CN1500806B - HIV-lgp160 membrane protein, preparing method and uses thereof - Google Patents
HIV-lgp160 membrane protein, preparing method and uses thereof Download PDFInfo
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Abstract
The present invention relates to one kind of glycosylated HIV-lgp160 membrane protein, the continuous perfusion culture process in bioreactor to prepare the said membrane protein and its application as antigen preparation. The said membrane protein has glycosylating modification and correct spatial structure and no pathogenetic danger, and may be prepared inside bioreactor in large scale.
Description
Technical field
The present invention relates to the eukaryotic system protein expression, the preparation and in the diagnostic reagent Application for Field, especially relate to a kind of through glycosylation modified HIV-1 gp160 membranin, also relate to a kind of preparation method who utilizes the bio-reactor continous pouring to cultivate described membranin, and as the application of antigen preparation.
Background technology
Known human immunodeficiency virus (HIV) is a kind of pathogenic agent that causes acquired immunodeficiency syndrome, can cause the immune response of human body behind its infection human body, produces corresponding antibody.By detecting in blood or the body fluid whether have HIV antibody, can judge whether this sample has infected HIV.The HIV coating is the main target position of humoral immunoresponse(HI), and the corresponding antibody of generation is referred to as anti-env domain antibodies, comprises anti-gp160 antibody, anti-gp120 antibody and anti-gp41 antibody.The detected by Western blot HIV that the World Health Organization, U.S. food and drug administration, ministry of Health of China etc. are formulated infects in the judging criterion, all regulation must have anti-env domain antibodies reaction band (Li Jingyun, the etiological diagnosis of acquired immune deficiency syndrome (AIDS), Beijing: military medicine Science Press, 1999,29-35).Big quantity research report also confirms all to contain in HIV the infected's blood sample anti-env domain antibodies.In view of the above, detecting HIV antibody is to detect anti-env domain antibodies to a great extent.
Existing immunology diagnosis technology general using antigen-antibody reaction detects anti-env domain antibodies.Can gp160, gp120, gp41 be arranged with anti-env domain antibodies bonded antigen, obtaining means has blood extraction, intestinal bacteria system gene engineering expression, chemical process polypeptide synthetic etc.
Select from fragment, gp41 uses maximum fragments at present, its detection be anti-gp41 antibody.But infect judging criterion according to the detected by Western blot HIV that the World Health Organization, U.S. food and drug administration, ministry of Health of China etc. are formulated, if there is a response line in the pol district, just be judged to be the HIV infection as long as a response line occurs among then anti-gp41, anti-gp120, the anti-gp160, that is to say to have anti-gp41 feminine gender fully, and anti-gp160 or anti-gp120 male HIV infected specimen.Therefore it is not enough only relying on gp41 in the HIV antibody test.And gp160 has comprised the most antigenic determinant in env district as the precursor of gp120 and gp41, is that the most suitable diagnostic reagent uses in the various antigens in env district.
From acquisition methods, wherein from HIV the infected's blood or from the metainfective lymphocytolysis liquid of HIV, separate the HIV antigen that obtains, owing to can remain the worry of 100% validity to the HIV inactivation technology, no matter to antigenic preparation person, still concerning antigenic user, all exist real or the potential pathogenic risk.This has seriously limited the use of this technology.
The method of having described in EP-A-O 326 490 with chemosynthesis prepares HIV-1 gp41, and US 5,800, and the method for having described chemosynthesis in 983 prepares HIV-1 gp160 fragment, and above-mentioned synthetic peptide all can be used for detecting HIV-1 antibody.Because the technology of chemically synthesized polypeptide is comparatively ripe, so this method can realize on a large scale, the production of good reproducibility.But with the synthetic polypeptide of chemical process, be subjected to the restriction of reaction efficiency and yield, fragment length can only have the dozens of amino-acid residue, can't comprise more antigenic determinant.What simultaneously existing industrialization synthetic technology obtained is linear peptides or branched peptide, lacks secondary structure, also can't realize necessary post transcriptional modificaiton, and immunologic competence is very limited, and has the possibility to the positive sample omission when being applied to diagnostic reagent.
US 5,834,267 and (cell and molecular immunology magazine such as Han Baoguang, 1999, the 15th volume, the 2nd phase) having described a kind of respectively is the technology that the host bacterium prepares genetically engineered gag-env fused antigen with e. coli bl21 or DH5 α. the intestinal bacteria system has very high expression efficiency, all kinds of HIV antigen costs that obtain are low, security is good, but with natural antigen compare lack transcribe the back glycosylation modified, and the antigenic space structure that obtains by the inclusion body mode, particularly there were significant differences for disulfide linkage position and natural antigen, cause antigenic activity to descend and non-specific rising. in addition, existing expression vector system all at expressing the tens thousand of daltonian protein designs of molecular weight, surpasses 100,000 daltonian protein when being used to express molecular weight, during as gp160, exist expression amount low, plasmid is unsettled may.
Eukaryotic expression system is the antigenic main path that obtains through glycosylation modified.The non-human archeocyte of present more use system.Use baculovirus expression system at insect cell inner expression gp160 (Wells DE, Compans RW, Virology 1990Jun as Wells DE; 176 (2): 575-86), Fenouillet E expressing cho cell env antigen (Fenouillet E, et.al, Virology 1997 Apr 28; 231 (1): 89-95).But with people's cell difference, the gp160 antigen and the natural antigen that obtain with above-mentioned inhuman source cell eukaryotic expression system have certain difference to above-mentioned non-human archeocyte on glycosylation structure and level on glycosylation mechanism.
Expressing gp160 antigen with human archeocyte is undoubtedly ideal.US 5,580, and 720 to have described a kind of be the technology of eukaryotic cell expression gp160 with bjab cell.US 6,074, and 646 to have described a kind of be the technology of eukaryotic expression gp160 with cem cell.That the former is used is a kind of CD4
-, the Epstein-Barr virus feminine gender, people Burkitt ' s lymphocyte series, belong to people B-lymphocyte series.But under natural situation, HIV enters can at first concentrate behind the human body and attacks the CD4T lymphocyte, and have 200,000,000 CD4T lymphocytes to be killed average every day, discharge simultaneously about 1,000,000,000 HIV viruses (Ho DD, et a1.Nature, 1996,373:123-126).Therefore, the T lymphocyte is more suitable in preparation gp160 as the main host cell under the natural situation of HIV virus.The latter is with the HIV gp160 gene fragment eukaryotic expression vector of packing into, transduction goes into to separate from the people T-of acute lymphoblastic leukemia patient peripheral blood lymphocyte series again, the gene engineering antigen that obtains is the part of HIV gp160 gene fragment, can not comprise whole antigenic determinants.
Sum up foregoing invention and achievement in research, all have the limitation of different aspect.
Therefore, the invention provides does not a kind ofly have pathogenic risk, glycosylation modified and correct space structure is arranged, comprises the antigenic preparation method of a large amount of antigenic determinants and be raw material with it, and preparation is used for the antigenic label of HIV antibody diagnosis, has overcome above-mentioned defective.
Summary of the invention
One of technical issues that need to address of the present invention provide a kind of HIV-1 gp160 membranin, its immunity specifically is in conjunction with HIV-1 antibody, its amino acid residue sequence is shown in SEQ ID NO.:1, be the HIV membranin gp160 of HIV-1 env district coding or the part of gp160, and the N-that has high mannose type or heterozygous is glycosylation modified, the molecular weight of measuring through SDS-PAGE is 130,000-140,000 dalton.Having overcome has pathogenic risk in the prior art, only contain the incomplete antigen determinant, or immunocompetence such as is suppressed at defective.
Two of the technical issues that need to address of the present invention provide a kind of in bio-reactor continous pouring cultivate to prepare the method for HIV-1 gp160 membranin, overcome the defective of not carrying out mass preparation in the prior art with bio-reactor.
Three of the technical issues that need to address of the present invention provide the application of HIV-1 gp160 membranin as antigen preparation.
Under native state, after HIV attacked target cell, at first synthetic gp160 membranin in target cell formed gp41 and gp120 then after enzyme is cut in born of the same parents, therefore the gp160 membranin has comprised each the major antigen determinant on gp41 and the gp120 substantially as the precursor of gp41 and gp120 membranin.Method of the present invention is promptly at the gp160 membranin.
Under natural situation, HIV virus is the main host cell with human T lymphocyte, in host cell, finish the synthetic and modification of the various viral proteins that comprise gp160, host cell cracking subsequently, comprising that the viral protein of gp160 and the virion that assembling finishes are released together. can think in view of the above to be in or approaching natural space structure with the gp160 membranin that obtains behind the HIV virus infection human T lymphocyte. as a kind of eukaryotic expression system, it can comprise the post transcriptional modificaiton of glycosylation fully to the gp160 membranin. the host that the present invention synthesizes gp160 with the eukaryotic cell conduct, and think that the human T lymphocyte is that HUT-78 is an optimized results, set up simultaneously and obtain secretion gp160, but lysis does not take place, do not discharge the method for infectious HIV particulate specific cells strain.
Because the polysaccharase of HIV is not read correct functioning, cause the HIV viral genome sudden change probability that obtains after duplicating higher.Present known HIV-1 exists M group and O group, and (Virol 1997 for Jonassen, T.O.et.al, and 231:43-47), both gene order differences comprise ten hypotypes of A-J greater than 45% in the M group, and gene order difference reaches 30% between each hypotype.The membranin antigen that obtains behind the HIV-1 infection of eukaryotic cells with viruses with a certain hypotype can not guarantee that the membranin antibody that produces with the HIV-1 of other each hypotypes all has good avidity.The present invention can obtain different gp160 antigen by using different HIV-1 subtype viral infection eukaryotic cells.On gene order, virulence, membranin structure, exist between HIV-2 and HIV-1 than big-difference, not within the scope that the present invention relates to.
Based on Ag-Ab in conjunction with this immunology principle, now set up the technology of the anti-HIV antibody of multiple detection, one of its core technology is antigenic mark, as horseradish peroxidase or alkali phosphatase enzyme mark HIV antigen are used for euzymelinked immunosorbent assay (ELISA) HIV antibody diagnosing reagent kit, the HIV antigen of colloid gold label is used for golden mark method antibody diagnosing reagent kit, the HIV antigen of lanthanide series rare-earth elements mark is used for the time-resolved fluorescence method antibody diagnosing reagent kit, the HIV antigen of acridinium ester mark is used for the chemoluminescence method antibody diagnosing reagent kit and the HIV antigen of tris (bipyridine) ruthenium mark is used for electrochemiluminescence method antibody diagnosing reagent kit.The invention provides embodiment at the marking method of the antigenic optimization of gp160 membranin.
The invention provides a kind of HIV-1 gp160 membranin, its immunity specifically is in conjunction with HIV-1 antibody, its amino acid residue sequence is shown in SEQ IDNO.:1, this sequence is found in Genbank, and it is numbered K03455, is the HIV membranin gp160 of HIV-1env district coding or the part of gp160, and have high mannose type or heterozygous is glycosylation modified, the molecular weight of measuring through SDS-PAGE is 130,000-140,000 dalton.
HIV-1 gp160 membranin of the present invention, it is further characterized in that described membranin is to the molecular weight of the glycosyl part of EndoH sensitivity about 20 under non-sex change condition, 000 dalton, and, under the sex change condition to about 40,000 dalton of molecular weight of the glycosyl part of Endo H sensitivity.Because, the glycosyl molecular weight characterization that records under the sex change condition total seminose type or the heterozygous level of glycosylation of antigen, the glycosyl molecular weight characterization that records under the non-sex change condition seminose type or the heterozygous level of glycosylation of antigenic surface glycosylation site.Because existing diagnostic reagent production technology generally adopts the bag quilt under the non-sex change condition or connects enzymatic process, so antigenic surface glycosylation situation has more significance under the non-sex change condition.
HIV-1 gp160 membranin of the present invention can further describe with reference to its preparation technology.For example, in one embodiment, technology comprises the cell with HIV-1 virus infection HUT78, and HIV-1 type human immunodeficiency virus is 1 with the ratio of cell count: 500-10000; Never filter out the cell strain of secretion membranin in the Po Sui cell; From above-mentioned cell strain, filter out in H9 cell synplasm formation experiment and find the infectious human immunodeficiency virus particulate cell strain of secretion; Identify the degree of glycosylation of above-mentioned cell strain with Glycosylase; Under appropriate condition, cultivate the cell strain that obtains; And purifying, collection can be in conjunction with the HIV membranin of HIV-1 antibody.
So-called synplasm forms experiment and is meant that this characteristic of cytogamy phenomenon can appear in the H9 cell that utilization is infected by HIV, with cell strain and the H9 cell co-cultivation that is screened, and, judge whether cell strain secretes the HIV virion that causes a disease by observing H9 cellular form and cracking situation.The H9 cell can obtain from American type culture collection (ATCC), and numbering HTB-176 is the cell strain (Chen TR.J.Natl.Cancer Inst.84:1922-1926,1992) that typically is used to cultivate HIV virus.
An eukaryotic example described here, that can express the HIV membranin is a HUT78 clone.This HUT78 clone can be passed through American type culture collection (ATCC) and obtain code T IB-161.This clone initial separation is from Sezary syndrome peripheral blood of patients, belong to the human T lymphocyte (Gazdar AF et al., Blood1980,55:409-417).With common T-clone different be that the cultivation of this cell does not also rely on external source T-cell growth factor (T-cell growth factor), is fit to scale operation antigen.
Although use the strain of HIV-1B subtype virus in the exemplary embodiments of the present invention described here, other also might be used to protein preparation of the present invention not in this HIV-1 virus strain of mentioning especially.Variant subtype virus strain can obtain from each hygiene control and research institution, as the Center for Disease Control.
The ratio of HIV virus consumption and HUT78 cell consumption is the important factor that influences the result when making up cell strain.Inventor's optimization data are provided in an embodiment.The person skilled in the art also can be adjusted according to experience separately.
The method that screening secretion membranin described here, while are not secreted the cell strain of the HIV virion that causes a disease again is that the infinite dilution method is obtained mono-clonal and ELISA screening.Its visible Hubei Province of general operation method levy and the monograph of Shen Guanxin (levy in Hubei Province, and tissue culture and molecular cell learn a skill, Beijing Publishing House, 1994,100-107; Shen Guanxin, modern immunological experiment technology, Hubei science tech publishing house, 1998,116-123) other multiple different technologies that are applicable to cell strain mono-clonalization and screening are known by the person skilled in the art, also can be applicable to this.
The present invention cuts back SDS-PAGE electrophoresis with the Glycosylase enzyme and judges degree of glycosylation and type.Enzyme is cut operation and is undertaken by the specification sheets requirement of used enzyme, and there is different requirements in each enzyme production company.ENDO H with New England Biolab company removes seminose type and heterozygous glycosyl in a particular embodiment.SDS-PAGE press the operation of the described method of Sambrook (Sambrook J, et.al, Molecular cloning:a laboratory manual, Cold Sring Harbor LaboratoryPress, 1989,880-887).
The preparation method of a kind of HIV-1 gp160 membranin provided by the invention comprises the steps:
1) with HIV-1 type HIV (human immunodeficiency virus) infection eukaryotic cell, HIV-1 type human immunodeficiency virus is 1 with the ratio of cell count: 500-10000;
2) from above-mentioned eukaryotic cell, filter out can secrete can be specifically immunity in conjunction with the proteinic cell strain of HIV-1 membranin antibody;
3) from above-mentioned eucaryotic cell strain, filter out the cell strain of in H9 cell synplasm formation experiment, finding the infectious human immunodeficiency virus of secretion;
4) cut the method for identifying molecular weight with the SDS-PAGE electrophoresis with the Glycosylase enzyme, from above-mentioned eucaryotic cell strain, filter out the sufficient cell strain of secreted membranin glycosylation;
5) the extensive above-mentioned cell strain of perfusion culture in bio-reactor is controlled level of glycosylation by regulating culture condition, collects culture supernatant;
6) purifying, collection from culture supernatant obtains HIV-1 gp160 membranin.
The invention provides a whole set of preparation technology based on 5 liters of bio-reactor scales, those skilled in the art can amplify technology or dwindle according to known model, to satisfy the production needs of different scales.
The present invention cultivates above-mentioned cell strain with RPMI 1640 substratum continous pourings in the 5L of B.Braun company bio-reactor, this cell strain also can be cultivated in the bio-reactor that B.Braun or other companies such as NBS or Bioengineer similarly design for cell cultures. be suitable for different substratum at different cultivation stages, the RPMI1640 that comprises GIBCO company, DMEM-HG, the HB104 of DuPont company etc., and can add different concns according to the difference of culture condition or do not add calf serum or foetal calf serum. the variation of substratum will exert an influence to gp160 antigen degree of glycosylation and molecular weight, also can influence simultaneously the time that continous pouring is cultivated. the scheme of optimization is to adopt the higher substratum of serum content in the cell amplification stage, antigen secretion and glycosylation stage adopt serum content lower or the cultivation of serum free medium continous pouring, progressively reduce the content of serum in the culture system, generally being controlled at the every day serum content is about the 50-60% of last one day. high-load serum can provide the sufficient nutrient composition, impel the cell rapid amplifying. and constantly reduce serum-concentration, but pair cell antigen expressed and glycosylation process provide persistent stimulation.
The multiple different technologies that is applicable to protein purification will be known by the person skilled in the art.For example ammonium sulfate precipitation, ion exchange chromatography, gel-filtration, and the combination of various technology.Embodiment described here is with anti-gp41 affinity chromatography purification, and it is according to being that gp160 antigen has the antigenic determinant that can be anti-gp41 antibody recognition in a large number, can be specifically with the affinity post on the aglucon combination.The scale of chromatography column can be as required amplified in the scope or is dwindled to several the liter at 1ml, and purification effect is not had obvious influence.
According to existing experience,, can obtain the turnout of the 50mg/ month based on the industrial scale of 5 liters of bio-reactors.Rise to 30 liters of technologies in the scope 5 and send out greatly, expression level is not had obvious influence.
The present invention also provides the antigen preparation that comprises HIV-1 gp160 membranin, comprises described membranin, or the enzyme labelling thing of described membranin, colloid gold label thing, chemiluminescent labels, fluorescent marker, rare earth element marker and electrochemical label thing.
Because the gp160 antigen among the present invention is the protein that a part amount is big, have glycosylation and complex space structure, with the polypeptide antigen commonly used and the gene engineering antigen of intestinal bacteria system very big difference is arranged, so provide in an embodiment through optimized conditions.
" antigen preparation " of indication is meant all kinds of antigenic labels among the present invention.The method of alkaline horseradish peroxidase-labeled gp160 and colloid gold label gp160 is provided in an embodiment.But the marker of gp160 also not only is confined to these two kinds, comprise enzyme labelling, colloid gold label, chemiluminescent labeling, electrochemiluminescence mark, fluorescent mark and rare earth element mark, its form can be liquid, as antigen-marker solution, also can be solid-state, as antigen-marker microballoon.
Description of drawings
Fig. 1. HIV-1gp160 membranin of the present invention SDS-PAGE collection of illustrative plates after the Glycosylase enzyme is cut, wherein strips A is a restriction analysis under the non-sex change condition, the HIV-1gp160 after the arrow points enzyme is cut; Band B is a restriction analysis under the sex change condition, the HIV-1gp160 after the arrow points enzyme is cut; Band C is molecular weight standard (the high molecular mark of Sigma company).
Fig. 2. the purification technique diagram of HIV-1gp160 membranin of the present invention, wherein Fig. 2 A is the chromatography collection of illustrative plates; Fig. 2 B is each sample SDS-PAGE calibrating collection of illustrative plates, and used molecular weight standard is the high molecular mark of Sigma company, and the arrow indication is the HIV-1gp160 membranin.
Embodiment
Embodiment 1 expresses the foundation of the HUT78 cell strain of gp160 membranin
Present embodiment has been described to make up and can have been expressed through glycosylation modified gp160 membranin, and the while forms experiment through H9 cell synplasm and finds again to secrete the HUT78 cell strain of infectious human immunodeficiency virus.
The HUT78 cell strain is incubated in RPMI 1640 perfect mediums that contain 10%FCS, to the logarithmic growth after date, by every 1*10
6Individual cell adds 10
2-10
3The ratio of copy number HIV virus adds HIV-1B subtype virus liquid, places 37 ℃ of C0
2Cultivate in the incubator after 1 hour, move in the T25 culturing bottle, add RPMI 1640,10%FCS, continue at 37 ℃ of CO to final volume 5ml
2Cultivate in the incubator, changed liquid 1 time in per three to four days.After infection, can occur a large amount of cell aggregations earlier and become synplasm, a large amount of cytoclasis death after several days, but still continued growth of individual cells is arranged.
Draw the cell of still continued growth, dilution line by line on 96 porocyte culture plates till every hole has only a cell, is cultivated with RPMI 1640,10%FCS.With gp41 antibody bag 96 hole enzyme plates, every hole adds 100 microlitre culture supernatant, hatches 1 hour for 37 ℃, wash gp41 antibody 100 microlitres that every hole behind the plate adds horseradish peroxidase-labeled, hatched 30 minutes for 37 ℃, wash plate after, add tmb substrate, detect the gp160 expression amount in the supernatant.Draw the clone who expresses gp160, dilution line by line on 96 porocyte culture plates once more is till every hole has only a cell.Change mono-clonal over to 24 orifice plates and continue to cultivate, collect culture supernatant.Measure the gp160 expression amount once more.Draw the high cell strain of gp160 expression amount, amplification back liquid nitrogen container is frozen, is cell one to be selected.
In 24 orifice plates, every hole adds HUT 78 cell 1ml and (contains 2.5*10
5Individual cell).Every hole adds 0.1ml cell one to be selected in 8 holes.3 holes add HIV-1 virus liquid 0.1ml respectively in addition, 0.001ml, 0ml (confirm that through quantitative PCR titre is 10
5Copy/ml), contain 10000,100,0 live viruses respectively, as positive control, sensitivity contrast and negative control.37 ℃ of CO
2Cultivate after 2 days in the incubator, every hole adds RPMI 1640 substratum 1ml.Draw the 1ml culture supernatant every 3 days later on, add fresh RPMI 1640 substratum of 1ml.Observation of cell form situation under the every day mirror, continuous 21 days.To not have synplasm appearance, the sucking-off of acellular cracked cell strain, the rearmounted liquid nitrogen container that increases is frozen, is cell two to be selected.
In the 25cm2 Tissue Culture Flask, cultivate cell two to be selected, the results culture supernatant, behind the centrifugal removal cell debris, (anti-gp41 is available from BiodesignIntemational to be splined on the anti-gp41-Sepharose4B affinity column of usefulness pH 7.2 phosphoric acid buffer equilibrated; Sepharsoe4B is available from Amersham Biosciences), after peak to be passed flows to end,, collect elution peak, to the pH 7.2 phosphoric acid buffers liquid of dialysing with 2M potassium sulfocyanate wash-out.Get 20 microlitre elution peaks, add G5 buffer 2 microlitres, Endo H 1 microlitre (New England BioLab company), 37 ℃ of enzymes were cut 3 hours.Sample after getting enzyme and cutting, 6% gum concentration SDS-PAGE electrophoresis.Other gets 20 microlitre elution peaks, adds Denature buffer 2 microlitres, and 100 ℃ were heated 10 minutes, added G5 buffer 2 microlitres, Endo H 1 microlitre (New England BioLab company) again, and 37 ℃ of enzymes were cut 3 hours.Sample after getting enzyme and cutting, 6% gum concentration SDS-PAGE electrophoresis.The sufficient gp160 membrane protein molecule of glycosylation amount 130 kilodaltons, ENDO H enzyme is cut about 110 kilodaltons of back molecular weight under the non-sex change condition, and ENDO H enzyme is cut back molecular weight about 94 and is done dalton under the sex change condition.Extrapolate antigenic surface glycosylation site high mannose type and heterozygous glycosyl molecular weight 20 kilodaltons, total high mannose type and heterozygous glycosyl molecular weight 40 kilodaltons.Choosing the cell strain that can secrete the sufficient gp160 membranin of glycosylation, both has been the HUT78 cell strain of required expression gp160 membranin.This cell strain excretory gp160 membranin Glycosylase restriction enzyme digestion and electrophoresis collection of illustrative plates is seen Fig. 1.
Embodiment 2 usefulness HUT78 cell strains prepare the HIV-1gp160 membranin
Present embodiment has been described with the HUT78 cell strain of expressing the gp160 membranin and has been prepared the antigenic technology of gp160, comprises cell recovery, cultivation, purifying.
The HUT78-gp160 cell strain generally is stored in the liquid nitrogen container.Take out from liquid nitrogen container during use, put 37 ℃ of warm water rapidly and melt, move in the 15ml centrifuge tube, and add 10ml RPMI 1640 perfect mediums (containing 10% calf serum and 0.5% penicillin streptomycin mixed solution), 1500rpm removed supernatant in centrifugal 15 minutes.
Add an amount of above-mentioned perfect medium, after evenly suspending, be seeded to the T-75 culturing bottle, put to incubator and cultivate, 37 ℃ of constant temperature, control CO
2Concentration 5%.Whenever cell density reaches 3～6 * 10
5/ ml, survival rate is greater than 90% o'clock, with the passage amplification, makes cell count after going down to posterity 1～2 * 10
5About/ml.
(for the 5L bio-reactor, total cellular score needs 2.5～4 * 10 approximately after obtaining the q.s cell
8Individual), 1000ml RPMI1640 perfect medium (containing 10% calf serum and 0.5% penicillin streptomycin mixed solution) is injected the 5L bio-reactor, be preheated to 37 ℃, regulating pH to 7.2. inserts cell in the aseptic inoculation bottle subsequently, insert bio-reactor, the temperature of controlling reactor is at 36.5～37.5 ℃, and dissolved oxygen is 40～70%, and pH is between 7.15～7.35. and the nutrient solution cumulative volume is that 3～4L. is when the nutrient solution residual sugar is lower than 3mg/ml, cell count reaches 4～6 * 10 in the inoculation post-reactor
8After individual; enter antigen presentation and glycosylation stage; the beginning perfusion culture; every day, slow stream added 2-3L RPMI 1640 low blood serum mediums (containing 2% calf serum and 0.5% penicillin streptomycin mixed solution); gather in the crops the 2-3L nutrient solution simultaneously, groundwater increment is as the criterion to guarantee that living cell rate and cell density are basicly stable.When living cell rate is lower than 50%, finish to cultivate.But general cultured continuously 20-30 day, results culture supernatant 50-60 liter.
To gather in the crops supernatant merges, remove by filter cell debris, with molecular weight cut-off 50000 daltonian Pillicon filters filter (Sartorius company) concentrated supernatants, until 30 times of volume-diminished, (anti-gp41 is available from Biodesign International to be splined on the anti-gp41-Sepharose4B affinity column of usefulness pH 7.2 phosphoric acid buffer equilibrated; Sepharsoe4B is available from Amersham Biosciences), after peak to be passed flows to end,, collect elution peak with 2M potassium sulfocyanate wash-out, pH 7.2 phosphoric acid buffers were dialysed behind the liquid, put-60 ℃ of preservations.Identify that through the SDS-PAGE electrophoresis general per 50 liters of culture supernatant can be obtained 50mg high purity gp160 antigen.Chromatography collection of illustrative plates and electrophoretogram are seen Fig. 2.
The antigenic horseradish peroxidase-labeled thing of embodiment 3gp160
This programme has been described the gp160 antigenic mark horseradish peroxidase technology through optimizing.The scale of mark is 20 gram horseradish peroxidases in the present embodiment.Those skilled in the art can amplify with the experience scale of carrying out or dwindle as required.
Get 20mgHRP (RZ＞3.0) and be dissolved in the 2ml distilled water, dispose HRP solution.The NaIO4 that gets 25mg is dissolved in the 2ml distilled water, dispose NaIO4 solution.NaIO4 solution is added in the HRP solution, place 4 ℃ of lucifuge reactions 30 minutes.Get 2ml 1% ethylene glycol solution and add in the above-mentioned enzyme reaction solution, place 4 ℃ of lucifuge reactions 30 minutes, make the activatory enzyme solution.Getting activatory enzyme solution 6ml and add the gp160 antigen that 20mg desires mark, mix and put into dialysis tubing, is 4 ℃ of dialysed overnight of carbonate buffer solution of 9.6 to pH.The NaBH4 solution that adds the 5mg/ml of 0.6ml next day, 4 ℃ of lucifuge reaction 2.5hr.Add isopyknic saturated ammonium sulphate solution again, 4 ℃ of precipitation 2hr.Centrifugal supernatant discarded, precipitation is splined on PBS (pH7.2) equilibrated Sephacryl S-200 gel-filtration column (AmershamBiosciences) with a small amount of PBS (pH7.2) dissolving, collects the macromolecule main peak, adds isopyknic glycerine ,-20 ℃ of preservations.
The antigenic colloid gold label thing of embodiment 4gp160
With 1%HAuCl
4Solution with distilled water be diluted to ten thousand/, be heated to boiling, adding 1% citric acid three sodium solution, to make its concentration be 1.3/ ten thousand, when the color of solution by faint yellow become redness after, continued to boil 10 minutes, stop heating, be cooled to room temperature, use 1%K
2CO
3Regulate pH to 5.55.
The gp160 antigen that embodiment two is obtained is with the phosphate buffered saline buffer dialysis of 20mM pH7.4 24 hours, changes one time dialyzate in per 8～10 hours.After dialysis finishes, be diluted to 0.12mg/ml with distilled water, and Sterile Filtration, by 1 volume gpl60 antigen: the ratio adding Radioactive colloidal gold of 10 volume Radioactive colloidal golds, stirred 10 minutes.Again in 1 volume gp160 antigen: the ratio of 0.25 volume 10%BSA adds 10%BSA, stirred 5 minutes, the centrifugal supernatant of abandoning, the Tris-HCl that contains 0.5%BSA (pH8.8) damping fluid that adds equal volume washs 4 times, the centrifugal supernatant of abandoning, with an amount of Tris-HCl (pH9.0) damping fluid that contains 0.2%BSA, 5% sucrose, 80mMNaCl with resolution of precipitate.Be stored in 2～8 ℃.
According to disclosure of the present invention; those skilled in the art need not too much test and can and use and implement the present invention HIV-1 gp160 required for protection membranin and preparation method thereof; and producing a desired effect. embodiment disclosed by the invention only describes the present invention; but be not to be construed as limiting the invention. those skilled in the art are with conspicuous similar surrogate or transformation; or substitute preparation described here at preparation relevant chemically or on the physiology with some; or related content of the present invention changed; but do not exceed spirit of the present invention; scope and thought all fall into the scope of protection of present invention.
Sequence table
(1) general information
(i) a kind of HIV-1 gp160 membranin and its production and application;
(ii) sequence number: 1
(2) information of SEQ ID NO.:1
(i) sequence signature:
(A) length: 856 amino acid;
(B) type: amino acid;
(D) topological framework: line style;
(ii) molecule type: polypeptide
(xi) sequence description: SEQ ID NO.:1:
Met?Arg?Val?Lys?Glu?Lys?Tyr?Gln?His?Leu?Trp?Arg?Trp?Gly?Trp?Arg?Trp?Gly?Thr?Met?Leu?Leu?Gly
Met?Leu?Met?Ile?Cys?Ser?Ala?Thr?Glu?Lys?Leu?Trp?Val?Thr?Val?Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys?Glu
Ala?Thr?Thr?Thr?Leu?Phe?Cys?Ala?Ser?Asp?Ala?Lys?Ala?Tyr?Asp?Thr?Glu?Val?His?Asn?Val?Trp?Ala
Thr?His?Ala?Cys?Val?Pro?Thr?Asp?Pro?Asn?Pro?Gln?Glu?Val?Val?Leu?Val?Asn?Val?Thr?Glu?Asn?Phe
Asn?Met?Trp?Lys?Asn?Asp?Met?Val?Glu?Gln?Met?His?Glu?Asp?Ile?Ile?Ser?Leu?Trp?Asp?Gln?Ser?Leu
Lys?Pro?Cys?Val?Lys?Leu?Thr?Pro?Leu?Cys?Val?Ser?Leu?Lys?Cys?Thr?Asp?Leu?Lys?Asn?Asp?Thr?Asn
Thr?Asn?Ser?Ser?Ser?Gly?Arg?Met?Ile?Met?Glu?Lys?Gly?Glu?Ile?Lys?Asn?Cys?Ser?Phe?Asn?Ile?Ser?Thr
Ser?Ile?Arg?Gly?Lys?Val?Gln?Lys?Glu?Tyr?Ala?Phe?Phe?Tyr?Lys?Leu?Asp?Ile?Ile?Pro?Ile?Asp?Asn?Asp
Thr?Thr?Ser?Tyr?Lys?Leu?Thr?Ser?Cys?Asn?Thr?Ser?Val?Ile?Thr?Gln?Ala?Cys?Pro?Lys?Val?Ser?Phe?Glu
Pro?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala?Gly?Phe?Ala?Ile?Leu?Lys?Cys?Asn?Asn?Lys?Thr?Phe?Asn?Gly
Thr?Gly?Pro?Cys?Thr?Asn?Val?Ser?Thr?Val?Gln?Cys?Thr?His?Gly?Ile?Arg?Pro?Val?Val?Ser?Thr?Gln?Leu
Leu?Leu?Asn?Gly?Ser?Leu?Ala?Glu?Glu?Glu?Val?Val?Ile?Arg?Ser?Val?Asn?Phe?Thr?Asp?Asn?Ala?Lys
Thr?Ile?Ile?Val?Gln?Leu?Asn?Thr?Ser?Val?Glu?Ile?Asn?Cys?Thr?Arg?Pro?Asn?Asn?Asn?Thr?Arg?Lys
Arg?Ile?Arg?Ile?Gln?Arg?Gly?Pro?Gly?Arg?Ala?Phe?Val?Thr?Ile?Gly?Lys?Ile?Gly?Asn?Met?Arg?Gln?Ala
His?Cys?Asn?Ile?Ser?Arg?Ala?Lys?Trp?Asn?Asn?Thr?Leu?Lys?Gln?Ile?Ala?Ser?Lys?Leu?Arg?Glu?Gln
Phe?Gly?Asn?Asn?Lys?Thr?Ile?Ile?Phe?Lys?Gln?Ser?Ser?Gly?Gly?Asp?Pro?Glu?Ile?Val?Thr?His?Ser?Phe
Asn?Cys?Gly?Gly?Glu?Phe?Phe?Tyr?Cys?Asn?Ser?Thr?Gln?Leu?Phe?Asn?Ser?Thr?Trp?Phe?Asn?Ser?Thr
Trp?Ser?Thr?Glu?Gly?Ser?Asn?Asn?Thr?Glu?Gly?Ser?Asp?Thr?Ile?Thr?Leu?Pro?Cys?Arg?Ile?Lys?Gln?Ile
Ile?Asn?Met?Trp?Gln?Lys?Val?Gly?Lys?Ala?Met?Tyr?Ala?Pro?Pro?Ile?Ser?Gly?Gln?Ile?Arg?Cys?Ser?Ser
Asn?Ile?Thr?Gly?Leu?Leu?Leu?Thr?Arg?Asp?Gly?Gly?Asn?Ser?Asn?Asn?Glu?Ser?Glu?Ile?Phe?Arg?Pro
Gly?Gly?Gly?Asp?Met?Arg?Asp?Asn?Trp?Arg?Ser?Glu?Leu?Tyr?Lys?Tyr?Lys?Val?Val?Lys?Ile?Glu?Pro
Leu?Gly?Val?Ala?Pro?Thr?Lys?Ala?Lys?Arg?Arg?Val?Val?Gln?Arg?Glu?Lys?Arg?Ala?Val?Gly?Ile?Gly
Ala?Leu?Phe?Leu?Gly?Phe?Leu?Gly?Ala?Ala?Gly?Ser?Thr?Met?Gly?Ala?Ala?Ser?Met?Thr?Leu?Thr?Val
Gln?Ala?Arg?Gln?Leu?Leu?Ser?Gly?Ile?Val?Gln?Gln?Gln?Asn?Asn?Leu?Leu?Arg?Ala?Ile?Glu?Ala?Gln
Gln?His?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile?Lys?Gln?Leu?Gln?Ala?Arg?Ile?Leu?Ala?Val?Glu?Arg
Tyr?Leu?Lys?Asp?Gln?Gln?Leu?Leu?Gly?Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile?Cys?Thr?Thr?Ala?Val
Pro?Trp?Asn?Ala?Ser?Trp?Ser?Asn?Lys?Ser?Leu?Glu?Gln?Ile?Trp?Asn?His?Thr?Thr?Trp?Met?Glu?Trp
Asp?Arg?Glu?Ile?Asn?Asn?Tyr?Thr?Ser?Leu?Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln?Glu
Lys?Asn?Glu?Gln?Glu?Leu?Leu?Glu?Leu?Asp?Lys?Trp?Ala?Ser?Leu?Trp?Asn?Trp?Phe?Asn?Ile?Thr?Asn
Trp?Leu?Trp?Tyr?Ile?Lys?Leu?Phe?Ile?Met?Ile?Val?Gly?Gly?Leu?Val?Gly?Leu?Arg?Ile?Val?Phe?Ala?Val
Leu?Ser?Ile?Val?Asn?Arg?Val?Arg?Gln?Gly?Tyr?Ser?Pro?Leu?Ser?Phe?Gln?Thr?His?Leu?Pro?Thr?Pro?Arg
Gly?Pro?Asp?Arg?Pro?Glu?Gly?Ile?Glu?Glu?Glu?Gly?Gly?Glu?Arg?Asp?Arg?Asp?Arg?Ser?Ile?Arg?Leu
Val?Asn?Gly?Ser?Leu?Ala?Leu?Ile?Trp?Asp?Asp?Leu?Arg?Ser?Leu?Cys?Leu?Phe?Ser?Tyr?His?Arg?Leu
Arg?Asp?Leu?Leu?Leu?Ile?Val?Thr?Arg?Ile?Val?Glu?Leu?Leu?Gly?Arg?Arg?Gly?Trp?Glu?Ala?Leu?Lys
Tyr?Trp?Trp?Asn?Leu?Leu?Gln?Tyr?Trp?Ser?Gln?Glu?Leu?Lys?Asn?Ser?Ala?Val?Ser?Leu?Leu?Asn?Ala
Thr?Ala?Ile?Ala?Val?Ala?Glu?Gly?Thr?Asp?Arg?Val?Ile?Glu?Val?Val?Gln?Gly?Ala?Cys?Arg?Ala?Ile?Arg
His?Ile?Pro?Arg?Arg?Ile?Arg?Gln?Gly?Leu?Glu?Arg?Ile?Leu?Leu
Sequence table
＜110〉Shanghai Kehua Bio-engineering Co., Ltd
＜120〉a kind of HIV-1 gp160 membranin and its production and application
<130>PCNKH021238
<140>02145275.X
<160>1
<210>1
<211>856
<212>PRT
<400>1
Met?Arg?Val?Lys?Glu?Lys?Tyr?Gln?His?Leu?Trp?Arg?Trp?Gly?Trp?Arg
1 5 10 15
Trp?Gly?Thr?Met?Leu?Leu?Gly?Met?Leu?Met?Ile?Cys?Ser?Ala?Thr?Glu
20 25 30
Lys?Leu?Trp?Val?Thr?Val?Tyr?Tyr?Gly?Val?Pro?Val?Trp?Lys?Glu?Ala
35 40 45
Thr?Thr?Thr?Leu?Phe?Cys?Ala?Ser?Asp?Ala?Lys?Ala?Tyr?Asp?Thr?Glu
50 55 60
Val?His?Asn?Val?Trp?Ala?Thr?His?Ala?Cys?Val?Pro?Thr?Asp?Pro?Asn
65 70 75 80
Pro?Gln?Glu?Val?Val?Leu?Val?Asn?Val?Thr?Glu?Asn?Phe?Asn?Met?Trp
85 90 95
Lys?Asn?Asp?Met?Val?Glu?Gln?Met?His?Glu?Asp?Ile?Ile?Ser?Leu?Trp
100 105 110
Asp?Gln?Ser?Leu?Lys?Pro?Cys?Val?Lys?Leu?Thr?Pro?Leu?Cys?Val?Ser
115 120 125
Leu?Lys?Cys?Thr?Asp?Leu?Lys?Asn?Asp?Thr?Asn?Thr?Asn?Ser?Ser?Ser
130 135 140
Gly?Arg?Met?Ile?Met?Glu?Lys?Gly?Glu?Ile?Lys?Asn?Cys?Ser?Phe?Asn
145 150 155 160
Lle?Ser?Thr?Ser?Ile?Arg?Gly?Lys?Val?Gln?Lys?Glu?Tyr?Ala?Phe?Phe
165 170 175
Tyr?Lys?Leu?Asp?Ile?Ile?Pro?Ile?Asp?Asn?Asp?Thr?Thr?Ser?Tyr?Lys
180 185 190
Leu?Thr?Ser?Cys?Asn?Thr?Ser?Val?Ile?Thr?Gln?Ala?Cys?Pro?Lys?Val
195 200 205
Ser?Phe?Glu?Pro?Ile?Pro?Ile?His?Tyr?Cys?Ala?Pro?Ala?Gly?Phe?Ala
210 215 210
Ile?Leu?Lys?Cys?Asn?Asn?Lys?Thr?Phe?Asn?Gly?Thr?Gly?Pro?Cys?Thr
215 230 235 240
Asn?Val?Ser?Thr?Val?Gln?Cys?Thr?His?Gly?Ile?Arg?Pro?Val?Val?Ser
245 250 255
Thr?Gln?Leu?Leu?Leu?Asn?Gly?Ser?Leu?Ala?Glu?Glu?Glu?Val?Val?Ile
260 265 270
Arg?Ser?Val?Asn?Phe?Thr?Asp?Asn?Ala?Lys?Thr?Ile?Ile?Val?Gln?Leu
275 280 285
Asn?Thr?Ser?Val?Glu?Ile?Asn?Cys?Thr?Arg?Pro?Asn?Asn?Asn?Thr?Arg
290 295 300
Lys?Arg?Ile?Arg?Ile?Gln?Arg?Gly?Pro?Gly?Arg?Ala?Phe?Val?Thr?Ile
305 310 315 320
Gly?Lys?Ile?Gly?Asn?Met?Arg?Gln?Ala?His?Cys?Asn?Ile?Ser?Arg?Ala
325 330 335
Lys?Trp?Asn?Asn?Thr?Leu?Lys?Gln?Ile?Ala?Ser?Lys?Leu?Arg?Glu?Gln
340 345 350
Phe?Gly?Asn?Asn?Lys?Thr?Ile?Ile?Phe?Lys?Gln?Ser?Ser?Gly?Gly?Asp
355 360 365
Pro?Glu?Ile?Val?Thr?His?Ser?Phe?Asn?Cys?Gly?Gly?Glu?Phe?Phe?Tyr
370 375 380
Cys?Asn?Ser?Thr?Gln?Leu?Phe?Asn?Ser?Thr?Trp?Phe?Asn?Ser?Thr?Trp
385 390 395 400
Ser?Thr?Glu?Gly?Ser?Asn?Asn?Thr?Glu?Gly?Ser?Asp?Thr?Ile?Thr?Leu
405 410 415
Pro?Cys?Arg?Ile?Lys?Gln?Ile?Ile?Asn?Met?Trp?Gln?Lys?Val?Gly?Lys
420 425 430
Ala?Met?Tyr?Ala?Pro?Pro?Ile?Ser?Gly?Gln?Ile?Arg?Cys?Ser?Ser?Asn
435 440 445
Ile?Thr?Gly?Leu?Leu?Leu?Thr?Arg?Asp?Gly?Gly?Asn?Ser?Asn?Asn?Glu
450 455 460
Ser?Glu?Ile?Phe?Arg?Pro?Gly?Gly?Gly?Asp?Met?Arg?Asp?Asn?Trp?Arg
465 470 475 480
Ser?Glu?Leu?Tyr?Lys?Tyr?Lys?Val?Val?Lys?Ile?Glu?Pro?Leu?Gly?Val
485 490 495
Ala?Pro?Thr?Lys?Ala?Lys?Arg?Arg?Val?Val?Gln?Arg?Glu?Lys?Arg?Ala
500 505 510
Val?Gly?Ile?Gly?Ala?Leu?Phe?Leu?Gly?Phe?Leu?Gly?Ala?Ala?Gly?Ser
515 520 525
Thr?Met?Gly?Ala?Ala?Ser?Met?Thr?Leu?Thr?Val?Gln?Ala?Arg?Gln?Leu
530 535 540
Leu?Ser?Gly?Ile?Val?Gln?Gln?Gln?Asn?Asn?Leu?Leu?Arg?Ala?Ile?Glu
545 550 555 560
Ala?Gln?Gln?His?Leu?Leu?Gln?Leu?Thr?Val?Trp?Gly?Ile?Lys?Gln?Leu
565 570 575
Gln?Ala?Arg?Ile?Leu?Ala?Val?Glu?Arg?Tyr?Leu?Lys?Asp?Gln?Gln?Leu
580 585 590
Leu?Gly?Ile?Trp?Gly?Cys?Ser?Gly?Lys?Leu?Ile?Cys?Thr?Thr?Ala?Val
595 600 605
Pro?Trp?Asn?Ala?Ser?Trp?Ser?Asn?Lys?Ser?Leu?Glu?Gln?Ile?Trp?Asn
610 615 620
Thr?Thr?Trp?Met?Glu?Trp?Asp?Arg?Glu?Ile?Asn?Asn?Tyr?Thr?Ser?Leu
625 630 635 640
His?Ile?His?Ser?Leu?Ile?Glu?Glu?Ser?Gln?Asn?Gln?Gln?Glu?Lys?Asn
645 650 655
Glu?Gln?Glu?Leu?Leu?Glu?Leu?Asp?Lys?Trp?Ala?Ser?Leu?Trp?Asn?Trp
660 665 670
Phe?Asn?Ile?Thr?Asn?Trp?Leu?Trp?Tyr?Ile?Lys?Leu?Phe?Ile?Met?Ile
675 680 685
Val?Gly?Gly?Leu?Val?Gly?Leu?Arg?Ile?Val?Phe?Ala?Val?Leu?Ser?Ile
690 695 700
Val?Asn?Arg?Val?Arg?Gln?Gly?Tyr?Ser?Pro?Leu?Ser?Phe?Gln?Thr?His
705 710 715 720
Leu?Pro?Thr?Pro?Arg?Gly?Pro?Asp?Arg?Pro?Glu?Gly?Ile?Glu?Glu?Glu
725 730 735
Gly?Gly?Glu?Arg?Asp?Arg?Asp?Arg?Ser?Ile?Arg?Leu?Val?Asn?Gly?Ser
740 745 750
Leu?Ala?Leu?Ile?Trp?Asp?Asp?Leu?Arg?Ser?Leu?Cys?Leu?Phe?Ser?Tyr
755 760 765
His?Arg?Leu?Arg?Asp?Leu?Leu?Leu?Ile?Val?Thr?Arg?Ile?Val?Glu?Leu
770 775 780
Leu?Gly?Arg?Arg?Gly?Trp?Glu?Ala?Leu?Lys?Tyr?Trp?Trp?Asn?Leu?Leu
785 790 795 800
Gln?Tyr?Trp?Ser?Gln?Glu?Leu?Lys?Asn?Ser?Ala?Val?Ser?Leu?Leu?Asn
805 810 815
Ala?Thr?Ala?Ile?Ala?Val?Ala?Glu?Gly?Thr?Asp?Arg?Val?Ile?Glu?Val
820 825 830
Val?Gln?Gly?Ala?Cys?Arg?Ala?Ile?Arg?His?Ile?Pro?Arg?Arg?Ile?Arg
835 840 845
Gln?Gly?Leu?Glu?Arg?Ile?Leu?Leu
850 855
Claims (4)
1. the preparation method of a HIV-1gp membranin comprises the steps:
(1) with HIV-1gp type HIV (human immunodeficiency virus) infection eukaryotic cell;
(2) from above-mentioned eukaryotic cell, filter out can secrete can be specifically immunity in conjunction with the proteinic cell strain of HIV-1 membranin antibody;
(3) from above-mentioned eucaryotic cell strain, filter out the cell strain of in H9 cell synplasm formation experiment, finding the infectious human immunodeficiency virus of secretion;
(4) cut the method for identifying molecular weight with the SDS-PAGE electrophoresis with Glycosylase, from above-mentioned eucaryotic cell strain, filter out the sufficient HUT78 cell strain of secreted membranin glycosylation;
(5) the extensive above-mentioned HUT78 cell strain of perfusion culture in bio-reactor is collected culture supernatant;
(6) purifying, collection from culture supernatant obtains the HIV-1gp membranin;
It is characterized in that: wherein the condition and the process of step (5) are as follows: after obtaining the q.s cell, the 1000mlRPMI1640 perfect medium is injected bio-reactor, this perfect medium is for containing 10% calf serum and 0.5% penicillin streptomycin mixed solution; Be preheated to 37 ℃, regulate pH to 7.2, subsequently cell is inserted in the aseptic inoculation bottle, insert bio-reactor, the temperature of controlling reactor is 36.5-37.5 ℃, and dissolved oxygen is 40-70%, and pH is 7.15-7.35; When the nutrient solution residual sugar is lower than 3mg/ml, cell reaches (4-6) * 10
8After individual, enter antigen presentation and glycosylation stage, the beginning perfusion culture, every day, stream added the low blood serum medium of 2-3L RPMI1640, and this low blood serum medium is collected the 2-3L nutrient solution simultaneously for containing 2% calf serum and 0.5% penicillin streptomycin mixed solution; When living cell rate is lower than 50%, finish to cultivate.
2. preparation method according to claim 1 is characterized in that the HUT78 cell strain of the eukaryotic cell behaviour T-lymphocyte series in the described step (1), and described HIV-1 type human immunodeficiency virus is 1 with the ratio of cell count: 500-10000.
3. preparation method according to claim 1, the purifying that it is characterized in that described step (6) is with anti-gp41 affinity chromatography purification.
4. preparation method according to claim 1 is characterized in that the perfusion culture time is continuous 20-30 day.
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| CN 02145275 CN1500806B (en) | 2002-11-14 | 2002-11-14 | HIV-lgp160 membrane protein, preparing method and uses thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5116740A (en) * | 1988-08-16 | 1992-05-26 | Akzo N.V. | Method for producing native hiv gp160 |
| US5766844A (en) * | 1995-03-07 | 1998-06-16 | Akzo Nobel N.V. | Human T-cell line infected with HIV-2 which secretes functionally intact HIV-2 GP160. |
-
2002
- 2002-11-14 CN CN 02145275 patent/CN1500806B/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5116740A (en) * | 1988-08-16 | 1992-05-26 | Akzo N.V. | Method for producing native hiv gp160 |
| US5766844A (en) * | 1995-03-07 | 1998-06-16 | Akzo Nobel N.V. | Human T-cell line infected with HIV-2 which secretes functionally intact HIV-2 GP160. |
Non-Patent Citations (2)
| Title |
|---|
| 作者:.标题:/organism="Human immunodeficiency virus 1"，/note="HTLV-III/LAV"，/name="envelope polyprotein".http://www.ncbi.nlm.nih.gov, GenBank database卷号： 期号：.2002,Accession No:AAB50262. |
| 作者:.标题:/organism="Human immunodeficiency virus 1",/note="HTLV-III/LAV",/name="envelope polyprotein".http://www.ncbi.nlm.nih.gov, GenBank database卷号：期号：2002,Accession No:AAB50262. * |
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