CN106267416B

CN106267416B - AIDS therapeutic equipment

Info

Publication number: CN106267416B
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: 2019-02-19
Anticipated expiration: 2036-07-01
Also published as: CN106267416A

Abstract

A kind of AIDS therapeutic equipment for medical domain, it is characterized in that the separator of preparation energy washed corpuscles and blood plasma, it prepares HIVgp120 and gp41 antibody and combines HIVgp120 the and gp41 antibody of goat-anti Ig, respectively with foreign gene transfection and hybridoma technology building not only retained archaeocyte characteristic again energy indeterminate growth CD4+T cell strain and hybridoma macrophage strain, it is formulated in agar gel after amplifying cells strain and clarifier is formed with high-biocompatibility material package, gel is set 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 mating type and sequestered gp120 and gp41 antibody, CD4+T cell strain, the strain of hybridoma macrophage is fixed on agar gel absorption HIV and is used , made clarifier is applied in combination with separator and regulatory process, and extracorporeally circulating blood is divided into blood plasma and haemocyte by separator, and the purified device of blood plasma, which filters out, converges feedback with haemocyte after HIV.

Description

AIDS therapeutic equipment

Technical field

The present invention relates to the preparation of AIDS therapeutic equipment in medical domain and applications, are mainly used for AIDS patient's blood plasma Chinese mugwort The removing of virus is grown, to achieve the purpose that treat AIDS.

Background technique

It after HIV enters human body, is swallowed first by macrophage, but HIV changes certain positions in macrophage quickly Acidic environment creates the condition for being suitble to it to survive, is not killed not only and breeds in it instead.Because CD4 be HIV by Body, so the HIV bred in macrophage is through its envelope protein gp120 and under the auxiliary of gp41, (gp41 plays bridge Effect, utilizes itself hydrophobic effect mediate retroviral cyst membrane and cell membrane fusion) entering CD4+ cell, (cell, monokaryon macrophage are thin Born of the same parents, Dendritic Cells etc.), it is proliferated rapidly in the cell, generates 10 daily⁹~10¹⁰Virion, and it is normal constantly to enter other And regenerated CD4+ replicate into the cell, manufacture more virus infected cells, make peripheral blood CD4+T cell sustaining breakdown, subtract It is few.The T cell of infected Apoptosis or even infected by HIV can be directly activated to express in conjunction with gp120 and the CD4 receptor of HIV Envelope antigen can also start normal T-cell, cause a large amount of broken of CD4+ cell indirectly by cell surface CD4 molecule cross-link Bad, as a result cause the severe immune deficiency centered on CD4+T cell defect, patient mainly shows: periphery lymphocyte is reduced, T4/T8 proportional arrangement disappears to the reaction of phytohemagglutin phytolectin and certain antigens, delayed allergy decline, NK cell, macrophage Cell activity weakens, and the synthesis of the cell factors such as IL2, interferon is reduced.CD4+T cell is most important immunocyte, infection Person once loses a large amount of CD4+T cells, and entire immune system will all lose the infection of various diseases by deathblow Go resistance.HIV can also show as hiding without showing clinical symptoms, genome for a long time after entering host's CD4+ cell RNA reverse transcription enters in host cell nuclear at double-stranded DNA with viral integrase enzyme, under the action of integrase, double-stranded DNA integration Into host cell gene group, the viral DNA being integrated is known as provirus, and the several months that can hide does not replicate even for many years, causes The incubation period of AIDS several months to many years.In the incubation period of AIDS, HIV is mainly in the macrophage and Dendritic Cells of lymph node Breeding, these cells are intracorporal HIV depots, and releasably the CD4+T cell into peripheral blood or transfection peripheral blood, there is silk point Splitting original, antigen, TNF, IL-2 and lymph plain (LT) can excite HIV provirus gene multiple in the CD4+T Intracellular transcription of infection System.After being largely proliferated, inhibition of HIV particle constantly discharges from the infection cell being destroyed and is free on blood, then enters back into Other cells continue course of infection, and inhibition of HIV particle is discharged from the cell of infection to before extracellular, are expressed in infection cell table Merging between gp120 and gp41 the energy mediated infection cell and non-infected cells in face, such as the CD4 of infected by HIV⁺T cell table The gp120 reached leads to cell fusion and forms multinucleate giant cell in conjunction with the CD4 of non-infected cells.

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

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

Ag-Ab precipitation reaction was initially applied to research Liesegang's phenomenon in 1905 in gel, applied within 1932 In identification bacterium bacterial strain, nineteen forty-six Oudin carries out immunodiffusion in test tube, for analyzing antigen mixture, 1948 Elek and Ouchterlony establishes the two-way double-diffusion process of agar respectively, for identifying simultaneously, more two or more antigens or anti- Body.The media gel agar or agarose of Ag-Ab precipitation reaction in gel are a kind of polysaccharide body containing sulfate, high temperature When can be dissolved in water, gel is congealed into after cold, inside forms a kind of porous reticular structure, and aperture is very big, allow macromolecular Substance (molecular weight is up to million or more) passes freely through.The size in aperture further depends on agar concentration, and agar concentration is big, aperture phase To smaller, agar concentration is small, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, since agar or agarose have Good chemical stability, water content is big after gel, and transparency is good, and convenient sources are easy to handle, therefore is a kind of diffusion well Medium.The molecular weight of antigen and antibody in gel from high concentration region to low concentration region generally all 200,000 hereinafter, spread When suffered resistance very little, be substantially in free diffusing form.Due to the molecular weight of different antigen molecules, structure, shape and electricity Lotus amount is different, therefore its diffusion coefficient is different, and diffusion velocity is also just different in gel.When antigen and corresponding antibodies are after spreading It meets in gel, forms antigen antibody complex if the two is appropriate in place's ratio of meeting and form maximum compound.By In the molecular weight increase of compound, particle increases, thus does not continue to spread and generate to precipitate, and shows threadiness or band-like, this Kind precipitating is formed one " specific barrier ", and all antigen or antibody molecule same in immunology cannot pass through, And those of property difference molecule can continue to spread by this barrier, until forming the compound of themselves. In this way, synantigen is not formed by each have their own position of precipitating.Such reaction is known as agar gel diffusion or AGP test, or Immune proliferation, linear or band-like " specific barrier " formed are known as immuning lines or immunoprecipitation band, referred to as precipitate Line or sealed Belt.It is at present with the routine experiment checkup item of known antibodies detection unknown quantity corresponding antigens, and " middle traditional Chinese medicines Allusion quotation " standard method of the regulation for the detection of influenza virus vaccine hemagglutinin content in 2010 editions.Usually by a certain amount of goat-anti people Ig antiserum ingredient is mixed in agar gel, is made containing the specificity sero-fast agar plate of goat-anti people Ig, is beaten after to be solidified Hole, and human serum to be checked (IgG, IgA, IgM etc.) is added in corresponding aperture, spread serum to be checked around in agar plate, Properly locate to combine in antigen and antibody concentration ratio, forms macroscopic white precipitate ring and no longer spread.Thus may be used See, when a kind of solution passes through semi-solid gel, the gel pore detention that macromolecular solute therein is just acted on by molecular sieve is solidifying In glue, antibody that antigen especially therein can be fixed in advance in gel in conjunction with 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 one kind of T lymphocyte, average Service life is generally 7 days or so, but certain T cells can long-term surviving, unlimited amplification especially after immortality is melted into cell line (strain). Foreign literature report, simian virus 40 (SV40) can be such that certain human cells immortalize.Poulin DL, Kung AL and Sullivan CS etc. is studies have shown that the importing of SV40T antigen gene can accelerate the growth rate of transformed cells, immortalized cells Repeatedly still there is metastable multiplication characteristic and functional status, while can also retain its initial cell after passage in vitro Many phenotypic differentiations.Reilly establishes vascular smooth muscle cells strain with the genetic transformation of simian virus large T antigen, constructs cell membrane Type is to study the inhibiting effect mechanism of heparin for vascular smooth muscle.Su etc. utilizes the superficial cell strain converted through SV40, structure Cell model is built to analyze the regulating and controlling effect of epithelial cell internal protein synthesis.Miquel etc. is thin with the cuticulated epithelium that SV40 is converted Born of the same parents' strain, the cell adhesion mediated as cell model research laminin 5.The forefront converted through SV40 such as Webber The physiological function and secreting function of prostate epithelial cell are studied in glandular epithelium strain as cell model.Racusen etc. is used Renal cells model is converted through Ad12-SV40 to study the damage and disease of proximal convoluted tubule.Hougton etc. is turned with SV40 Change establishes Bone marrow Stromal cell as cell model to study under certain condition of culture, cell with to fat cell and at The potential of the two-way differentiation of osteocyte further studies the mechanism of osteoporosis.Foreign study, which is also shown that, imports exogenous human end Human telomerase reverse transcriptase (hTERT) can make cell keep normal phenotype and differentiating characteristic.It is successfully established using hTERT in recent years The immortalized cell lines of certain cells, substantially holding chromosome stabilityX, differentiation be normal, contact inhibition, opposite without oncogenicity etc. Normal growth characteristics.In dentistry field, Japanese scholars Kamata, Fujita and Fujii transfection hTERT establish immortality Change people's Gingival Fibroblasts, periodontal cell, pulp cells system and Dental Follicle Cells system, cell population doublings number up to 150 times or more, Cell shows original biological characteristics, and the GAP-associated protein GAP of derived cell can be expressed after Fiber differentiation.Kitagawa etc. Transfection hTERT establishes people cementoblast system, and cell multiplication is up to 200 times or more, cell differentiation marker such as alkaline phosphatase The expression such as enzyme, type i collagen are stablized.Because of the needs of research work, almost 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, brain edema cell model etc..So CD4+ can be prepared T cell strain is used to prepare the clarifier for the treatment of AIDS after massive amplification, is adsorbed and is removed in blood plasma with CD4+T cell HIV, while AIDS is treated by being defeated by the cell factor that CD4+T cell generates.

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

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

Summary of the invention

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

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

The object of the present invention is achieved like this: preparation can washed corpuscles and blood plasma separator, preparation gp120 and Gp41 antibody, then goat-anti gp120 and gp41 antibody is prepared as corresponding antigens, building CD4+T cell is transfected with foreign gene Strain is simultaneously expanded by cell growth stimulator of its peculiar molecular antibody, has not only retained former macrophage feature but also energy with hybridoma technology preparation The hybridoma macrophage strain of indeterminate growth and amplification cultivation, take cell strain with high-biocompatibility material package and preparing can prevent Only cell and its fragment filter out and can provide the clarifier in place to remove HIV, take gp120 and gp41 antibody and goat-anti The mixing of gp120 and gp41 antibody is fully tied goat-anti gp120 and gp41 antibody but remains the gp120 and gp41 that have high titre Mixed antibody is incorporated the agar kept the temperature after 100 DEG C dissolve in 39~42 DEG C of gradient concentration with reversed gradient titre by antibody It is sequentially added clarifier from down to height by antibody titer by sugar, is cooled to after semi-solid gel followed by adding next time, is made Gel in clarifier forms the layer distributed of antibody titer from high to low and agarose concentration from low to high from top to bottom, Be conducive to the effect of plasma perfusion and molecular sieve and immune clearance, wherein in conjunction with goat-anti HIV antibody and unbonded gp120 And gp41 antibody, CD4+T cell strain, macrophage strain are fixed in Ago-Gel, are played an absorption HIV and are used, it is made net Change device to merge Additional regulatory program with separator group and AIDS therapeutic equipment is made, the blood in extracorporal circulatory system is divided by separator Blood plasma and haemocyte, the purified device of blood plasma converge rear and then feed back after filtering out HIV with haemocyte.

Technological core of the invention is made of separator and clarifier, separator energy washed corpuscles and blood plasma, clarifier In cleanser it is thin by HIV antibody, the HIV antibody in conjunction with goat-anti Ig, CD4+T cell strain and the macrophage for being fixed on agar gel Born of the same parents' strain is made, wherein the molecular weight of HIV antibody its conjugate with goat-anti Ig ining conjunction with is bigger than the HIV antibody molecular weight being not associated with, Not easily pass through gel molecular sieve and contained goat-anti Ig easily with agar gel secure bond the characteristics of, HIV antibody is also easier to therewith Be fixed in agar gel, the HIV in blood plasma and the HIV antibody for being fixed in agar gel can occur association reaction when meeting and Antigen antibody complex is formed, so that agar gel is fixed in by HIV antibody, because the CD4 molecule of CD4+T cell surface is The receptor of HIV and become HIV permissive cell, HIV can be adsorbed when meeting with HIV, the HIV being adsorbed is consolidated with CD4+T cell It can be fixed therewith by phagocytosis when HIV meets therewith due to agar gel because of the natural phagocytosis characteristic of hybridoma macrophage In agar gel, and agar gel is formed by filter opening and reduces with increasing for agarose concentration, clarifier entrance fine jade Lipolysaccharide concentration is low, and filter opening is just big, is conducive to the association reaction of plasma perfusion and high titre antibody or cell and HIV；And exit Concentration is high, and filter opening is just small, is easy to detention HIV or Large molecular conjugates, it is clear that clarifier is combined with a variety of special and non-specific HIV Except composition, in case extraordinary strain, because of the futile treatment caused by immunity difference, the present invention is substituted in the method for external mechanical removal HIV The conventional anti-reverse transcription drug treatment in vivo of HIV can not be effectively killed for a long time.

Specific embodiment

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

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

Fig. 3 is the schematic diagram of internal structure of the clarifier 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) it is connected with plasma separator (4), plasma separator (4) purification in parallel with two through blood plasma pump (6) and circulation line (7) Device (8), clarifier (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is another End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4 Being cannot be by the haemocyte of micropore (3), and 5 be the blood plasma and chemical analysis that can pass through micropore (3), and 6 be plasma separator exocoel, 7 be blood plasma outflux, and 8 be that there is the haemocyte of switchable valve to export.

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

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

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

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

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

2, cell culture and the adherent preliminary sorting of macrophage

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

3, cd4 cell sorts

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

4, CD14 cell (macrophage) sorts

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

Basic skills is analogous to cd4 cell, using immunomagnetic beads method.1. reagent: people's CD14 immunomagnetic beads kit (Miltenyi Biotec, Germany), RPMI-1640 culture medium (Hyclone, the U.S.)；2. immunomagnetic beads method: (A) magnetic bead and spy Anisotropic one monocyte of target cell combines: every 1 × 10⁸The magnetic bead and 800uL buffering of 200uL coupling CD14 antibody is added in a PBMC Liquid (contains 10% bovine serum albumin(BSA) 2.5mL and 2mol/L EDTA0.5mL, 4 DEG C of refrigerator pre-coolings), in 15mL centrifuge tube It mixes well, 4 DEG C of incubation 15min, centre can slightly shake 1 time.Take out centrifuge tube after 15min, every 1 × 10⁷A cell is added 1 Buffer is pre-chilled in~2mL, and 1 000r/min is centrifuged 8min, abandons supernatant, and 0.5mL buffer is added and blows and beats into single cell suspension. (B) it collects the monocyte of marked by magnetic bead: cell splitter is placed on MACS magnetic frame, 1mL buffer statocyte is added Splitter drips to no liquid, immediately adds above-mentioned cell suspension in people's cell splitter, rinses cell with 0.5mL buffer Splitter 3 times.After to be rinsed, 1mL buffer is added, the emigrated cells splitter from magnetic frame is quickly pushed with needle column, Go out the cell combined in splitter with one magnetic bead of CD14 antibody, the as macrophage of CD14+.

In addition, following 2 kinds of methods sorting, including 1. adherent method also can be used: PBMC being placed in and is cultivated containing RPMI-1640 In the culture dish of base, in 37 DEG C, contain 5%C0: cell incubator (Themo electro corporation CLASS 100, The U.S.) in be incubated for 2h.It after adherent mononuclear cells, inhales and abandons upper layer suspension cell, PBs buffer gently washs 3 times, is added a small amount of Mono- 1640 culture medium of RPMI, scrapes attached cell with cell scraper.1 000r/min is centrifuged 5min, abandons supernatant.2. fluidic cell Art method: CD14 label: PBMC is taken, with buffer (bovine serum albumin(BSA) 2.5mL and the 2mol/LEDTA 0.5mL containing 10%) Adjusting cell density is 1 × 10⁸CD14+-FITC antibody 100uL is added in/mL in every milliliter of cell suspension, and 4 DEG C are protected from light label 18min, then 1mL streaming buffer is added to terminate dyeing into centrifuge tube, PBs is washed 3 times, with the PBS for containing 2% mycillin Adjustment cell density is 2 × 107/mL.Selected by flow cytometry apoptosis: by the cell of preparation in flow cytometer (BD FAcsAria II, the U.S.) on sort, according to the fluorescence intensity of CD14 antibody, the relative particle of the relative size of cell and cell and interior The complexity of portion's structure collects the cell of CD14+.

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

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

(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/ that a pipe freezes out of liquid nitrogen container 0), be immediately placed in hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not Any heavy chain immunoglobulin or light chain are secreted, the highest growth scale of cell is 9 × 10⁵/ ml, the doubling time is usually 10~ 15h；Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC The NCI-H929 human myeloma cell strain that cell bank is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min； It is repeated 1 times.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3-4 days In generation, expands culture, and fusion adjusts cell state in first 24 hours, guarantees that cellular morphology is good before merging, growth is vigorous.It is added Appropriate basal medium gently beats 1000r/m centrifugation 5-10min after mixing, washes repeatedly cell 2 times into centrifuge tube.

(3) CD14 cell (macrophage) to be hybridized prepares: the mononuclear macrophage that the present invention sorts is with basal medium Total cell number is adjusted to 1 × 10⁸~2 × 10⁸For cell fusion.Blue dyeing phase-contrast microscopy, viable count are expected with platform It should be higher than that 80% is qualified.

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

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

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

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

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

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

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

6, hybridoma macrophage strain treatment cell preparation

That is the amplification cultivation of hybridoma macrophage strain.Above-mentioned cell precipitation is gently resuspended using complete culture solution and is moved back Enter in culture bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Amplification cultivation is passed on repeatedly, until required hybridoma cell strain In quantity, every 10 generation of secondary culture positive hybridoma cell strain, detect the function of macrophage hybridoma cell strain, see whether steady It is fixed.Continuation carries out extensive industrialization preparation in several bottles, saves backup.

(2) preparation of CD4+T cell strain

1, the source of primary lymphocyte

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

2, the preparation of CD4+T cell

1. main agents and instrument: CD4, CD8 immunomagnetic beads (German Mei Tian Ni Bioisystech Co., Ltd)；Isothiocyanic acid Fluorescein CD4-FITC, CD8-FITC, IgG1-FITC (Immunotech company)；(perseverance letter in Shanghai is biochemical for lymphocyte separation medium Reagent Co., Ltd)；(the raw work biotechnology service in Shanghai is public for ethylenediamine tetra-acetic acid (EDTA), 0.2% Trypan Blue liquid Department)；Newborn bovine serum (Hyclone company)；MiniMACS magnetic separation system (German Mei Tian Ni Bioisystech Co., Ltd)； EpicsXL type flow cytometer (BeckmanCoulter company of the U.S.).2. separation (the density gradient of mononuclearcell (PBMC) Centrifugal process): it is sterile to take 20mL blood sample (500IU/mL2mL heparin sodium is anticoagulant)；PBS liquid mixes well 2~3 times of hemodilution The anticoagulant venous blood of 6mL is slowly superimposed in the 10mL centrifuge tube that 4mL lymphocyte separation medium has been added with dropper along tube wall afterwards Horizontal centrifugal (400r/min, 20 DEG C) 35min in horizontal centrifuge；It is divided into 3 layers after centrifugation in pipe, upper layer is blood plasma and PBS liquid, Lower layer is mainly red blood cell and granulocyte, and middle layer is lymphocyte separation medium, is had in upper, middle layer interface one with mononuclearcell Based on white cloud and mist layer narrow band be PBMC, be inserted into cloud and mist layer with capillary syring, draw PBMC and be placed in another 50mL centrifuge tube In, it is added 5 times and (300r/min, 20 DEG C) 10min is centrifuged with upper volume PBS, abandon supernatant 50mLPBS and cell, centrifugation is resuspended (350r/min, 20 DEG C) 15min abandons supernatant, is added Buffer (PBS+0.5% newborn bovine serum+2mmol/LEDTA, pH7.2) Cell is resuspended in 2mL, takes 15uL cell suspension that the cell (PBMC) counted in 4 block plaids under microscope is added on blood counting chamber Sum.3. CD4+T cell and CD8+T cell isolate and purify: PBMC cell suspension, which is divided equally to two 1.5mLEppendorf, manages, It is centrifuged (300r/min, 20 DEG C) 10min, is discarded supernatant, the every 80uLBuffer of cell is resuspended and contains cell number 10⁷It is a, every 10⁷It is a thin Born of the same parents add 20uLCD4MicroBeads or CD8MicroBeads, mix well, and in 4~8 DEG C of hatching 15min, are washed with 1mLBuffer Cell is washed, (300r/min, 20 DEG C) 10min is centrifuged, 500uLBuffer is discarded supernatant and cell is resuspended, MS splitter is placed on It in the magnetic field of MACS separator, is rinsed with 500uLBuffer, by 500uL cell suspension by splitter, uses 500uLBuffer It rinses splitter repetitive operation 3 times, collects efflux, contain non-CD4+T lymphocyte or non-CD8+T lymphocyte in efflux, Splitter is taken out from separator, with 1000uLBuffer pressure flush splitter, collects efflux, this is thin for CD4+T lymph Born of the same parents or CD8+T lymphocyte (cell viability detection: take 15uL cell suspension and isometric trypan blue molten respectively before and after cell purification Liquid mixing, the not colored shinny person of microscopically observation are living cells, and the coloring person of swelling is dead cell, are calculated living in 200 cells The percentage of cell).

3, amplification in vitro CD4+T cell

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

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

It is in the method that hTERT immortalizes CD4+T cell: with restriction endonuclease EcoR I and Xho I double digestion plasmid pCIneo- HTERT and carrier pLXSNneo, the hTERT and pLXSNneo separated with Ligation Mix connection through PCR amplification, gel electrophoresis Digestion products construct pLXSNneo-hTERT recon, and it is green to expand, purify simultaneously picking resistant to ammonia benzyl to convert DH5a competent cell Mycin bacterium colony extracts plasmid, imports the T lymphocyte that in vitro passage is in logarithmic growth with lipofection, makes recon and thin The DNA of born of the same parents is integrated, and expands the clone for the positive recombinant that culture is screened through G418, screens cellular morphology, growth curve, dyeing It is body caryogram, the test of nude mice tumorigenesis, transfection Cell Telomerase Activity, hTERT mRNA expression product, immunohistochemical staining, thin Born of the same parents' proliferating cycle and apoptosis rate meet immortalized cells characteristic and person same or similar with primary cell is as hTERT immortality The CD4+T cell of change.

It is in the method that SV40 immortalizes CD4+T cell: is connected simultaneously with T4DNA ligase through BamHI digestion The SV40LTag DNA of pcDNA3.1 (-) DNA and PCR amplification, agarose gel electrophoresis separation, construct SV40LTag- PcDNA3.1 (-) recombinant plasmid, it is amp-R to expand, purify simultaneously picking to convert DH5a competent escherichia coli cell Bacterium colony extracts plasmid, and the T lymphocyte of in vitro culture is imported with lipofection, integrates the DNA of recon and cell, with The cell containing positive recombinant of G418 screening passes on, expands culture, screens cellular morphology, cell growth curve, chromosome Caryogram, the test of nude mice tumorigenesis transfect the big T genetic test of SV40 in cell DNA, the measurement of mRNA expression product and determined dna sequence As a result meet immortalized cells characteristic and CD4+T cell that person same or similar with primary cell immortalizes as SV40.

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

(I) extraction of hTERT: (i) digestion pClneo-hTERT:hTERT be located at the EcoRI of plasmid pClneo-hTERT with Between the site SalI, pLXSNneo vector multiple cloning site (MCS) is containing EcoRI and XhoI restriction enzyme site.Commercially available purchase pCIneo- HTERT plasmid is dissolved in suitable ultra-clean H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O adds limitation Property restriction endonuclease EcoR I and Xho each 0.5ul of I, 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator, be added 5uL electrophoresis sample-adding Buffer terminates reaction, routinely after PCR method amplification hTERT, collects amplified matter in case electrophoresis.(ii) hTERT electrophoresis: electrophoresis is taken Grade agarose is made into 10% Ago-Gel with electrophoretic buffer, pours on the gel casting platform sealed, plugs sample comb, Envelope band is removed from glue platform after being gelled admittedly, comb is extracted, is put into the electrophoresis tank added with enough electrophoretic buffers, is buffered Liquid is higher by gel surface about 1mm, prepares hTERT digestion sample with suitable 10 × sample loading buffer, then will with pipettor Sample is added in sample well, and does suitable standard control object simultaneously, connects electrode, keeps the hTERT Ghandler motion that faces south dynamic, then in 1- Under the voltage of 10V/cm (80V) gel electrophoresis to be sufficiently separated hTERT segment apart from when (30min), close power supply.(iii) HTERT purifying is with recycling: from separating hTERT band in agarose: the coagulating the segment of hTERT containing target under long wave ultraviolet light source Adhesive tape band, which is cut, to be fitted into bag filter, and 2ml electrophoretic buffer is added into bag filter, is allowed to submerge gel, and empty steam bubble, will Bag filter level is put into electrophoresis tank (length direction is parallel with electrophoresis), and appropriate amount of buffer solution is added by bag filter and submerges (about 6-7mm), Power on, 150 volts of electricity are washed, and observe all remove gel to hTERT in the UV lamp, are changed direction of an electric field and are continued to be powered 1 point 1.5 times of volume n-butanols are added from buffer is sucked out in bag filter in 1-5ml Eppendorf pipe in clock, mix extracting and go EB, most high speed 2 minutes on desk centrifuge, sucks upper layer butanol solution, so repeat it is secondary, from the molten of lower layer hTERT Isometric phenol chloroform (2) are added in liquid to extract 2 times, supernatant is transferred to 1/10 times of volume 3M of addition in another Eppendorf pipe Dehydrated alcohol is pre-chilled in NaAc, 2 times of volumes, stays overnight in 20 DEG C, 12000g, is centrifuged 10 minutes at 4 DEG C, obtains hTERT and precipitates, in abandoning Clearly, dry ethyl alcohol is abandoned after being added 70% ethanol washing 2 times, and 50 μ l TE are added and dissolve hTERT.In addition, low melting-point agarose also can be used Purpose hTERT segment is separated from gel, is purified by gel method, hTERT filter membrane inserted sheet method etc..

(II) hTERT composition (0.1-5 μ g), the 1 μ l of the 9 above-mentioned purifying of μ l the connection of hTERT and pLXSNneo carrier: are taken 10mmol/L ATP, 10ul Ligation Mix or e. coli dna ligase, the mixing of 2ul pLXSNneo empty carrier, 15 DEG C It incubates for 24 hours, constructs pLXSNneo-hTERT recon.

(III) purifying, amplification, the identification of pLXSNneo-hTERT recon: the preparation of (i) E. coli competent: its Basic skills is ice-cold CaCl₂Or a variety of divalent cations etc. handle bacterium, feed them into competence and are converted, and use CaCl₂Fresh or freezing competent escherichia coli cell is prepared, preparation in quantity competent bacteria is usually used in, this law is suitable for big Most coli strains, operating process are summarized as follows.(ii) with competent E.coli purifying, amplification pLXSNneo-hTERT Recon: 200 μ l are taken to be transferred to sterile microcentrifugal tube from every kind of competent cell suspension with cooling sterile pipette tip In, every pipe adds DNA or connection reaction mixture (volume≤10 μ l, DNA≤50ng), gently rotates to mix content, in ice Centrifuge tube is put into pre-heating to the rack for test tube in 40 DEG C of circulator bath by middle placement 30min, places 90s~2min, should not Test tube is shaken, quickly pipe is transferred in ice bath, makes the cooling 1~2min of cell, every centrifuge tube adds 800 μ lSOC culture mediums, uses water Culture medium is warmed to 37 DEG C by bath, and then pipe is transferred on 37 DEG C of shaking tables, and incubating 45min makes bacteria resuscitation, and expresses matter Antibiotic-resistance marker's gene of grain coding, the competent cell that proper volume (every 90mm plate is up to 200 μ l) has been converted It is transferred on the SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, plate is placed in room temperature to liquid and is absorbed, Horizontalization ware is cultivated in 37 DEG C, may occur in which bacterium colony after 12~16h.(iii) it screens, expand recon: being connect with sterile toothpick or sterilizing Kind needle is selected single bacterium colony and is inoculated in 5mL sterile LB culture medium or rich medium, after overnight incubation, is then added to In the 2L flask of 500mL culture medium containing LB, then at 37 DEG C of cultures to saturation state (OD₆₀₀≈ 4), what addition 1mL newly matched contains Precipitating is resuspended in the GTE solution of 25mg/mL lysozyme, in being placed at room temperature for 10min, 10mL is added and newly matches NaOH/SDS solution, Yu Bing 7.5mL acetic acid solution is added in upper placement 10min, is gently mixed with suction pipe until viscosity declines and forms big precipitating, Yu Bing Upper placement 10min, in 4 DEG C, 20 000g are centrifuged 10min, supernatant are gently poured into another clean centrifuge tube, if There is visible drift that can use several layers of filtered through gauze, the isopropanol of 0.6 times of volume is added, is mixed by inversion, it is placed at room temperature for 5~ 10min, in room temperature, 1 500g is centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs precipitating, then of short duration rapid centrifugation, inhales Go ethyl alcohol, vacuum drying (precipitating can be in 4 DEG C of long-term preservations).(iv) identification and amplification of recombinant plasmid: the list on picking plate Bacterium colony is inoculated in the 3ml LB culture medium of ampicillin containing 100ug/ml, 37 DEG C, is cultivated in 250r/min shaking table, is received after 14h Collect culture, 4 DEG C, 10000r/min centrifugation 5min are extracted in a small amount by kit specification and purified recombinant plasmid.

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

(V) CD4+T cell preculture: by above-mentioned cell inoculation in containing 5~10nmol/L insulin, 20% fetal calf serum In RPMI1640 liquid, or be inoculated in containing 20% fetal calf serum, 5~10nmol/L insulin low sugar DMEM cell culture medium in, Generally it is inoculated in culture medium (the 1.6%1M HEPES buffer solution of 3ml Fresh；15% fetal calf serum (FBS)；1% penicillin And streptomysin；PRMI 1640 is supplied to 100%), being placed in 37 DEG C, in volume fraction 5%CO2 incubator, is cultivated 1-2 days, from The heart removes supernatant, spare.

(VI) pLXSNneo-hTERT recon imports T lymphocyte and expands culture: making in 1.5ml microcentrifugal tube Standby following solutions: pLXSNneo-hTERT recon is dissolved in 100 μ l serum-free mediums by pipe A；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, and pipe A and pipe B is mixed, 45min is stood at room temperature, is trained with serum-free Nutrient solution washing above-mentioned T lymphocyte 2 times.1ml serum-free is added in Lipofectamine-pLXSNneo-hTERT mixture Culture solution mixes gently, and is added dropwise in above-mentioned T lymphocyte, and 1ml serum-free medium is added, and (fetal calf serum concentration is 20ml/ L), in CO₂Transfection liquid is sucked out in incubator culture 10h, adds 4ml complete culture solution (fetal calf serum concentration is 20%), continues to cultivate 20h, discards culture solution, and replacement concentration is 400mgL^-1G418 culture solution continue to cultivate, after 8 days select living cells work expand After culture, then G418 concentration is increased to 800mgL^-1, will stablize in the G418 environment of high concentration growth cell continue into Row amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If cell increases Slowly or cell density is low or medium pH value is in acidity, and half amount culture solution is sucked out, carries out equivalent oil changing.When total amount reaches 14ml When be transferred in 75ml culture bottle, every 2-3 weeks addition 5-10ml fresh culture.Cell culture to 9-10 weeks (the about the 75th generation), Still in logarithmic growth phase, i.e. cell is accelerated with incubation time in multiplication relation, and dead cell is less than 10%.When total amount reaches It when to 45ml, sets in 50ml centrifuge tube, is centrifuged 1500 turns, 10 minutes, after abandoning supernatant, 3ml freezing media (5% diformazan is added Base Asia maple (dimethyl sufoxide), 95%FBS) it mixes, at cell suspending liquid, (cell concentration is about 10⁵/ml).It freezes Pipe packing, 1ml/ pipe, sets -20 DEG C of 2h, then set -70 DEG C of 2h, then freeze in -196 DEG C of liquid nitrogen (or immediately 80 degree under zero setting, It is moved into liquid nitrogen container after 1-2 hours).

(VII) immortalize the identification of CD4+T characteristics of cell biology: (i) observes cellular morphology: visible lymphocyte is obvious Increase, clustering phenomena occur, there is lymphocyte blastogenesis feature.(ii) cell growth curve is observed: using incubation time as horizontal axis, Cell quantity is the longitudinal axis (logarithm), be depicted in after curve is made on semi-logarithmic scale the cell growth curve, immortalize Cell line is in that typical " S " feature or " arched roof " are formed；(iii) chromosome is checked: by analyzing karyotype, if dyeing Body caryogram is diploid " 46, XX " or " 46, XY " then illustrate that there is no vicious transformations for the cell line.(iv) flow cytometry Detection: the cell proportion of synthesis, division in the 19th continuous cell line of detection, if its proliferative capacity is not obviously than building be normal thin Born of the same parents' enhancing, explanation are the results of hTERT integration, expression.(vii) determined dna sequence: routinely sequenator detects, and shows hTERT Gene order.(v) hTERT detection in cell DNA is transfected: such as with Immunohistochemical detection, the interior dye of the nucleus of hTERT transfection The visible a large amount of brown particles of color, show that hTERT has been integrated into the cell；(vi) mRNA expression product measures: taking 100 μ l systems Pcr amplification product is taken 2 μ l DNA solutions to dilute 100 times, is surveyed dense with gel reclaims kit (Takara, Japan) recovery product Degree, remaining DNA and each 10 μ l of upstream and downstream primer are sequenced.

(VIII) hTERT mediates CD4+T cell bank: screening and continue passage, expansion culture meets forever after above-mentioned identification OEG cell characteristic and the cell same or similar with primary cell, take that growth conditions are good, the difference in logarithmic growth phase The cell of generation is centrifuged (1 200r/min, 6min), and cell is resuspended with 0.5~1ml of frozen stock solution containing dimethyl sulfoxide, Cell density is 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enter -196 DEG C of liquid nitrogen It freezes, it is spare to construct the stable immortalization CD4+T cell bank of biological characteristics in this way.

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

(I) extraction of SV40 large T antigen DNA: (i) SV40DNA digestion: contain large T antigen gene from commercially available purchase SV40 freeze dried powder or SV40 plasmid, are dissolved in suitable H₂In O or TE buffer, add 2uL10 × enzyme cutting buffering liquid and 18uL H₂O, adds restriction enzyme BamH I (1-5U/ugDNA), and 5uL is added in 37 DEG C of incubation 1h, 75 DEG C of heating 15min, inactivator Electrophoresis sample loading buffer terminates reaction in case electrophoresis.(ii) SV40DNA electrophoresis: electrophoresis grade agarose is taken to be made into electrophoretic buffer 10% Ago-Gel pours on the gel casting platform sealed, plugs sample comb, removes from glue platform after being gelled admittedly Envelope band is removed, comb is extracted, is put into the electrophoresis tank added with enough electrophoretic buffers, buffer is higher by gel surface about 1mm, with suitable 10 × sample loading buffer of amount prepares DNA sample, and then sample is added in sample well with pipettor, and does simultaneously suitably DNA molecular amount standard control object connects electrode, keeps the DNA Ghandler motion that faces south dynamic, and electrophoresis is to enough under the voltage of 1-10V/cm gel Isolation of DNA fragments apart from when, close power supply.(iii) about 2600bp SV40 large T antigen DNA is separated from agarose: The gel-tape containing target DNA fragments is cut under 300-360nm long wave ultraviolet light source and is fitted into bag filter, is added into bag filter Enter 2ml electrophoretic buffer, be allowed to submerge gel, and empty steam bubble, bag filter level is put into electrophoresis tank, appropriate amount of buffer solution is added Bag filter is submerged into (about 6-7mm), is powered on, 150 volts of electricity are washed, and observe all remove gel to DNA in the UV lamp, are changed Direction of an electric field continues to be powered 1 minute, and from buffer is sucked out in bag filter in 1-5ml Eppendorf pipe, 1.5 times of volumes are added N-butanol mixes extracting and removes EB, and most high speed 2 minutes on desk centrifuge suck upper layer butanol solution, and so repeatedly two It is secondary, isometric phenol chloroform (2) are added from the solution of lower layer speech DNA and is extracted 2 times, and supernatant is transferred in another Eppendorf pipe 1/10 times of volume 3M NaAc, 2 times of volume pre-cooling dehydrated alcohols is added, stays overnight in 20 DEG C, 12000g, is centrifuged 10 minutes at 4 DEG C, DNA precipitating is obtained, supernatant is abandoned, dry ethyl alcohol is abandoned after being added 70% ethanol washing 2 times, 50 μ l TE dissolving DNAs are added.In addition, also can be used 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 the above-mentioned DNA composition of 9 μ l (0.1-5 μ g), 10 2 × connection of μ l buffers, 1 μ l 10mmol/L ATP, T4DNA ligase (20~500 cohesive end unit) or large intestine bar Bacterium DNA ligase, the mixing of pcDNA3.1 empty carrier, 15 DEG C incubate for 24 hours, are built into SV40T/pcDNA3.1 recon.

(III) amplification, separation and identification of SV40T/pcDNA3.1 recon: the preparation of (i) E. coli competent: from One single colonie (such as bacillus coli DH 5 2) of picking or the fresh 16~20h of 1ml in the fresh plate of 37 DEG C of 16~20h of culture Overnight culture is gone in 1L the or 500ml flask containing 100mlLB culture medium, in 37 DEG C acutely shaking culture about 2~ 3h measures OD600 value ≈ 0.4 every 20~30min, bacterium is aseptically transferred to one, ice-cold 50ml In polypropylene centrifuge tube, 10~20min is placed on ice, and 10min is centrifuged with 4000r/min with SorvallGS2 rotary head in 4 DEG C, To recycle cell, culture solution reciprocal is pre-chilled with 10ml with ice by pipe inversion 1min so that the trace culture solution of final residual is flow to end 0.1mMCaCl₂Every part of precipitating is resuspended, is put on ice, 10min is centrifuged with 4000r/min with SorvallGS3 rotary head in 4 DEG C, To recycle cell, culture solution is poured out, by pipe inversion 1min so that the trace culture solution of final residual is flow to end, every 50ml initial incubation The 0.1M CaCl that object 2ml ice is pre-chilled₂It is resuspended every part and sinks and determine, at this point it is possible to which cell is distributed into aliquot, ice in liquid nitrogen rapidly Freeze, -70 DEG C of storages are spare, take 200 μ l to be transferred to from every kind of competent cell suspension with cooling sterile pipette tip sterile In microcentrifugal tube, should every Guan Zhongjia DNA or connection reaction mixture (volume≤10 μ l, DNA≤50ng), gently rotate with Content is mixed, 30min is placed in ice, centrifuge tube is put into pre-heating to the rack for test tube in 40 DEG C of circulator bath, is put 90s~2min is set, test tube is not shaken, quickly pipe is transferred in ice bath, makes the cooling 1~2min of cell, every centrifuge tube adds 800 Culture medium is warmed to 37 DEG C with water-bath, then pipe is transferred on 37 DEG C of shaking tables by μ lSOC culture medium, and incubating 45min makes bacterium Recovery, and antibiotic-resistance marker's gene of expression plasmid coding, the competent cell that proper volume has been converted are transferred to On SOB culture medium containing 200mmol/LMgSO4 and corresponding antibiotic, plate is placed in room temperature to liquid and is absorbed, is inverted flat Ware is cultivated in 37 DEG C, may occur in which bacterium colony after 12~16h.(ii) it the screening, amplification and extraction of recon: with sterile toothpick or goes out Bacterium transfer needle selects single bacterium colony and is inoculated in the sterile LB culture medium of 5mL or rich medium (the super meat of such as super broth or TB Soup culture medium) in, after overnight incubation, it is then added in the 2L flask of 500mL culture medium containing LB (containing appropriate antibiotic), then at 37 DEG C are cultivated to saturation state (OD₆₀₀≈ 4), the GTE solution for the lysozyme containing 25mg/mL that 1mL newly matches is added, precipitating is resuspended, in It is placed at room temperature for 10min, 10mL is added and newly matches NaOH/SDS solution, and is mixed gently until liquid becomes uniform, limpid and viscous It is thick, in placing 10min on ice, 7.5mL acetic acid solution is added, is gently mixed with suction pipe until viscosity declines and forms big sink It forms sediment, in placing 10min on ice, in 4 DEG C, 20 000g are centrifuged 10min, and supernatant is gently poured into another clean centrifuge tube In, if there is visible drift can use several layers of filtered through gauze, the isopropanol of 0.6 times of volume is added, is mixed by inversion, is placed at room temperature for 5~10min, in room temperature, 1 500g is centrifuged 10min, and 70% ethyl alcohol of 2mL is added and gently washs precipitating, then it is of short duration quickly from The heart sucks ethyl alcohol, and is dried in vacuo (precipitating can be in 4 DEG C of long-term preservations).(iii) identification of recon: above-mentioned big from competence The DNA (containing recon SV40T/pcDNA3.1) extracted in enterobacteria, ibid method carries out digestion with restriction enzyme BamH I, The identification of 10g/L agarose gel electrophoresis, obtains 2 bands of size about 2600bp and 5600bp, the former meets in GenBank The size of SV40T segment.

(VI) importing and expansion culture of SV40T/pcDNA3.1: following solutions are prepared in 1.5ml microcentrifugal tube: pipe SV40T/pcDNA3.1 is dissolved in 100 μ l serum-free mediums (fetal calf serum concentration is 20ml/L) by A；Pipe B, by 20 μ l Lipofectamine is dissolved in 80 μ l serum-free mediums, pipe A and pipe B is mixed, the underlying 45min of room temperature.Use free serum culture Liquid washing 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 is added dropwise in above-mentioned T lymphocyte, and 1ml serum-free medium is then added, and (fetal calf serum concentration is 20ml/L), in CO₂Transfection liquid is sucked out in incubator culture 10h, adds 4ml complete culture solution (fetal calf serum concentration is 20%), after Continuous culture 20h, discards culture solution, and replacement concentration is 400mgL^-1G418 culture solution continue to cultivate, select living cells after 8 days Make after expanding culture, then increases G418 concentration to 800mgL^-1, the cell of growth will be stablized in the G418 environment of high concentration Continue amplification cultivation.Culture 9 days or so is under the microscope, it is seen that lymphocyte significantly increases, and clustering phenomena occurs.If thin Born of the same parents increase slowly or cell density is low or medium pH value is in acidity, are sucked out half and measure culture solution, carry out equivalent oil changing.When total amount reaches It is transferred in 75ml culture bottle when to 14ml, every 2-3 weeks addition 5-10ml fresh culture.Cell culture about 6-8 weeks 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 It when reaching 45ml, sets in 50ml centrifuge tube, is centrifuged 1500 turns, 10 minutes, abandons supernatant, 3ml freezing media (5% diformazan is added Base Asia maple (dimethyl sufoxide), 95%FBS) it mixes, at cell suspending liquid, (cell concentration is about 10⁵/ml).It freezes Pipe packing, 1ml/ pipe, sets -20 DEG C of 2h, then set -70 DEG C of 2h, then freeze in -196 DEG C of liquid nitrogen (or immediately 80 degree under zero setting, It is moved into liquid nitrogen container after 1-2 hours).

(VII) immortalize the identification of CD4+T characteristics of cell biology: (i) observes cellular morphology: visible lymphocyte is obvious Increase, clustering phenomena occur, there is lymphocyte blastogenesis feature.(ii) cell growth curve is observed: using incubation time as horizontal axis, Cell quantity is the longitudinal axis (logarithm), be depicted in after curve is made on semi-logarithmic scale the cell growth curve, immortalize Cell line is in that typical " S " feature or " arched roof " are formed；(iii) chromosome is checked: by analyzing karyotype, if dyeing Body caryogram is diploid " 46, XX " or " 46, XY " then illustrate that there is no vicious transformations for the cell line；(iv) flow cytometry Detection: the cell proportion of synthesis, division in the 19th continuous cell line of detection, if its proliferative capacity is not obviously than building be normal thin Born of the same parents' enhancing, explanation are the results of the integration of SV40 large T antigen, expression.(viii) determined dna sequence: routinely sequenator detects, and shows Show SV40 large T antigen DNA sequence dna.(v) the big T genetic test of SV40 in cell DNA is transfected: such as with Immunohistochemical detection, The visible a large amount of brown particles of dyeing, show that SV40T antigen has been integrated into the cell in the nucleus of SV40 transfection；(vi) mRNA table It is measured up to product: the sequencing of T antigen mRNA RT-PCR product: taking the amplified production of 100 μ l systems, use gel reclaims kit (Takara, Japan) recovery product, takes 2 μ l DNA solutions to dilute 100 times, surveys concentration, remaining DNA and upstream and downstream primer are each 10 μ l are sequenced.

(VIII) SV40LT gene mediated CD4+T cell bank: screening and continues passage, expands culture accords with after above-mentioned identification Close immortalized cells characteristic and the cell same or similar with primary cell, take growth conditions it is good, in logarithmic growth phase The cell of different generations is centrifuged (1 200r/min, 6min), is resuspended with 0.5~1ml of frozen stock solution containing dimethyl sulfoxide thin Born of the same parents, cell density are 5 × 10⁵Cryopreservation tube, through 4 DEG C, 0.5h is added in a/ml；- 20 DEG C, 2h；- 70 DEG C, overnight, enter -196 DEG C of liquid Nitrogen freezes, and it is spare to construct the stable CD4+T cell bank of biological characteristics in this way.

(4) with the preparation of CD4+T cell identity function particle: can be by CD4 molecule, the CD4 molecule of genetic recombination and similar The molecule of function is coupled by conventional chemical, is crosslinked, for being made and being coated with CD4 molecule is fixed on carrier in affine absorption etc. Grain, or directly take intimate particle substitution CD4+ cell application.It is thin that CD4+T cell of the invention represents other CD4+ Born of the same parents, including prepared with other methods and immortalize CD4+T cell.

(3) preparation of HIV-1gp120 antibody

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

1, it is converted using lymphocyte Epstein-Barr virus and combines preparation HIV-1gp120 monoclonal antibody with hybridoma technology

Specimen origin have it is following it is several by way of: take the lymphocyte strain frozen in Infectious Diseases Lab sample database (through inactivating The immune lymphocyte with EBV transfection of HIV)；The fresh White Blood Cells Concentrate in blood station is bought, inactivation HIV infection strain was then carried out and exempts from The lymphocyte of epidemic disease；It is derived from the cord blood lymphocytes cell (immune through inactivation HIV) saved as scientific research；Directly it is derived from HIV-1 The peripheral blood lymphocytes (being used for itself) of the infected itself, it is thin to separate single core using Histopaque lymphocyte separation medium Born of the same parents (PBMC), adjusting concentration are 2x 10⁶After suitable Epstein-Barr virus (EBV) stoste is added, be placed in 370C, 5%C02 overnight incubation, B cell to be hybridized is prepared, with the positive hole of ELISA method screening anti-HIV-1 outer membrane protein (gp120), metastatic cells to 24 orifice plates Continue culture 2 weeks, repeats to measure anti-gp120 confirmation positive with ELISA method, continuously clone secondary and massive amplification culture.It will After positive cell strain mixes (3: 1) with the heterogeneous myeloma cell of people mouse (being bought by Zhejiang University's siberian crabapple), 1ml50% is added PEG merges the two, and cell is then resuspended and cultivated liquid in IMDM culture solution, addition Peritoneal Cells of Mice is (by Zhejiang within second day Jiang great Xue siberian crabapple is bought) it is used as trophocyte, anti-gp120 antibody is screened with ELISA after continuing culture 3 weeks, selects strong positive Hole hybrid tumor cell amplification culture, and repeatedly clone is carried out until obtaining stable cell line, with this cell line culture, preparation HIV-1 antibody, using ELISA detection kit, by specification operation measures the Ig subclass of antibody, and with the survey of conventional ELISA method The potency and specificity for determining antibody select the high antibody of high specificity, potency.

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

(1) reagent and recombinant antigen: it is related to reagent: HIV-1gp120 genetic fragment, HIV-1gp120 antigen, HIV antigen Nitrocellulose membrane item provides BamH I restriction endonuclease Xho I restriction endonuclease, nucleic acid coprecipitator, T4DNA by Beijing Wan Tai Pharma Inc. Ligase is purchased from precious biological Co., Ltd；Liagen plastic recovery kit is purchased from QIAquick company；1640 dry powder culture of RPMI Base is purchased from Gibco company；Top grade newborn bovine serum is purchased from bright marine growth Engineering Co., Ltd；SupersignalWest Pico Trial Kit, TMB Substrate Kit are purchased from Pierce company；Mouse 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 Magnificent biology Co., Ltd, the goat anti-mouse IgG antibody of horseradish peroxidase (HRP) label are purchased from doctor's moral Co., Ltd. Construction of recombinant plasmid and identification: vector plasmid PEGX-4T-2 BamH I, Xho I digestion, T4DNA Ligase connection gp120 Genetic fragment, recombinant plasmid transformed enter E.colistrain XL1 blue, are sequenced.The inducing expression and identification of recombinant protein: weight Group plasmid is transformed into XL1-Blue Escherichia coli, 25 DEG C under the effect of IPTG inducer, 190r/min concussion, overnight, 4 000r/min is centrifuged 10min, collects bacterium, SDS-PAGE testing goal protein expression situation.Fusion protein purification and identification: table Precipitating is collected by centrifugation up to product to hang through PBS, after cracking bacterium with 30W Ultrasonic Pulverization instrument, supernatant filtering, filter is collected by centrifugation Liquid AKTA PURIFYER100 protein purification instrument, GST column purification, obtain fusion protein GST-HIV, and concentration centrifuge tube carries out dense Contracting, S21 type biology spectrophotometer measurement concentration, SDS-PAGE identify purifying protein.

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

(3) foundation of Detection of Monoclonal Antibody: latter all tail vein bloods are immunized for the third time, determine positive serum with square matrix method Best effort concentration and GST-HIV fusion protein best peridium concentration.It operates as follows: according to 1: 1000,1: 500,1: 200,1: 100 four dilution dilutes antigen using coating buffer, longitudinal to be coated with 96 hole elisa Plates, every 100 μ L of hole, 4 DEG C of packets It is stayed overnight, is washed 3 times, every minor tick 3 minutes.Positive serum and negative serum are made into doubling dilution by 1: 1000 respectively, It is laterally loaded onto the 10th hole, every 100 μ L of hole is placed in 37 DEG C of incubation 1h in wet box, washs 3 times, every minor tick 3 minutes.Enzyme mark is anti- Body HRP- sheep anti-mouse igg makees 1: 10000 dilution to specifications, and every hole 100 μ L, 37 DEG C of incubation 1h are washed 3 times.People is added now to match OPD substrate solution, 100 μ L of every hole, 37 DEG C are protected from light appropriate time, and every hole adds 100 μ L terminate liquids to terminate reaction, detects it OD492 value.Positive hybridoma cell screening technique is established.According to experiment condition and method in square matrix method, melted respectively with GST-HIV Hop protein is experimental group, and recombinant bacterium (containing plasmid pET-32a) albumen is control group, screening positive clone after inducing expression.Operation Steps are as follows: with most suitable peridium concentration envelope antigen in 96 hole elisa Plates, 100 holes μ l/, 4 DEG C of refrigerator coatings are overnight.Take out packet It is washed 3 times by cleaning solution is added after plate, washs 3min every time；Every hole adds cell conditioned medium to be measured (1: 5 dilution) 100 μ L, 37 DEG C of temperature Case is incubated for 50min, washs 3 times later, washs 3min every time；Every hole adds 100 μ l of secondary antibody, and 37 DEG C of incubation 30min are washed It washs 3 times, washs 3min every time；The 100 μ L of OPD substrate solution now matched is added in every hole, and room temperature is protected from light 10-15min；In every hole 100 μ l of 2mol/L H2SO4 terminate liquid is added for terminating reaction；It will test plate and be placed on survey OD492 value in microplate reader.Control group Set up: positive controls are appropriate diluted positive serum, and negative control group is the unrelated list for having identical dilution with primary antibody Anti- cell conditioned medium.Indirect ELISA the selection result determines.Every group of detection OD492 value, with P (sample value)/N (feminine gender value) >=2.0 Person is judged to positive value.Screening positive clone standard: cell conditioned medium is reacted with positive screening group (purifying rear fusion protein coating) in sun Property, while it is positive sample that the detection hole being negative is reacted with negative selection group (mycoprotein of the plasmid containing pET-32a after induction) Product.

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

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

(6) preservation of hybridoma cell strain and secondary culture: saving and recovery: saving preceding 12 hour adjustment cell and grows shape State.Take one bottle of growth vigorous, cell suspension is made in the good cell of form after appropriate digestion, 1000r/m is centrifuged 5min, goes Clear liquid, flicking tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and dispenses cell cryopreservation tube, Cryopreservation tube is put into liquid nitrogen container after taking-up and saves backup by 1mL/ pipe, -70 DEG C of refrigerator overnights.40 DEG C of left sides are got out before recovery Right hot water, cryopreservation tube is carefully taken out from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, after defrosting 1000r/m is centrifuged 5min, opens cryopreservation tube under aseptic condition in superclean bench, the cell after defrosting is washed with complete culture solution It washs once, is then centrifuged 5min in 1000r/m, discards supernatant, cell precipitation moves into training after being gently resuspended using complete culture solution It supports in bottle, sets 37 DEG C, cultivated in 5%CO2 incubator.Secondary culture positive hybridoma cell continuously passed for 10 generations, using indirect The method of ELISA measures culture supernatant antibody titer, observes the variation of potency, 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 culture, dilute by basal medium Releasing cell number is 1 × 10⁷/mL.Mouse peritoneal injects 0.2mL/ only, and mouse ascites production is observed after injection, bright to abdomen Aobvious distension rises, and punctures abdominal cavity with syringe needle, centrifuge tube collects ascites.Acquisition finishes, and injects appropriate basal medium to mouse peritoneal, Every 2-3 days, same method took ascites again.The ascites being collected into, 10000r/m are centrifuged 5min, and Aspirate supernatant dispenses, -20 DEG C It saves.

(8) CHARACTERISTICS IDENTIFICATION of monoclonal antibody: specificity: Weston-Blotting experiment is carried out referring to " molecular cloning " method.It is single Anti- subgroup identification selects mouse monoclonal Ig class/subgroup identification ELIAS secondary antibody to use kit measurement, grasps by kit specification Make.

(9) HIV-1gp120 monoclonal antibody is after further refining using (professional businessman can be entrusted to complete).

(4) preparation of HIV-1gp41 antibody

With the preparation of HIV-1gp120 antibody

(5) preparation of goat-anti people-Ig

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

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

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

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

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

5, the measurement of antiserum titre: antiserum titre is using ELISA measuring method: being to be coated with sample when coating After liquid is diluted to 1: 1000 concentration, every hole adds 100 μ L on ELISA Plate, is then placed in aluminium box and is put into 4 DEG C of refrigerators overnight. The next morning, which takes out, pats dry coating buffer, is washed three times with PBST, and every minor tick five minutes pats dry enzyme with gauze for the last time The confining liquid of 10% serum is added in target, and every hole adds 100UL, is put into 37 DEG C, 1~2h of water-bath.Then it takes out again and pats dry closing Liquid washs 3 every minor tick of ELISA Plate five minutes with PBST, is patted dry for the last time with gauze, and 100 hole μ L/ primary antibodies (1: 2000 are added Dilution is diluted with the PBST of 4% cow's serum, is put into 37 DEG C, 1~2h of water-bath).Then it takes out again and pats dry primary antibody liquid, PBST is washed Wash ELISA Plate three times, every minor tick 5 minutes pats dry ELISA Plate with gauze for the last time, it is added secondary antibody liquid (rabbit-anti sheep 1: 1000), Every hole adds 100 μ L, is put into 37 DEG C, 1~2h of water-bath.It takes out again and pats dry secondary antibody liquid and wash the every minor tick five of ELISA Plate 3 with PBST Minute, it is patted dry for the last time with gauze, the H SO of 2M is added in every hole after adding 50 μ L substrate developing solutions, black out to develop the color 10-20 minutes 50 μ L of solution terminate reaction, after with microplate reader survey OD value (in half an hour).

Two, the preparation of AIDS blood purification

1, the preparation of blood purification cell column

With the made hybridoma macrophage and CD4+T cell of the sterile saline cleaning present invention, 1000r/min from The heart is centrifuged 5min again after cleaning with 1000r/min, is 1: 0.5~3 by the ratio between the strain of hybridoma macrophage and CD4+T cell strain Ratio take sedimentation cell, be packed into 200ml hydrostatic column made of acrylate etc high-biocompatibility material, filling To 4/5, cell column is sealed spare.

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 It is fully tied with gp41 antibody, but the surplus free gp120 and gp41 antibody for having enough high titres, then will contain combination The mixed antibody of type and sequestered gp120 antibody and mixed antibody containing mating type and sequestered gp41 antibody again with etc. Potency mixing, is made into final mixed antibody, prepares 5 kinds of gel mixed antibodies respectively with the agarose C1-4B solution of gradient concentration, The potency for making HIVgp120 and gp41 antibody is 1: 700,1: 500,1: 300,1: 200,1: 100 and corresponding agarose concentration It is followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, by antibody titer from low to high spare blood to be perfused in equal volume Liquid daf molecule column and the clarifier containing cleanser is made.Agar gel be formed by filter opening with increasing for agarose concentration and It reduces, clarifier entrance agarose concentration is low, and filter opening is just big, is conducive to plasma perfusion and high titre antibody or cell and HIV Association reaction；And exit concentration is high, filter opening is just small, is easy to detention HIV or Large molecular conjugates.

Formula one: gp120 and gp41 antibody is kept the temperature after 100 DEG C dissolve at 39~42 DEG C with carrier function 0.7%, 0.8%, 0.9%, 1.0%, 1.1% agarose C1-4B physiological saline is made into reversed corresponding 1: 700,1: 500,1 : 300, the gel antibody of 1: 200,1: 100 gradient titre successively takes 40ml gel antibody that blood is added by low titre to high titre In liquid daf molecule column, it is desirable that under the gel antibody being formerly added is cooled to 37 DEG C as just then adding after semi-solid agar gel Once, so that the blood purification cell column is formed antibody titer from high to low (from import to outlet) from top to bottom and from low to high The layer distributed of agarose concentration, wherein in conjunction with goat-anti HIV (gp120 and gp41) antibody and unbonded gp120 antibody, Gp41 antibody and hybridoma macrophage and/or CD4+T cell, which are fixed in gel, to be play a part of to adsorb HIV.

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

Formula three: antibody prepared by different HIV infection strains and its corresponding titre being made by multiple proportions are indicated respectively The valence value of gradient, such as 1 titre 1: 100 of HIV infection strain pipe, 1: 200 pipe ...；2 titre 1: 100 of HIV infection strain pipe, 1: 200 pipes ..., and so on, it then will infect the liquid agarose C1- of 1 titre 1: 100 of strain pipe with 10ml39~42 DEG C heat preservation 4B is put into blood purification cell column after mixing, it is to be placed be cooled to semi-solid gel after, then will infection 1 titre 1: 200 of strain pipe with The liquid agarose C1-4B of 10ml39~42 DEG C heat preservation is mixed, and mixing liquid is put into the gel upper layer of blood purification cell column, according to this Analogize.It to be prepared in practical applications by combination, for example the HIV infection strain in somewhere period has totally 3 plants of A, B, C, preparation Antibody has the pipe of A strain 1: 100,1: 200 pipe ...；The pipe of B strain 1: 100,1: 200 pipe ...；The pipe of C strain 1: 100,1: 200 pipe ...；Through Be exactly after combination ABC strain 1: 100 pipe, ABC strain 1: 200 manage ... ABC strain 1: 1000 manage, then by ABC strain 1: 1000 manage and The liquid agarose C1-4B of 10ml39~42 DEG C heat preservation is put into blood purification cell column after mixing, to be placed to be cooled to semisolid After gel, then by the liquid agarose C1-4B mixing of the pipe of ABC strain 1: 900 and 10ml39~42 DEG C heat preservation, mixing liquid is put into blood Semisolid gel upper layer has been cooled in daf molecule column, and so on, centre can be spaced selection, for example then select ABC 1: 500 pipe of strain and the liquid agarose C1-4B of 10ml39~42 DEG C heat preservation are mixed, and mixing liquid is put into blood purification cell column It is cooled to semisolid gel upper layer, the dosage of liquid agarose C1-4B can also adjust in 1ml~10ml (to be made by At blood purification cell column, form gradient antibody content from low to high from import to outlet, only have in view of antigen and antibody Immune response can be just played in proper ratio, forms precipitation line and prevents moving ahead for comparator antibody or antigen, so difference HIV contains When the blood plasma of amount passes through cleanser, HIV is just adsorbed detention in different levels, and HIV infection strain of the invention can be with In conjunction with goat-anti Ig and unbonded gp120 and gp41 antibody in the almost all of infection strain at that time of letter lid and adsorbent It is all the HIV aglucon being fixed in Agar Gel in advance, HIV antibody is tied especially in conjunction with the HIV antibody and HIV for having goat-anti HIV It is bigger that conjunction is formed by immune complex molecular weight, is entirely capable of being delayed in Agar Gel and being removed, about along with aperture For 85nm Agar Gel inherently can detention diameter be 100~120nm HIV, hardly had so theoretically inferring Treat invalid AIDS patient).

3, the specification and material of blood purification

Blood purification cell column or blood purification are the cylinder or rectangular, infundibulate that bottom diameter is small, top diameter is big, volume For 200~300ml, inlet and outlet are equipped with cell screen clothes, and entrance top diameter sieve mesh number is 800 mesh；Exit bottom diameter sieve mesh Number be 2.0~5.0 mesh (2.5~5.0 mesh are equivalent to 0.1~0.2 micron or 100~200 nanometers), specifically can be made into 2.0 mesh, 2.5 mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh 7 kinds of different sizes, to stop 120 nanometers inhibition of HIV or Bigger bacterium；The cell strainer that mesh number is 100 mesh (being equivalent to 4 microns) is arranged in liquid outlet, may filter out to stop Cell；It is equipped with buffer area between liquid entrance and mesh screen, is conducive to the stability of system circulation.

Blood purification selects the high molecular material of acrylate etc, it is desirable that good biocompatibility, hardly activation are mended Body, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.Can by covalently, The methods of grafting, polymerization improve the structure of material, the inhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxidative stress Influence, to improve biocompatibility, reduce complication generation.Certain substances with anticoagulation are solidificated in carrier Or on the material of absorber inner surface, blood clotting can inhibit, improve biocompatibility, can also reduce heparin dosage, and having can It is able to achieve no-rod tractor.Such as heparin is aggregated on polyacrylonitrile-polyethyleneimine film, effect may be more preferable, and can reduce Allergic reaction during absorption, the polyacrylonitrile surface for solidifying chitosan and heparin covalent object also show good blood compatibility Property, and can inhibit the activity of pseudomonas aeruginosa, reduce cell-cytotoxic reaction.

Three, the preparation of plasma separator

1, it preparation principle: is prepared according to the molecular size of haemocyte and blood plasma components.Such as visible component (blood in blood of human body Cell) size are as follows: normocyte is about 7 microns (μm), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, neutrality About 12 μm of granulocyte, eosinophil is more bigger, and basophilic granulocyte and neutrophil leucocyte are close, and 6-8 μm of small lymphocyte, Approximate with red blood cell, monocyte is maximum, and about 15-20 μm.Blood platelet is disc, 1~4 micron of diameter to 7~8 microns not Platelet mean diameter Deng, people is 2-4 micron, 0.5~1.5 micron of thickness.

2, material: poly-vinegar non-woven fabrics, acetate fiber, absorbent cotton etc. can be selected, it is desirable that good biocompatibility hardly activates Complement, the change for not causing inflammatory reaction, not causing leucocyte, blood platelet, blood oxygen pressure, complement C 3, C5a.It can be by altogether The methods of valence, grafting, polymerization improve the structure of material, the microinhomogeneities for adjusting surface, hydrophily, reduction to blood coagulation and oxygen Change stress influence, the generation to improve sieving adequacy and biocompatibility, reduce complication.

3, type and spec: for the shape of separator, filter core preparation can be made with materials such as acetate fiber or absorbent cotton The shapes such as flat structure are prepared into as filter core at column construction, with materials such as poly-vinegar non-woven fabrics；By haemocyte and blood to be separated The molecular size of slurry composition determines aperture.It is plasma separator according to the present invention property stabilization, good biocompatibility, penetrating Property high high molecular polymer hollow fibre type filter is made, hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm, Aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm.The hole only permit blood plasma filtration, but can stop all cells at Point.

Four, the component of AIDS therapeutic equipment

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

2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.(1) blood pump (Blood Pump): for pushing blood Circulation is to maintain going on smoothly for blood purification treatment, and usual blood pump part often has rotary test speed function, to monitor patient Blood circumstance, therefore blood pump runner and the setting of groove spacing are accurate, and need often adjustment, according to the feelings of bloody path pump line Spacing is generally set as 3.2~3.3mm by condition, can not be too loose, and it is inaccurate otherwise to will cause blood flow detection；Also can not be too tight, otherwise It will cause pipe breakage.(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, and uses It to continue the injecting heparin in sieving pipeline (patient blood), contacts, is easy with air since the blood of patient recycles in vitro Blood coagulation phenomenon occurs, anticoagulative can be occurred using heparin pump.(3) sound pulse pressure monitor: arterial blood pressure monitoring mainly to The stopping state of dynamic monitoring blood separator micropore, in addition to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When When blood flow deficiency, angiosthenia will be reduced；When having blood coagulation, thrombosis, especially separator blockage of the micro orifice, angiosthenia will It increases；Vein pressure monitoring is used to monitor the pressure of pipeline blood reflux, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow When insufficient and venous return syringe needle falls off, vein pressure will decline, if bloody path return pipe distortion blocking or reflux syringe needle When blocking, vein pressure will be increased.(4) air monitering (Air Detector): the air gas for monitoring blood pathway Bubble, the general principle for using ultrasonic listening, in order to avoid air embolism occurs for patient and is arranged.When having monitored air bubble When, detection system can drive artery and vein bloody path folder to carry out blocking blood flow, prevent dangerous generation.

In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional member Property, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warned Report the blood purifying therapeutical instrument of the micro computers such as reason and ring off signal processing.

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

1, it installs: such as Fig. 1, with sterile working connecting components, including plasma separator, clarifier and each circulation line.

2, it is vented: with sterile saline filling liquid separator, clarifier and each circulation line, excluding separator, clarifier And its gas, bubble in circulation line, it goes through, confirmation after gas, bubble without using.

3, lead to liquid: arterial blood line pipe (1) being connected to the arteries of AIDS patient, in operation the row of going through again Whether gas is complete, and whether liquid stream is unobstructed, and flow liquid in pipe is avoided to pollute.

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

5, start: one end of arterial blood line pipe (1) is connected with arteries, venous line (5) are connected into venous blood Then pipe opens blood pump (2), blood flow is 100~150ml/min, and such as Fig. 1, arterial blood is through arterial blood line pipe (1) entrance Plasma separator (4), the blood plasma separated reach clarifier (8) through circulation line (7) under the action of blood plasma pump (6), to Full of blood plasma, about 10 minutes, begin releasing blood plasma, flowed out through circulation line (10), it is synchronous that blood plasma is perfused to clarifier (9), When blood plasma in clarifier (8) has nearly flowed, perfusion blood plasma is started again at, clarifier (9) begins releasing blood plasma at this time, and two simultaneously Clarifier (8), the clarifier 9 of connection) alternately, purified blood plasma divides through circulation line (10) and plasma separator (4) From haemocyte converge after through venous line (5) feed back.Such as Fig. 2, when blood to be separated enters the inner cavity of plasma separator (1) (2) when, the effect through valve (8) can enter the exocoel (6) of separator, so by the small molecule blood plasma components (5) of micropore (3) It flows out, and cannot be flowed out through valve (8) by the haemocyte (4) of micropore (3) by plasma outlet port (7).Such as Fig. 3, when containing HIV (1) when blood plasma enters clarifier, HIV (1) therein is fixed on HIV antibody (2), macrophage in agar gel (8) respectively Cell (4), CD4+T cell (6) are combined into antigen antibody complex (3), macrophage phagosome (5), CD4+T cell conjugates (7), the HIV after being combined no longer moves down, and the HIV for 100~120nm not in addition being combined is again by because of concentration more Gao Erkong The bottom agar gel molecular Screen Out of smaller about 1% concentration of 85nm of diameter stays at (9).HIV is so removed, until in advance The plasma circulation amount (usually 9L) of setting, treatment just end.Entire therapeutic process is controlled by computer, and can be examined at any time Survey working condition, easy to use, automation and safety.

Six, the verifying of AIDS therapeutic equipment effect

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

The effect of in order to verify CD4+T cell clearance HIV, the present invention devises easy test method: taking the 2.5 of sterilizing × 300mm Westergren's blood sedimentation tube 5, draw respectively be centrifuged (1000r/min, 5min) precipitating CD4+T cell to 200mm carve Degree then draws the heat preservation after 100 DEG C dissolve and reaches about 10mm long quarter in 39~42 DEG C of 0.9% spare agarose C1-4B Degree, after setting blood sedimentation stand cooling, agarose becomes semisolid, and blood sedimentation tube inner cell can be prevented to flow out but not prevent the water of small molecule Pass through with the substance of chemical analysis etc.The 5 of the AIDS patient that Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database save Example blood plasma, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube upper end blank pipe, blood sedimentation tube lower layer to be flowed through in batches CD4+T cellular layer and out of blood sedimentation tube flow out after, collect efflux, referred to as AIDS filter after blood plasma.Take AIDS filter before blood plasma and Blood plasma after filter is operated according to HIV-1p24 antigen detection kit (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai), With the p24 of known concentration 0pg/ml, 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml Antigen is lower than 5pg/ml, 0~400pg/ml of measurement range, range of linearity 0.5pg/ml~80pg/ as control, minimum detection limit 450nm measures absorbance (OD) in ml, 15min, and blank control calibration object absorbance value is not higher than 0.050,0pg absorbance value It is not less than 1.000 higher than 0.100,1000pg/ml absorbance, is considered as positive, testing result (table as absorbance > 0.12 1) illustrate, after AIDS blood plasma filters the simple purification device of the cell containing CD4+T, part HIV is adsorbed by CD4+T cell, through the 1st After secondary filtration, HIV total body clearance is 22.84%, after the 2nd filtration, total body clearance 35.31%, and after the 3rd filtration, Total body clearance is 41.9%.Illustrate the increase with filtration number, HIV can be removed constantly, to reach treatment AIDS mesh 's.

1 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (pg/ml) of the cell containing CD4+T

2, hybridoma macrophage removes the verifying of HIV effect

The effect of removing HIV for check cross tumor macrophage, the present invention devises easy test method: taking sterilizing 2.5 × 300mm Westergren's blood sedimentation tube 5, draw respectively be centrifuged (1000r/min, 5min) precipitating macrophage hybridoma Cell strain then draws after 100 DEG C dissolve heat preservation in 39~42 DEG C of 0.9% spare agarose C1-4B to 200mm scale, Reach about 10mm long scale, set blood sedimentation tube it is cooling after, agarose becomes semisolid, can prevent blood sedimentation tube inner cell flow out but not The substance of the water and chemical analysis etc that prevent small molecule passes through.What Ling Qu Disease Control and Prevention Center and Infectious Diseases Lab sample database saved 5 blood plasma of AIDS (AIDS) patient, respectively about 10mL, respectively takes 9mLAIDS to filter preceding blood plasma and injects blood sedimentation tube in batches (simply only Makeup is set) upper end blank pipe, wait flow through the hybridoma macrophage strain layer of blood sedimentation tube lower layer and out of blood sedimentation tube after outflow, collection stream Blood plasma after the filter of liquid out, referred to as AIDS.Blood plasma and blood plasma after filter before taking AIDS to filter, with human macrophage migration inhibitory factor (MIF) ELISA detection kit (hundred stamen Biotechnology Co., Ltd of Shanghai) pairing detection, by specification operation, detection range be 0~ 800pg/ml, susceptibility 1.0pg/ml, can be under white background, and directly detect by an unaided eye: color is deeper in reacting hole, positive Stronger, to be colourless or extremely shallow, the depth of the be in color of foundation is indicated negative reaction with "+", "-" number.OD value can also be surveyed: On ELISA detector, at 450nm (if developing the color with ABTS, 410nm), each hole OD value is surveyed after returning to zero with blank control wells, if It is as positive greater than 2.1 times of defined negative control OD value.As a result such as table 1, MIF testing result is yin in blood plasma before filtering Property (or because of content lower than detection sensitivity, blood plasma long-term preservation cause degradation etc.), and testing result is the positive in blood plasma after filtering. Illustrate that macrophage hybridoma cell strain produces MIF cell factor in this process.MIF is collection cell factor, growth factor, swashs Element and the multi-effect protein molecular of enzyme characteristic play central as inherent immunity and the regulatory factor of inflammatory reaction Effect plays panimmunity function in various infection and active chronic inflammation disease.Making the simple purification of AIDS blood plasma filtration Before and after device while MIF pairing detection, the present invention has also done the pairing detection of HIV-1p24, is detected and is tried according to HIV-1p24 antigen Agent box (Biotechnology Co., Ltd is inspired in enzyme-linked immunization, Shanghai) operation, with known concentration 0pg/ml, 0.5pg/ml, 1pg/ The p24 antigen of ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are lower than as control, minimum detection limit 5pg/ml, 0~400pg/ml of measurement range, 450nm measures absorbance in the range of linearity 0.5pg/ml~80pg/ml, 15min (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ml absorbance not higher than 0.050,0pg absorbance value It is considered as the positive as absorbance > 0.12 not less than 1.000, as a result (table 2) illustrates the simple purification dress of AIDS blood plasma filtration It postpones, part HIV is swallowed by macrophage hybridoma cell strain to be adsorbed, and the blood plasma HIV after filtration is significantly reduced, through the 1st time After filtration, HIV clearance rate is 20.55%, and after the 2nd filtration, HIV clearance rate is 42.83%, p < 0.01, is had apparent Effect illustrates constantly be removed with the increase HIV of filtration number, to reach treatment AIDS purpose.

1 AIDS blood plasma of table filter MIF testing result before and after the simple purification device of the macrophage containing hybridoma (it is quantitative: pg/ml)

2 AIDS blood plasma of table filters the simple purification device front and back p24 testing result (p24:pg/ of the macrophage containing hybridoma ml)

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

The present inventor's basic skills according to the invention has done following simple confirmatory experiment: take HIV-1gp120 antibody, Gp41 antibody (Shanghai Rui Qi Biotechnology Co., Ltd) is added to and keeps the temperature after 100 DEG C dissolve in 50 DEG C of 1.0% spare fine jades In lipolysaccharide C1-4B, titre is 1: 300~500 after mixing, 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 agarose becomes semisolid after cooling.Ling Qu Disease Control and Prevention Center and infectious disease are real Test the sample of 5 AIDS patients of room sample database preservation, each about 10mL of blood plasma after removing cell, blood before respectively taking 9mLAIDS to filter Slurry injects blood sedimentation tube upper end blank pipe in batches, the 1.0% agarose C1-4B antibody-containing of blood sedimentation tube lower layer to be flowed through and from blood In immersed tube after outflow, efflux, blood plasma after referred to as AIDS filter are collected.Blood plasma and blood plasma after filter before taking AIDS to filter, according to HIV- 1p24 antigen detection kit (enzyme-linked immunization, Shanghai inspire Biotechnology Co., Ltd) operation, with known concentration 0pg/ml, The p24 antigen of 0.5pg/ml, 1pg/ml, 2.5pg/ml, 5pg/ml, 20pg/ml, 40pg/ml, 80pg/ml are minimum as control Detection limit is lower than 5pg/ml, 0~400pg/ml of measurement range, and 450nm is surveyed in the range of linearity 0.5pg/ml~80pg/ml, 15min Determine absorbance (OD), blank control calibration object absorbance value is not higher than 0.100,1000pg/ not higher than 0.050,0pg absorbance value Ml absorbance is not less than 1.000, is considered as the positive as absorbance > 0.12, and testing result (table 1) illustrates, the filter of AIDS blood plasma After crossing simple purification device, part HIV is adsorbed by corresponding antibodies, and after the 1st filtration, HIV total body clearance is 20.01%, After the 2nd filtration, total body clearance 27.99%, after the 3rd filtration, total body clearance 37.36%.Illustrate with filtration The increase HIV of number can be removed constantly, to reach treatment AIDS purpose.

1 AIDS blood plasma of table filters p24 testing result (pg/ml) before and after simple purification device

Above-mentioned confirmatory experiment show the HIV to dissociate in blood plasma can by blood purification agent (HIVgp120 antibody, HIVgp41 are anti- Body, CD4+T cell strain, the strain of hybridoma macrophage, agar gel micropore) it removes, show AIDS therapeutic equipment tool of the invention There is the significant therapeutic efficiency for removing blood plasma HIV.

Claims

1. a kind of AIDS therapeutic equipment for medical domain, which is characterized in that described net comprising plasma separator and clarifier Change device is made of high-biocompatibility material and exit is provided with sieve, and inside is filled with equipped with HIV antibody, combines goat-anti The HIV antibody of Ig, the strain of hybridoma macrophage and CD4+T cell strain agar gel, make its entrance to exit formed from High to Low antibody titer and the layer distributed of agarose concentration from low to high.

2. AIDS therapeutic equipment according to claim 1, which is characterized in that the clarifier by its exit sieve, Serve fixed and molecular sieve agar gel, preparation hybridoma macrophage strain therein, CD4+T cell strain, HIV antibody, The HIV antibody for combining goat-anti Ig collectively forms molecular sieve mechanical removal and cell and antibody mediated immunity removes the barrier of blood plasma HIV.

3. according to claim 1,2 any AIDS therapeutic equipment, which is characterized in that the volume of the clarifier is 200 ~300ml, inlet and outlet are equipped with cell screen clothes, and entrance top diameter cell screen clothes mesh number is 800 mesh, exit bottom diameter cell screen clothes Mesh number is 2.0~5.0 mesh, and cell strainer mesh number is 100 mesh.

4. AIDS therapeutic equipment according to claim 3, which is characterized in that 2.0 mesh, 2.5 are made in the exit sieve 7 kinds of specifications of mesh, 3.0 mesh, 3.5 mesh, 4.0 mesh, 4.5 mesh and 5.0 mesh.

5. AIDS therapeutic equipment according to claim 1, which is characterized in that the layering of the antibody titer from high to low Distribution refers to the mixed antibody potency from the entrance of clarifier to exit successively with 1: 700,1: 500,1: 300,1: 200,1: 100 points 5 layers.

6. AIDS therapeutic equipment according to claim 1, which is characterized in that point of the agarose concentration from low to high Layer distribution refer to the agarose concentration from the entrance of clarifier to exit successively with 0.7%, 0.8%, 0.9%, 1.0%, 1.1% point 5 layers.

7. according to claim 1,2,5 any AIDS therapeutic equipment, which is characterized in that the HIV antibody includes HIV- 1gp120 antibody, HIV-1gp41 antibody.

8. AIDS therapeutic equipment according to claim 1, which is characterized in that the separator hollow-fiber film diameter is 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 be stopped all thin Born of the same parents' filtration.

9. the preparation method of the cleanser in AIDS therapeutic equipment according to claim 1, which is characterized in that with sterile life It manages salt water and cleans made hybridoma macrophage and CD4+T cell, 1000r/min centrifugation is centrifuged again with 1000r/min after cleaning 5min takes cell precipitation to be packed into high biology in the ratio that the ratio between the strain of hybridoma macrophage and CD4+T cell strain are 1: 0.5~3 In 200ml hydrostatic column made of compatibility material, cell column is made, separately by gp120 and gp41 antibody respectively with goat-anti Gp120 and the mixing of goat-anti gp41 antibody, are fully tied goat-anti gp120 and goat-anti the gp41 antibody in mixed liquor, but surplus There is a large amount of gp120 and gp41 antibody, then two kinds of mixed liquors are mixed again, it is equal to be made into gp120 and gp41 antibody titer Final mixed antibody prepares 5 kinds of gel mixed antibodies respectively with the agarose C1-4B solution of gradient concentration, make HIVgp120 and The potency of gp41 antibody be 1: 700,1: 500,1: 300,1: 200,1: 100 and corresponding agarose concentration be followed successively by 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, purification is made so that spare cell column to be perfused in equal volume from low to high by antibody titer Device makes to be formed the layer distributed of antibody titer from high to low and agarose concentration from low to high from import to outlet, wherein Hybridoma macrophage, CD4+T cell and HIV antibody, which are fixed in gel, to be play a part of to adsorb HIV.

10. -8 any AIDS therapeutic equipments are preparing the application in purging in vitro device, feature according to claim 1 Be, the connection type of the purging in vitro device are as follows: one end of arterial blood line pipe (1) through heparin pump (2) and blood pump (3) with Plasma separator (4) be connected, plasma separator (4) through blood plasma pump (6) and circulation line (7) clarifier in parallel with two (8, 9) it is connected, then successively converges through circulation line (10), venous line (5).