water-bath 1～2h).Taking out and pat dry an anti-liquid, PBST washes Wash ELISA Plate three times, every minor tick 5 minutes, pat dry ELISA Plate with gauze for the last time, add two anti-liquid (the anti-sheep of rabbit 1: 1000), Every hole adds 100 μ L, puts into 37 DEG C, water-bath 1～2h.Take out again and pat dry the two anti-every minor ticks five of liquid PBST detersive enzyme target 3 Minute, patting dry with gauze for the last time, every hole adds 50 μ L substrate nitrite ions, and black out adds the H SO of 2M after developing the color 10-20 minute Solution 50 μ L terminate reaction, after with microplate reader survey OD value (in half an hour).

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

1, the preparation of blood purification cell column

Clean the made hybridoma macrophage of the present invention and CD4+T cell with physiological saline solution, 1000r/min from The heart, is centrifuged 5min with 1000r/min again after cleaning, is 1: 0.5～3 by the ratio of hybridoma macrophage strain and CD4+T cell strain Ratio take sedimentation cell, load in the 200ml hydrostatic column that acrylate etc high-biocompatibility material is made, filling To 4/5, cell column is sealed standby.

2, the outfit of blood purification gel antibody

Gp120 and gp41 antibody is mixed with goat-anti gp120 and gp41 antibody respectively, makes the goat-anti gp120 in mixed liquor All it is fully tied with gp41 antibody, but surplus free gp120 and the gp41 antibody having sufficiently high titre, then will be containing combining Type and the mixed antibody of sequestered gp120 antibody and the mixed antibody containing conjunction type and sequestered gp41 antibody again with etc. Titer mixes, and is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies with the agarose C1-4B solution of gradient concentration respectively, The titer making HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and the agarose concentration of correspondence It is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, irrigates standby blood with equal equal-volume from low to high by antibody titer Liquid daf molecule post and make the depurator containing cleanser.The filter opening that agar gel is formed is along with increasing of agarose concentration Reducing, depurator import department agarose concentration is low, and filter opening is just big, beneficially plasma perfusion and high titer antibody or cell and HIV Association reaction；And exit concentration is high, filter opening is the least, it is easy to detention HIV or Large molecular conjugates.

Formula one: by gp120 and gp41 antibody with play being incubated at 39～42 DEG C after 100 DEG C dissolve of carrier function 0.7%, the agarose C1-4B normal saline of 0.8%, 0.9%, 1.0%, 1.1% is made into the most corresponding 1: 700,1: 500,1 : 300, the gel antibody of the gradient titre of 1: 200,1: 100, take 40ml gel antibody successively by the paramount titre of low titre and add blood In liquid daf molecule post, it is desirable to the gel antibody being initially charged be cooled to 37 DEG C become semi-solid agar gel after the most then add under Once, make the blood purification cell column form antibody titer from high to low from top to bottom (from import to outlet) and from low to high The layer distributed of agarose concentration, wherein with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120 antibody, Gp41 antibody and hybridoma macrophage and/or CD4+T cell are all fixed in gel the effect playing absorption HIV.

Formula two: by gp120 and gp41 antibody with rise carrier function after 100 DEG C dissolve, be cooled to 39～42 DEG C After the agarose C1-4B of 1% content is made into the titre gradient of 1: 50～1000 by multiple proportions, Reperfu-sion blood purification cell column, make Blood purification cell column forms antibody titer from high to low and agarose from low to high from top to bottom (from import to outlet) The layer distributed of concentration, wherein resists with goat-anti HIV (gp120 and gp41) antibodies and unconjugated gp120 antibody, gp41 Body and hybridoma macrophage and/or CD4+T cell are all fixed in gel the effect playing absorption HIV.

Formula three: indicate the titre being made into by multiple proportions of the different antibody prepared by HIV strain and correspondence thereof respectively The valence value of gradient, such as HIV strain 1 titre 1: 100 pipe, 1: 200 pipe ...；HIV strain 2 titre 1: 100 pipe, 1: 200 pipes ..., the rest may be inferred, then will infect strain 1 titre 1: 100 pipe and 10ml39～the liquid agarose C1-of 42 DEG C of insulations Put into blood purification cell column after 4B mixing, to be placed be cooled to semi-solid gel after, then will infect strain 1 titre 1: 200 pipe with The liquid agarose C1-4B mixing of 10ml39～42 DEG C of insulations, mixing liquid puts into the gel upper strata of blood purification cell column, according to this Analogize.A, B, C totally 3 strain is had in actual applications by combination preparation, the HIV strain in such as somewhere period, preparation Antibody has A strain 1: 100 pipe, 1: 200 pipe ...；B strain 1: 100 pipe, 1: 200 pipe ...；C strain 1: 100 pipe, 1: 200 pipe ...；Warp After combination be exactly ABC strain 1: 100 pipe, ABC strain 1: 200 pipe ... ABC strain 1: 1000 manage, then by ABC strain 1: 1000 pipe with Put into blood purification cell column after the liquid agarose C1-4B mixing of 10ml39～42 DEG C of insulations, to be placed be cooled to semisolid After gel, then the liquid agarose C1-4B mixing by ABC strain 1: 900 pipe with 10ml39～42 DEG C of insulations, mixing liquid puts into blood Being cooled to semisolid gel upper strata in daf molecule post, the rest may be inferred, and centre can be spaced selection, the most then selects ABC Strain 1: 500 pipe mixes with the liquid agarose C1-4B of 10ml39～42 DEG C of insulations, and mixing liquid has put into blood purification cell column Being cooled to semisolid gel upper strata, the consumption of liquid agarose C1-4B (can also be made by adjusting in 1ml～10ml The blood purification cell column become, from import to going out interruption-forming gradient antibody content from low to high, in view of antigen and antibody only Just can play immunoreation when proper ratio, form precipitation line and stop moving ahead of comparator antibody or antigen, so different HIV contains When the blood plasma of amount is by cleanser, HIV just adsorbed detention in different aspects, and also the HIV strain of the present invention is permissible The most almost all of infection strain of letter lid, and be combined with goat-anti Ig in adsorbent and unconjugated gp120 and gp41 antibody Being all the HIV aglucon being fixed in advance in Agar Gel, HIV antibody is tied with HIV especially in conjunction with the HIV antibody having goat-anti HIV Close the immune complex molecular weight that formed bigger, be entirely capable of being delayed in Agar Gel and be eliminated, add aperture about For 85nm Agar Gel inherently can the detention a diameter of 100～HIV of 120nm, so inferring theoretically and having hardly The HIV sufferers failed to respond to any medical treatment).

3, the specification of blood purification and material

Blood purification cell column or blood purification are the cylinder that footpath, the end is little, footpath, top is big, or square, infundibulate, volume Being 200～300ml, import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh；Footpath sieve mesh at the bottom of exit Number is 2.0～5.0 mesh (2.5～5.0 mesh are equivalent to 0.1～0.2 micron or 100～200 nanometers), specifically can be made into 2.0 mesh, 7 kinds of different sizes of 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh, in order to stop 120 nanometers inhibition of HIV or Bigger antibacterial；Liquid outlet arranges the cell strainer that mesh number is 100 mesh (being equivalent to 4 microns), may leach in order to stop Cell；Relief area, the beneficially stability of system circulation it is provided with between liquid entrance and mesh screen.

Blood purification selects the macromolecular material of acrylate etc, it is desirable to good biocompatibility, activates benefit hardly Body, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can pass through covalency, The methods such as grafting, polymerization improve the structure of material, the regulation inhomogeneities on surface, hydrophilic, minimizing to blood coagulation and oxidative stress Impact thus improve biocompatibility, reduce complication generation.The material that some has anticoagulation is solidificated in carrier Or on the material of adsorber inner surface, blood coagulation can be suppressed, improve biocompatibility, also can reduce heparin consumption, and have can No-rod tractor can be realized.As being aggregated in by heparin on polyacrylonitrile-polymine film, effect may be more preferable, and can reduce Anaphylaxis during absorption, the polyacrylonitrile surface of solidifying shell polysaccharide and heparin covalent thing also show good blood compatibility Property, and the activity of pseudomonas aeruginosa can be suppressed, reduce cell-cytotoxic reaction.

Three, the preparation of plasma separator

1, preparation principle: prepare according to the molecular size of hemocyte and blood plasma components.Such as visible component (blood in blood of human body Cell) size be: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leukocyte is divided into 5 kinds, neutral Granulocyte about 12 μm, eosinophilic granulocyte is more bigger, and basophilic granulocyte is close with neutrophilic granulocyte, small lymphocyte 6-8 μm, Approximating with erythrocyte, mononuclear cell is maximum, about 15-20 μm.Platelet is disc, and diameter 1～4 microns are to 7～8 microns not Deng, the platelet mean diameter of people is 2-4 micron, thick 0.5～1.5 micron.

2, material: can be selected for poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc., it is desirable to good biocompatibility, activate hardly Complement, do not cause inflammatory reaction, do not cause the change of leukocyte, platelet, blood oxygen pressure, complement C 3, C5a.Can be by altogether Valency, be grafted, the method such as polymerization improves the structure of material, the regulation microinhomogeneities on surface, hydrophilic, minimizing be to blood coagulation and oxygen Change stress impact thus improve sieving adequacy and biocompatibility, the generation of minimizing complication.

3, type and spec: for the profile of separator, can prepare as filter element with the material such as acetate fiber or absorbent cotton Become column construction, be prepared as the shapes such as flat structure with materials such as poly-vinegar non-woven fabrics as filter element；By hemocyte to be separated and blood The molecular size of slurry composition determines aperture.Plasma separator stable in properties involved in the present invention, good biocompatibility, penetrating Hollow fibre type filter made by the high molecular polymer that property is high, hollow-fiber film a diameter of 270～370 μm, and film thickness is 50 μm, Aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm.This hole is only permitted blood plasma and is filtered, but can stop that all of cell becomes Point.

Four, the component of AIDS therapeutic equipment

1, key member: (1) plasma separator: for washed corpuscles and blood plasma；(2) blood purification: be used for adsorbing HIV in blood plasma.

2, additional member: include blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The part compositions such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring.(1) blood pump (Blood Pump): be used for promoting blood Circulating to maintain being smoothed out of blood purification treatment, usual blood pump part often has rotary test speed function, to monitor patient Blood circumstance, therefore blood pump runner and flute pitch set and want accurately and it needs to often adjust, according to the feelings of bloody path pump line Condition, is typically set as 3.2～3.3mm by spacing, can not be the most loose, and blood flow detection otherwise can be caused to be forbidden；Also can not be too tight, otherwise Pipe breakage can be caused.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump applied clinically, uses To continue injecting heparin in sieving pipeline (patient blood), owing to the blood of patient circulates and air contact, easily in vitro There is blood coagulation phenomenon, use heparin pump to be possible to prevent the generation of blood coagulation.(3) sound pulse pressure monitoring: arterial blood pressure monitoring mainly in order to The stopping state of dynamic monitoring blood separator micropore, additionally in order to monitor the change of extracorporeal circulation thrombosis, solidification and pressure.When During blood flow deficiency, arterial pressure will reduce；When having blood coagulation, thrombosis, particularly during separator blockage of the micro orifice, arterial pressure will Raise；Venous pressure monitoring is used for monitoring the pressure of pipeline blood backflow, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow When deficiency and venous return syringe needle come off, venous pressure will decline, if the distortion blocking of bloody path return duct or backflow syringe needle When there is blocking, venous pressure will raise.(4) air monitering (Air Detector): be used for monitoring the air gas of blood pathway Bubble, the principle of general ultrasonic listening, in order to avoid patient occurs air embolism to arrange.When having monitored air bubble Time, detecting system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent the generation of danger.

In a word, on the basis of key member of the present invention and additional member, Import computer regulates and controls and makes the people of operation Property, the personalization for the treatment of, the safety of design, and modularity, automatically monitoring and regulation and control, liquid crystal display, judge voluntarily to warn The blood purifying therapeutical instrument that the report micro computer such as reason and ring off signal processes.

Five, the connecting path of AIDS therapeutic equipment and using method

1, install: such as Fig. 1, with sterile working's connecting components, including plasma separator, depurator and each circulation line.

2, aerofluxus: with physiological saline solution topping up separator, depurator and each circulation line, gets rid of separator, depurator And gas in circulation line, bubble, go through, confirm without use after gas, bubble.

3, logical liquid: arterial blood line pipe (1) is connected the arteries of HIV sufferers, the row of going through the most again Gas is the most complete, and liquid stream is the most unobstructed, and avoids managing the pollution of interior flow liquid.

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

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) is connected venous blood Pipe, then opens blood pump (2), and blood flow is 100～150ml/min, and such as Fig. 1, arterial blood enters through arterial blood line pipe (1) Plasma separator (4), the blood plasma separated arrives depurator (8) through circulation line (7) under the effect of blood plasma pump (6), treats Be full of blood plasma, about 10 minutes, begin paying out blood plasma, through circulation line (10) flow out, synchronize to depurator (9) irrigate blood plasma, When blood plasma in depurator (8) has nearly flowed, starting again at perfusion blood plasma, now depurator (9) begins paying out blood plasma, and two also The depurator (8) of connection, depurator 9) alternately, the blood plasma after purification is divided through circulation line (10) and plasma separator (4) From hemocyte converge after through venous line (5) feed back.Such as Fig. 2, when blood to be separated enters the inner chamber of plasma separator (1) (2), time, through the effect of valve (8), the little molecule blood plasma components (5) that can pass through micropore (3) enters the exocoel (6) of separator, so Flow out by plasma outlet port (7), and the hemocyte (4) that can not pass through micropore (3) flows out through valve (8).Such as Fig. 3, when containing HIV (1), when blood plasma enters depurator, HIV antibody (2) that HIV therein (1) is fixed in agar gel (8) respectively, huge bite Cell (4), CD4+T cell (6) are combined into antigen antibody complex (3), macrophage phagocytic body (5), CD4+T cell conjugates (7), combined after HIV no longer move down, the most combined 100～HIV of 120nm are again by because of concentration more Gao Erkong The bottom agar gel molecular Screen Out of 1% concentration being about 85nm that footpath is less stays (9) place.So remove HIV, until in advance The plasma circulation amount (usually 9L) set, treatment just ends.Whole therapeutic process is by computer control, and can examine at any time Survey duty, easy to use, automatization and safety.

Six, the checking of AIDS therapeutic equipment effect

1, the checking of CD4+T cell clearance HIV effect

In order to verify effect of CD4+T cell clearance HIV, the present invention devises easy method of testing: take the 2.5 of sterilizing × 300mm Westergren's blood sedimentation tube 5, draws by centrifugation the CD4+T cell that (1000r/min, 5min) precipitate respectively and carves to 200mm Degree, then draws and is incubated after 100 DEG C dissolve at 39～42 DEG C of 0.9% standby agarose C1-4B, reaches about 10mm length and carves Degree, after putting blood sedimentation stand cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not stop the water of little molecule Pass through with the material of chemical analysis etc.The 5 of the HIV sufferers that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve Example blood plasma, the most about 10mL, before respectively taking 9mLAIDS filter, blood plasma injects blood sedimentation tube upper end blank pipe, blood sedimentation tube lower floor to be flowed through in batches CD4+T cellular layer and in blood sedimentation tube flow out after, collect effluent, referred to as AIDS filter after blood plasma.Take AIDS filter before blood plasma and Blood plasma after filter, operates according to HIV-1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai), With concentration known 0pg/ml, the p24 of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml Antigen is less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ as comparison, lowest detectable limit In ml, 15min, 450nm measures absorbance (OD), and blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not Higher than 0.100,1000pg/ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, testing result (table 1) illustrating, after AIDS blood plasma filters the simple purifier containing CD4+T cell, part HIV is adsorbed by CD4+T cell, through the 1st After secondary filtration, HIV total body clearance is 22.84%, and after the 2nd time filters, total body clearance is 35.31%, after the 3rd time filters, Total body clearance is 41.9%.Illustrate that HIV can constantly be removed along with the increase filtering number of times, thus reach to treat AIDS mesh 's.

Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after the simple purifier containing CD4+T cell

2, hybridoma macrophage removes the checking of HIV effect

In order to check cross tumor macrophage removes effect of HIV, the present invention devises easy method of testing: take sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draw by centrifugation the macrophage hybridoma that (1000r/min, 5min) precipitates respectively Cell strain, to 200mm scale, is then drawn and is incubated at 39～42 DEG C of 0.9% standby agarose C1-4B after 100 DEG C dissolve, Reaching the long scale of about 10mm, after putting blood sedimentation tube cooling, agarose becomes semi-solid, blood sedimentation tube inner cell can be stoped to flow out but will not The material of the water and the chemical analysis that stop little molecule etc passes through.Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab Sample Storehouse preserve 5 example blood plasma, the most about 10mL of acquired immune deficiency syndrome (AIDS) (AIDS) patient, before respectively taking 9mLAIDS filter, to inject blood sedimentation tube in batches (the cleanest for blood plasma Gasifying device) upper end blank pipe, wait flowing through the hybridoma macrophage strain layer of blood sedimentation tube lower floor and after flowing out in blood sedimentation tube, collect stream Go out liquid, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, with MIF (MIF) ELISA detection kit (stamen bio tech ltd, Shanghai hundred) pairing detection, by specification operate, detection range be 0～ 800pg/ml, sensitivity is 1.0pg/ml, directly can detect by an unaided eye: in reacting hole, color is the deepest under white background, positive The strongest, negative reaction is colourless or the most shallow, according to the depth in color, with "+", "-" number represents.Also OD value can be surveyed: On ELISA detector, in 450nm (if developing the color with ABTS, then 410nm) place, after returning to zero with blank control wells, survey each hole OD value, if More than 2.1 times of the negative control OD value of regulation, it is the positive.Result such as table 1, before filter, in blood plasma, MIF testing result is the moon Property (or because content preserves cause degraded etc. for a long time less than detection sensitivity, blood plasma), and after filtering, in blood plasma, testing result is the positive. Illustrate that macrophage hybridoma cell strain creates MIF cytokine in this process.MIF is collection cytokine, somatomedin, swashs Element and enzyme characteristic multi-effect protein molecular, the regulatory factor as inherent immunity and inflammatory reaction plays central Effect, plays panimmunity function in various infection and active chronic inflammation disease.Simple purification is filtered making AIDS blood plasma Before and after device while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, tries according to HIV-1p24 Detection of antigen Agent box (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operates, with concentration known 0pg/ml, 0.5pg/ml, 1pg/ The p24 antigen of ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is less than as comparison, lowest detectable limit 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, in 15min, 450nm measures absorbance (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ml absorbance Being not less than 1.000, be considered as positive as absorbance ＞ 0.12, result (table 2) illustrates that AIDS blood plasma filters and simply purifies dress Postponing, part HIV is by the phagocytosis absorption of macrophage hybridoma cell strain, and the blood plasma HIV after filtration significantly reduces, through the 1st time After filtration, HIV clearance rate is 20.55%, and after the 2nd time filters, HIV clearance rate is 42.83%, p ＜ 0.01, has significantly Effect, illustrates that the increase HIV along with filtering number of times can constantly be removed, thus reaches to treat AIDS purpose.

Table 1 AIDS blood plasma filter MIF testing result before and after the simple purifier containing hybridoma macrophage (quantitatively: pg/ml)

Table 2 AIDS blood plasma filters p24 testing result (p24:pg/ before and after the simple purifier containing hybridoma macrophage ml)

3, HIV-1gp120 antibody, the checking of gp41 cleaning antibody HIV effect

The present inventor, according to the basic skills of the present invention, has done following simple confirmatory experiment: take HIV-1gp120 antibody, Gp41 antibody (Rui Qi bio tech ltd, Shanghai), joins and is incubated after 100 DEG C dissolve at 50 DEG C of 1.0% standby fine jades In lipolysaccharide C1-4B, after mixing, titre is 1: 300～500, takes 2.5 × 300mm Westergren's blood sedimentation tube 5 of sterilizing, draws respectively 1.0% agarose C1-4B solution is to 200mm scale, and after cooling, agarose becomes semi-solid.Ling Qu Disease Control and Prevention Center and infectious disease are real Test the specimen of the 5 example HIV sufferers that room Sample Storehouse preserves, remove each about 10mL of the blood plasma after cell, respectively take blood before 9mLAIDS filter Slurry injects blood sedimentation tube upper end blank pipe in batches, the 1.0% agarose C1-4B containing antibody of blood sedimentation tube lower floor to be flowed through from blood After flowing out in immersed tube, collect effluent, blood plasma after referred to as AIDS filter.Take blood plasma after the front blood plasma of AIDS filter and filter, according to HIV- 1p24 antigen detection kit (euzymelinked immunosorbent assay (ELISA), inspiration bio tech ltd, Shanghai) operate, with concentration known 0pg/ml, The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml is as comparison, minimum Detection limit is less than 5pg/ml, measurement range 0～400pg/ml, range of linearity 0.5pg/ml～80pg/ml, and in 15min, 450nm surveys Determining absorbance (OD), blank calibration object absorbance is not higher than 0.050, and 0pg absorbance is not higher than 0.100,1000pg/ Ml absorbance is not less than 1.000, is considered as positive as absorbance ＞ 0.12, and testing result (table 1) illustrates, AIDS blood plasma is filtered After crossing simple purifier, part HIV is adsorbed by corresponding antibodies, and after the 1st time filters, HIV total body clearance is 20.01%, After the 2nd time filters, total body clearance is 27.99%, and after the 3rd time filters, total body clearance is 37.36%.Illustrate along with filtration The increase HIV of number of times can constantly be removed, thus reaches to treat AIDS purpose.

Table 1 AIDS blood plasma filters p24 testing result (pg/ml) before and after simple purifier

Above-mentioned confirmatory experiment shows, HIV free in blood plasma can (HIVgp120 antibody, HIVgp41 be anti-by blood purification agent Body, CD4+T cell strain, hybridoma macrophage strain, agar gel micropore) remove, show the AIDS therapeutic equipment tool of the present invention There is the therapeutic efficiency significantly removing blood plasma HIV.

Claims

1. the AIDS therapeutic equipment for medical domain, it is characterised in that by made HIV antibody, combine goat-anti Ig's HIV antibody, hybridoma macrophage strain and CD4+T cell strain mixed preparing, in agar gel, irrigate high-biocompatibility material The exit made arranges the depurator of screen cloth, make import department to exit form antibody titer from high to low and from low to high Agarose concentration layer distributed but daf molecule accounts for 4/5, with can the separator of separated plasma and hemocyte constitute external only The main part of gasifying device, for removing the HIV in blood plasma.

AIDS therapeutic equipment the most according to claim 1, it is characterised in that described depurator by the screen cloth in its exit, Play the fixing and agar gel of molecular sieve effect, prepare hybridoma macrophage strain therein, CD4+T cell strain, HIV antibody, The HIV antibody combining goat-anti Ig collectively forms molecular sieve mechanical removal and cell and the barrier of antibody mediated immunity removing blood plasma HIV.

3. according to the arbitrary described AIDS therapeutic equipment of claim 1,2, it is characterised in that the volume of described depurator is 200 ～300ml, to import and export and be equipped with cell screen cloth, footpath, import department top sieve number is 800 mesh, and at the bottom of exit, footpath sieve number is 2.0～5.0 mesh, liquid outlet arranges the cell strainer that mesh number is 100 mesh, arranges promotion system between liquid entrance and screen cloth The relief area of system stable circulation.

AIDS therapeutic equipment the most according to claim 3, it is characterised in that described exit screen cloth make 2.0 mesh, 2.5 7 kinds of specifications of mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh.

AIDS therapeutic equipment the most according to claim 1, it is characterised in that the layering of described antibody titer from high to low Distribution refers to from the mixed antibody titer of the import department of depurator to exit successively with 1: 700,1: 500,1: 300,1: 200,1: 100 points 5 layers.

AIDS therapeutic equipment the most according to claim 1, it is characterised in that dividing of described agarose concentration from low to high Layer distribution refer to from the agarose concentration of the import department of depurator to exit successively with 0.7%, 0.8%, 0.9%, 1.0%, 1.1% point 5 layers.

7. according to the arbitrary described AIDS therapeutic equipment of claim 1,2,5, it is characterised in that described HIV antibody includes HIV- 1gp120 antibody, HIV-1gp41 antibody.

AIDS therapeutic equipment the most according to claim 1, it is characterised in that described separator hollow-fiber film is a diameter of 270～370 μm, film thickness is 50 μm, and aperture is 0.2～0.6 μm, and fibre length is 13.5～26 μm, can stop all of carefully Born of the same parents filter.

9. the preparation for the AIDS therapeutic equipment cleanser of medical domain, it is characterised in that clear with physiological saline solution Washing made hybridoma macrophage and CD4+T cell, 1000r/min is centrifuged, and is centrifuged 5min with 1000r/min again, presses after cleaning The ratio that ratio is 1: 0.5～3 of hybridoma macrophage strain and CD4+T cell strain takes cell precipitation and loads high-biocompatibility material In the 200ml hydrostatic column made of material, make cell column, separately by gp120 and gp41 antibody respectively with goat-anti gp120 and gp41 Antibody mixes, and makes the goat-anti gp120 in mixed liquor and gp41 antibody all be fully tied, but surplus has the free of sufficiently high titre Gp120 and gp41 antibody, then by containing conjunction type and the mixed antibody of sequestered gp120 antibody and containing conjunction type and trip The mixed antibody of release gp41 antibody again with etc. titer mixing, be made into final mixed antibody, respectively with the agar of gradient concentration Sugar C1-4B solution 5 kinds of gel mixed antibodies of preparation, make the titer of HIVgp120 and gp41 antibody be 1: 700,1: 500,1: 300,1: 200,1: 100 and the agarose concentration of correspondence is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, imitate by antibody Valency irrigates standby cell column with equal equal-volume from low to high and makes depurator, makes from import to going out interruption-forming from high to low anti- Body titre and the layer distributed of agarose concentration from low to high but daf molecule accounts for 4/5, wherein hybridoma macrophage, CD4+ T cell and HIV antibody are all fixed in gel the effect playing absorption HIV.

10. according to the application in preparing purging in vitro device of the claim 1-8 arbitrary described AIDS therapeutic equipment, its feature Being, described purging in vitro device includes that one end of arterial blood line pipe (1) separates with blood plasma with blood pump (3) through heparin pump (2) Device (4) is connected, and plasma separator (4) is through blood plasma pump (6) and circulation line (7) depurator (8) in parallel with two, depurator (9) it is connected, confluxes through circulation line (10), venous line (5) the most successively.