CN101161287A - A anastomosing protein contraception vaccine carrying human molecule adjuvant and its preparing method - Google Patents
A anastomosing protein contraception vaccine carrying human molecule adjuvant and its preparing method Download PDFInfo
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- CN101161287A CN101161287A CNA2006101170912A CN200610117091A CN101161287A CN 101161287 A CN101161287 A CN 101161287A CN A2006101170912 A CNA2006101170912 A CN A2006101170912A CN 200610117091 A CN200610117091 A CN 200610117091A CN 101161287 A CN101161287 A CN 101161287A
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Abstract
The present invention relates to biopharmaceutical field, relates to a contraception vaccine cross-linking with human molecular adjuvant hC3d3, which is hCGbeta-hC3d3 fusion protein contraception vaccine. The present invention selects eukaryotic expression carrier pCI, building eukaryotic expression plasmid with purification label hCGbeta-hC3d3 fusion protein and hCG beta protein, to obtain high performance expression through animal host, to obtain high purity protein by identifying and purifying, to stimulate human PBMC test by vitro, and to approve human molecular adjuvant hC3d3 reinforcing hCG beta protein vaccine body fluid immunity efficacity and antifertility potential.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to a kind of and human molecule adjuvant hC3d3 contraception vaccine cross-linked, i.e. hCG β-hC3d3 anastomosing protein contraception vaccine.
Background technology
Human chorionic gonadotropin (hCG) is by the excretory a kind of glycoprotein hormones of Placenta Hominis syneytiotrophoblast, and its main physiological function is to prolong the corpus luteum life-span, and making it to increase becomes corpus luteum of pregnancy, and the secretion that increases steroid hormone is to keep gestation.HCG is a temporary transient species specificity hormone that occurs of trimester of pregnancy, existing studies confirm that, in and the biologic activity of hCG can interruption of pregnancy under the situation of not disturbing other biology of reproduction function, so hCG becomes optimal target antigen in the pregnancy vaccine design.Based on the specificity of β subunit in the hCG structure, entered II phase clinical experiment based on the synthetic peptide vaccine of hCG β.Experimental result shows that hCG β pregnancy vaccine has gratifying application prospect, but the contraceptive effect of the diversity between the immune effect individuality and present only 80% is far apart apart from clinical practice.Therefore, how significantly to strengthen the humoral immunization effect that it has antifertility action, weaken the goal in research that the cell immunoreceptor that might cause the damage of system genitale autoimmune has become hCG β pregnancy vaccine.In order to break the immunologic tolerance of body to this micromolecule autoantigen of hCG β, adopt diphtheria toxoid (DT) or tetanus toxoid (TT) to strengthen its immunogenicity in the former studies mostly, but DT and TT exist carrier mediated epi-position depression effect as carrier.
In immune system, C3d is one of last pyrolysis product of complement C3, and the receptor on it and B cell and the dendritic cells,follicular (FDC) (CR2, induce and keep most important for the normal body fluid immunne response by interaction CD21).Dempsy reported first C3d in 1996 is one can significantly strengthen the molecule adjuvant that humoral immunization is renderd a service, and C3d is merged the C3d-antigenic compound that forms with antigen can make 1000～10000 times of immunogenicity of antigens enhancings.After this, many C3d of studies confirm that are the vaccine adjuvants with tremendous potential.Reports such as Thomas D.Green three of film combining form copy C3d (mC3d3) and influenza virus hemagglutinin (HA) link the mHA-C3d3DNA vaccine that forms and Mitchell JA etc. three copy C3d (sC3d3) of secreted form and Measles virus hemagglutinin (H) link the sH-C3d3 dna vaccination that forms the maturation of the appearance of protective immunity, affinity of antibody and in and all obviously be better than HA and H aspect the titre.They have adopted similar methods that I type HIV (human immunodeficiency virus) peplos surface protein (HIV-1gp120) has been fused to the c-terminus of C3d3 recently, and have verified once more that in rodent C3d3 strengthens the ability of humoral immunization effect.For hCG β pregnancy vaccine, what determine its contraceptive effect is that humoral immunization is renderd a service, but not cellular immunization.During cellular immunization does not only have and the ability of hCG β biologic activity, also exist the danger that causes the damage of system genitale target cell autoimmune to a certain extent.Therefore, with technique for gene engineering development hCG β pregnancy vaccine, and significantly strengthen its Th2 type with antifertility action and humoral immunization effect, weakening Th1 type and CTL effect is the goal in research of hCG β pregnancy vaccine.
Research in the past utilizes technique for gene engineering to make up the eukaryon expression plasmid pcDNA3-hCG β-C3d3 of hCG β and 3 copy C3d molecules, and resulting fusion rotein confirms both had the CD21 molecule in conjunction with activity through identifying, also has the hCG beta antigen.On above research basis, hCG β-C3d3cDNA is built into the higher eukaryotic vector pCMV4 of expression efficiency, by genetic immunization to BALB/c mouse, show that molecule adjuvant C3d3 makes the immunogenicity of hCG β strengthen more than 200 times, and realized immunological effect from Th1 type cellular immunization to Th2 type humoral immunization bias.Since gene vaccine in host cell the expression restriction and gene vaccine with host cell gene group integration process in, might activate proto-oncogene or destroy antioncogene, exist carcinogenic danger in theory.Therefore, vivoexpression, purification hCG β-C3d3 fusion rotein, the preparation recombinant vaccine, and at immunological effect, antifertility effect and the mechanism of action thereof of this fusion rotein research hCG β-C3d3 vaccine will be more reasonable, safety.
Summary of the invention
The purpose of this invention is to provide a kind of efficient, stable, cheap and the crosslinked hCG β-hC3d3 anastomosing protein contraception vaccine of human molecule adjuvant C3d3.
Another object of the present invention provides the method for a kind of hCG of preparation β-hC3d3 fusion rotein.
Pregnancy vaccine of the present invention contains the hCG β-hC3d3 fusion rotein crosslinked with human molecule adjuvant C3d3, prepare by following method: select carrier for expression of eukaryon pCI for use, structure has the hCG β-hC3d3 fusion rotein and the proteic eukaryon expression plasmid of hCG β of purification tag, be pCI-6His-hCG β-hC3d3, by in Chinese hamster ovary (CHO) system, obtaining to efficiently express, get high-purity destination protein fusion rotein hCG β-hC3d3 through evaluation, purification.
The present invention prepares described pregnancy vaccine and comprises the steps:
1, hC3d gene clone and structure reorganization pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 eukaryon expression plasmid;
2. destination protein is expressed, evaluation, purification;
3.hCG β-C3d3 promotes B cellular expression CD86 and CD80 molecule;
4.hCG β-C3d3 promotes PBMC, B+T cell CD86, CD80, CD154, the CD25 developed by molecule of cultivation altogether;
5.hCG β-C3d3 promotes PBMC, the B+T emiocytosis IL-2 of cultivation altogether;
The present invention selects carrier for expression of eukaryon pCI for use, makes up hCG β-hC3d3 fusion rotein and the proteic eukaryon expression plasmid of hCG β, i.e. the pCI-6His-hCG β-hC3d3 that has 6 histidine (6His) purification tag respectively.The present invention has cloned people source C3d (hC3d), has made up eukaryon expression plasmid; Having realized obtaining in Chinese hamster ovary (CHO) system fusion rotein hCG β-hC3d3 efficiently expresses.Through the destination protein of identifying, purification obtains higher degree.By the stimulated in vitro human PBMC, confirmer's molecule adjuvant hC3d3 strengthens hCG β protein vaccine humoral immunization and renders a service and antifertility potential.Than prior art, its antifertility effect and the mechanism of action thereof will be more reasonable, safety.
Description of drawings
Fig. 1 is pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 construction of eukaryon expression plasmid for expressing sketch map.
Fig. 2 is pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 carrier for expression of eukaryon enzyme action evaluation figure
Wherein: 1: λ-Hind III Marker; 2:pCI-gs-signal-6His-hCG β-hC3d3; 3:pCI-gs-signal-6His-hCG β; 4:pCI-gs-signal-6His-hCG β-hC3d3/EcoR I; 5:pCI-gs-signal-6His-hCG β/EcoR I
Fig. 3 is that Western blotting analyzes pCI-gs-signal-6His-hCG β-hC3d3 and pCI-gs-signal-6His-hCG β expression product,
Wherein: 1,3:Biotinylated Protein Ladder; 2:There are three protein bandsof 136Ka (hCG β-hC3d3), 100KDa (hCG β-hC3d2), 64KDa (hCG β-hC3d); 4:One protein band of hCG β (24KDa).
Fig. 4 is that pCI-gs-signal-6His-hCG β-hC3d3 and pCI-gs-signal-6His-hCG β expression product are identified in the cytochemical staining of Raji cellular immunization.
Fig. 5: the expression of human peripheral immunologically competent cell CD80, CD86, CD154, CD25,
Wherein: * P＜0.05and**P＜0.01.
Fig. 6: PBMC is through variable concentrations antigenic stimulus 48h culture supernatant IL-2 output,
Wherein: * P＜0.05and**P＜0.01.
The specific embodiment
1) hC3d gene clone and structure reorganization pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 eukaryon expression plasmid
Have the hC3d fragment of restriction enzyme site with pcr amplification, the result shows the fragment of a treaty 0.9K b size.Sequencing result and target gene fragment are in full accord.By getting plasmid pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 (Fig. 1) after enzyme action, the coupled reaction.Enzyme action result (Fig. 2) confirms to make up correct.
2) destination protein is expressed, evaluation, purification
Two kinds of pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 plasmid transfection Chinese hamster ovary celIs of liposome method mediation.Obtain high-expression clone respectively by screening.After cultivating 24h, 48h, 72h, the output of pCI-gs-signal-6His-hCG β high-expression clone culture supernatant destination protein hCG β is respectively 11155,24337,45057mIu/ml/1 * 10
5Cells; And high-expression clone culture supernatant recombiant protein hCG β-hC3d3 output of pCI-gs-signal-6His-hCG β-hC3d3 is respectively 523.36,716.07,1140mIu/ml/1 * 10
5Cell.
Western blotting analyzes demonstration, and the expression product of pCI-gs-signal-6His-hCG β-hC3d3 positive colony is three bands, and the molecular weight size is equivalent to be about 136Ka, 100KDa respectively, (Fig. 3 a) for 64KDa.PCI-gs-signal-6His-hCG β positive colony expression product molecular weight is about 24KDa (Fig. 3 b).Raji cellular immunization cytochemistry result shows, pC-gs-signal-6His-hCG β-hC3d3 positive colony expression product the Raji cell membrane is positive painted (Fig. 4 a); PCI-gs-signal-6His-hCG β positive colony expression product then can not make Raji cell membrane painted (Fig. 3 b).
Under non-degeneration condition, with next step purification of His.Bind post pCI-gs-signal-6His-hCG β positive colony culture supernatant results hCG β albumen, purified product has higher degree.The purified product of the metal chelate affinity chromatography of pCI-gs-signal-6His-hCG β-hC3d3 positive colony culture supernatant is the mixture that contains three kinds of different molecular weight 136KDa, 100KDa, 64KDa.According to the difference of molecular weight size, to use Sephadex G150 column and carry out hCG β-hC3d3 fusion rotein that gel permeation chromatography is isolated 136Ka, described product has higher degree.
3) hCG β-C3d3 promotes B cellular expression CD86 and CD80 molecule
The negative isolating B cell of magnetic bead identifies that through flow cytometry its purity reaches 76%.After hCG β-C3d3 albumen stimulated, CD86+ cell table was 70.55% ± 29.32, with other each groups significant difference (P＜0.05) was arranged more all.The CD80+ cell is 32.64% ± 13.53, organizes comparison but there was no significant difference (P＞0.05) with matched group and hCG β.Adding under the CD21Ab situation, then the CD86+ cell reduces to 15.15% ± 8.38, with other each groups relatively there are no significant difference (P＞0.05).After PWM stimulated cultivation, CD80+ cell 62.96% ± 23.56 more all had significant difference (P＜0.05) with other each groups.The result shows that hCG β-hC3d3 albumen obviously strengthens the expression of B cell surface CD86 molecule; PWM is then to the B cell CD80 molecule regulating action that hoists.
4) hCG β-C3d3 promotes PBMC, B+T cell CD86, CD80, CD154, the CD25 developed by molecule of cultivation altogether
Under T and B co-culture of cells condition, hCG β-hC3d3 albumen is the obviously expression (P＜0.05) of rising tune B cell surface CD86 molecule also, the effect of CD21Ab this kind rising tune capable of blocking joint; And PWM mainly strengthens B cell CD80 developed by molecule.
After hCG β-C3d3 and PWM stimulation, CD154+ cell, CD25+ cell obviously increase, and with hCG β and matched group significant difference (P＜0.05) are arranged more all.If add CD21Ab in hCG β-C3d3 cultivates, then CD154+ cell, CD25+ cell reduce, and stimulate with independent hCG β and matched group relatively there are no significant difference (P＞0.05).Above results suggest, hCG β-hC3d3 albumen and PWM can obviously strengthen the expression (Fig. 5) of T cell surface CD154, CD25 molecule.
5) hCG β-C3d3 promotes PBMC, the B+T emiocytosis IL-2 of cultivation altogether
With 1nM, 10nM, 100nM 1000nM hCG β, hCG β-hC3d3, PWD and PBMC, B+T co-culture of cells, get the 48h culture supernatant, IL-2 secretion level behind the application ELISA detection T cell activation.The result shows that hCG β stimulating group only detects micro-IL-2 (32.5 ± 6.89) when 100nM concentration; And with after hCG β-hC3d3, the PWM processing, IL-2 content obviously raises, and with hCG β processed group and matched group significant difference (P＜0.05) is arranged relatively; The PWM processed group is respectively organized the IL-2 level than other and is significantly raise (P＜0.01) (Fig. 6).
Claims (4)
1. the anastomosing protein contraception vaccine of a carrying human molecule adjuvant, it is characterized in that containing the hCG β-hC3d3 fusion rotein crosslinked with human molecule adjuvant C3d3, prepare by following method: select carrier for expression of eukaryon pCI for use, structure has the hCG β-hC3d3 fusion rotein and the proteic eukaryon expression plasmid of hCG β of purification tag, obtain to efficiently express by animal reservoir's expression system, get the high-purity destination protein through evaluation, purification, comprise the steps:
1) hC3d gene clone and structure reorganization pCI-gs-signal-6His-hCG β, pCI-gs-signal-6His-hCG β-hC3d3 eukaryon expression plasmid;
2) express evaluation, purification destination protein fusion rotein hCG β-hC3d3;
3) hCG β-C3d3 promotes B cellular expression CD86 and CD80 molecule;
4) hCG β-C3d3 promotes PBMC, B+T cell CD86, CD80, CD154, the CD25 developed by molecule of cultivation altogether;
5) hCG β-C3d3 promotes PBMC, the B+T emiocytosis IL-2 of cultivation altogether.
2. the anastomosing protein contraception vaccine of carrying human molecule adjuvant according to claim 1 is characterized in that described fusion rotein adds 6 histidine purification tags at aminoterminal.
3. the anastomosing protein contraception vaccine of carrying human molecule adjuvant according to claim 1 is characterized in that the described hCG of having β-its molecular weight of hC3d3 fusion rotein is 136Ka.
4. the anastomosing protein contraception vaccine of carrying human molecule adjuvant according to claim 1 is characterized in that described animal reservoir's expression system is the Chinese hamster ovary system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191272A (en) * | 2010-03-18 | 2011-09-21 | 中国科学院动物研究所 | Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof |
| CN101709306B (en) * | 2009-12-16 | 2014-07-09 | 孔道春 | Fusion protein containing single-stranded DNA binding protein, expression and purification methods thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709306B (en) * | 2009-12-16 | 2014-07-09 | 孔道春 | Fusion protein containing single-stranded DNA binding protein, expression and purification methods thereof |
| CN102191272A (en) * | 2010-03-18 | 2011-09-21 | 中国科学院动物研究所 | Antifertility recombinant plasmid and genetic vaccine and preparation method and applications thereof |
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Open date: 20080416 |