CN102559693A - Vaccine adjuvant CCL28 as well as preparation method and application thereof - Google Patents
Vaccine adjuvant CCL28 as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a vaccine adjuvant CCL28 as well as a preparation method and an application thereof. The vaccine adjuvant has the advantages of simple preparation, easiness for quality control, easiness for mass production, easiness for storage and transportation and low cost. Experiment results show that with the adoption of the vaccine adjuvant, the level of cellular immunity and humoral immunity of an organism can be remarkably improved, and the antibody level of mucosal surface antigen specificity can be enhanced.
Description
Technical field
The present invention relates to genetically engineered, protein engineering and field of immunology, specifically, the present invention relates to a kind of vaccine adjuvant CCL28, also relate to the preparation method of a kind of vaccine adjuvant CCL28 simultaneously, also relate to the purposes of a kind of vaccine adjuvant CCL28.
Background technology
Resist in the process of disease the mankind, vaccine is being brought into play crucial effects.Since first vaccine---since the invention of antismallpox vaccine, many once lethal diseases all are able to capture because of succeeding in developing of corresponding vaccine or its mortality ratio are reduced greatly.Then; So far still there is the anxious appearance of treating effective vaccine of numerous disease, like AIDS, influenza etc., and some newborn virus diseases; Like (Rappuoli such as bird flu, SARS, lassa fevers; R., C.W.Mandl, et al. (2011). " Vaccines for the twenty-first century society. " Nat Rev Immunol.).Improve or develop an effective vaccine and can set about from a lot of aspects, wherein selecting suitable adjuvant for use is a kind of effective and easy method of going.Suitable adjuvant can enhancement antigen antigenicity and immunogenicity, thereby make body produce enough strong immunizing power (comprising humoral immunization, cellular immunization and mucosa-immune etc.), the invasion of opposing infective pathogen body and/or pathogenic.
An ideal adjuvant should have stimulates body to produce stronger immune response to antigen, and adjuvant itself does not have or have more weak antigenicity and immunogenicity, body is not produced other spinoff.Cytokine, because its special nature: on the one hand, it participates in immune adjusting; Closely related with the immunological competence of body, on the other hand, because it is the body oneself protein; Thereby itself does not have antigenicity and immunogenicity, in recent years, becomes the focus object of vaccine adjuvant research.IFN-α, IFN-γ, IL-6, IL-10, IL-15, IL-18 and IL-21 etc. have proved to have adjuvant effect (Bolesta preferably; E.; A.Kowalczyk, et al. (2006). " Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery. " J Immunol 177 (1): the 177-191.) (patent No.: 200510119721.5; 200710306039.6; 200610147664.6; 200510119720.0; 200480036307.1).CC chemokine ligand 28 (CCL28) is called the relevant epithelial cell chemokine (MEC) of mucous membrane again, mainly is expressed in mucous membrane epidermic cells such as tracheae, rectum, colon and vagina, and exocrinosity body of gland such as sialisterium.CCL28 is through chemotactic attracts IgA secretion plasmocyte to mucous membrane surface or body of gland (Lazarus with the relevant mutual effect of its acceptor CCR3 and CCR10; N.H.; E.J.Kunkel, et al. (2003). " A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts. " Journal of Immunology 170 (7): 3799-3805; Hieshima; K.; Y Kawasaki, et al. (2004). " CC Chemokine Ligands 25 and 28Play Essential Roles in Intestinal Extravasationof IgA Antibody-Secreting Cells. " J Immunol 173 (6): 3668-3675; Eksteen; B.; A.Miles, et al. (2006). " Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10. " Journal of Immunology 177 (1): 593-603; Sundtrom; P., S.B.Lundin, et al. (2009). " Human IgA-secreting cells induced by intestinal; but systemic; immunization respond to CCL25 (TECK) and CCL28 (MEC) (vol 38, pg3327,2008). " European Journal of Immunology 39 (11): 3267-3267.).Kutzler etc. find when the research influenza vaccines; After the mixed antigen with the CCL28 of plasmid form and plasmid form; Through the mode immune mouse of intramuscular injection, not only can improve IgG, the IgA level of antigen-specific in the serum, periphery IFN-γ level; And can strengthen the antibody-secreting (Kutzler of gut associated lymphoid tissue and lung; M.A., K.A.Kraynyak, et al. (2009). " Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo. " Gene Ther.).In addition; In the research of HIV-1; HIV-1 specific anti bulk concentration and CCL28 concentration positive correlation (Castelletti in discovery such as Castelletti HIV-1 infection women's breast milk and the saliva; E., S.Lo Caputo, et al. (2007). " The Mucosae-Associated Epithelial Chemokine (MEC/CCL28) Modulates Immunity in HIV Infection. " PLoS ONE 2 (10): e969.).
Summary of the invention
The objective of the invention is to be to provide a kind of adjuvant CCL28 of vaccine; This adjuvant can make low vaccine of replying produce strong and effectively immunoreation, and this by force and effectively immunoreation comprises that body is directed against effective humoral immunization, cellular immunization and mucosal immunization response reaction that antigen produces.
Another object of the present invention is the preparation method who has been to provide a kind of adjuvant CCL28 of vaccine, and this preparing method's process is simple, and quality is easy to control, is suitable for scale operation, and the adjuvant of processing is easy to store and transportation.
A further object of the present invention is the application of adjuvant CCL28 in intramuscular injection type, collunarium immunologic pattern (two types) vaccine medicine that has been to provide a kind of vaccine; Significantly enhancing body is to the immune response of vaccine generation for this adjuvant, and these immune responses comprise humoral immunization, cellular immunization and mucosa-immune reaction.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
Adjuvant of the present invention is meant the adjuvant that includes CCL28 gene (genotype adjuvant), albumen (protein type adjuvant) or bioactive fragment (polypeptide type adjuvant).
The preparation method of a kind of vaccine adjuvant CCL28 the steps include:
1, obtain spleen cell or PMBC (PBMC) from the separation of immunization acceptor, and its total mRNA of extracting (test kit is available from invitrogen company);
2, through RT-PCR technology (Molecular Cloning:ALaboratory Manual, the third edition), be strand cDNA (related reagent is available from invitrogen company) with gained mRNA rt;
3, the PCR primer of designs C CL28 specific amplification is right, and through primer to introducing suitable restriction enzyme site respectively at the gene two ends, obtain the CCL28 gene fragment through PCR (Molecular Cloning:A Laboratory Manual, the third edition) amplification then; Described primer is to being (primer is introduced restriction enzyme site and indicated sequence by underscore, and the restriction enzyme site title marks in the bracket behind primer):
Mouse:
The positive-sense strand primer: 5 '-GAT
GAATTCATGCAGCAAGCAGGGCTCACACTC-3 ' (EcoRI)
The antisense strand primer: 5 '-TTA
CTCGAGCTAACGAGAGGCTTCGTGCCTGTG-3 ' (XhoI)
The people:
The positive-sense strand primer: 5 '-AT
GAATTCATGCAGCAGAGAGGACTCGC-3 ' (EcoRI)
The antisense strand primer: 5 '-CG
CTCGAGCTAATAAGGAGTTTTATGGC-3 ' (XhoI)
4, to CCL28 gene fragment and pcDNA3.1 (+) (invitrogen company), carry out double digestion (NEB company) respectively, enzyme is cut the sticky end fragment that obtains and is connected through T4 ligase enzyme (NEB company), obtains recombinant expression plasmid;
5, will connect product transformed into escherichia coli DH5 α (TaKaRa company), with intestinal bacteria a large amount of amplification cultivation in the LB substratum, extract recombinant plasmid (plasmid extraction kit is available from Omega company) from cultured intestinal bacteria at last then;
6, behind the recombinant plasmid sequencing (Shanghai Sani company), the recombinant plasmid that sequence is correct is genotype adjuvant CCL28-pcDNA3.1 of the present invention.Wherein a kind of isolating gene (mouse source genotype adjuvant CCL28), its sequence is a nucleotide sequence shown in the SEQ ID 1, a kind of isolating gene (human source gene type adjuvant CCL28), its sequence is a nucleotide sequence shown in the SEQ ID 3.
7, sequence is correct CCL28 full length sequence or partial sequence; Behind pcr amplification; Subclone is transformed into intestinal bacteria Rosetta bacterial strain (Novagen company), behind prokaryotic expression and purifying to prokaryotic expression carrier pET-28a (Novagen company); (method is identical with the patent " polypeptide of recombination fusion protein CLD, encoding sequence and preparation method and application " that the applicant has applied for, and concrete steps are referring to this patent to obtain protein type or polypeptide type CCL28 adjuvant.The patent No.: 201110030466.2).Wherein a kind of expression and purified proteins matter (mouse source protein type adjuvant CCL28), its sequence is the aminoacid sequence shown in the SEQ ID 2, a kind of expression and purified proteins matter (people's source protein type adjuvant CCL28), its sequence is an aminoacid sequence shown in the SEQ ID 4.
The preparation method of the aminoacid sequence of recombination fusion protein CLD (number of patent application: 201110030466.2), the steps include:
The expression and purification of recombination fusion protein:
It is (commercially available that A, expression vector transform bacterial strain E.coli Rosetta bacterial strain; Novagen company), picking list bacterium colony is 0.6-1.0 to the test tube overnight cultures according to 1: 100 ratio enlarged culturing to OD value; Add IPTG to final concentration be 1mM; Induce centrifugal collection thalline after 4-5 hour, ultrasonication under the condition of ice bath, centrifugal collection inclusion body.Inclusion body is with containing the solution washing twice of 1M Guanidinium hydrochloride, then with the guanidine hydrochloride solution dissolving that contains 5mM DTT, and room temperature (20-25 ℃, below identical) 100rpm concussion 4 hours, 4 ℃, centrifugal 30 minutes of 12000rpm, supernatant cross 0.45 μ m membrane filtration.Supernatant is diluted in and contains 10% (volume, below identical) glycerine, 20mM Tris, and in the 500mM NaCl renaturation buffer, 1mM reduced glutathion/0.2mM Sleep-promoting factor B is as redox couple, under 4 ℃ of conditions, renaturation 24 hours.
B, recombinant protein are behind nickel chelating His-albumen affinity chromatography resin purification, and (0.2MNaCl) imidazoles is removed in dialysis for 5% volume glycerine, 20mM Tris, and after the balance, product concentrates with the ultrafiltration pipe with dialyzate.Each renaturation fusion rotein is concentrated into finite concentration, and purity detecting adopts SDS-PAGE, shows that the purity of protein of acquisition can reach more than 90%.
The application of a kind of vaccine adjuvant CCL28 in intramuscular injection type, collunarium immunologic pattern (two types) vaccines (medicine), it uses step to be:
1. intramuscular injection type vaccine: the recombinant plasmid CCL28-pcDNA3.1 that will from intestinal bacteria, extract and the antigen of plasmid form are dissolved in the saline water, mix.Then the antigen adjuvant mixture that mixes is injected in the mouse back leg quadriceps muscle of thigh, and with 100V, the positive antipole of the pulsed voltage of 50ms respectively shocks by electricity three times, promote plasmid transfection to go into muscle cell.
2. collunarium immunologic pattern vaccine: the antigen of albumen form and 1% Chitosan mix, and are prepared into antigen collunarium sample; The recombinant plasmid CCL28-pcDNA3.1 and the PEI (in vivo-jet PEI, Polyplus transfection company) that are dissolved in the ultrapure water mix, and are prepared into adjuvant collunarium sample (concrete steps are referring to product description).During immunity,, then sample is dropwise splashed into the mouse nostril earlier with mouse anesthesia.
Adjuvant of the present invention has following advantage:
1. adjuvant is the immunity receptor autogene, thereby adjuvant itself can not cause the immune response of body as antigen, and is little to the spinoff of body;
2. the preparation method of adjuvant is simple, and quality is easy to control, is prone to scale operation, is prone to storage and transport, and cost is low;
3. prove through related experiment, adjuvant CCL28 of the present invention, can enhancing body to antigenic immune response.Compare with the control group mice of not using adjuvant; Through CCL28 enhanced experimental mice; Antigen-specific IgG and IgA level have remarkable rising (wherein antigen-specific IgG and IgA level rise to 2168815.42 and 2070.7016 by 619753.075 and 915.61832 respectively in the mice serum of intramuscular injection immunization ways, raise 3.5 times and 2.3 times respectively in its serum; Antigen-specific IgG and IgA level rise to 1257901.74 and 3833.0124 by 359839.26 and 2061.8026 respectively in the mice serum of collunarium immunization ways; Raise 3.5 times and 1.9 times respectively); Simultaneously; The secretion of gamma-IFN antigen-specific spleen lymphocyte of reflection CTL level also have showed increased (wherein in the mouse spleen of intramuscular injection immunization ways secretion of gamma-IFN antigen-specific spleen lymphocyte by in each 1,000,000 cell 28.9 rise in each 1,000,000 cell 157.9, raise 5.5 times; In the mouse spleen of collunarium immunization ways secretion of gamma-IFN antigen-specific spleen lymphocyte by in each 1,000,000 cell 2.3 rise in each 1,000,000 cell 12.4, raise 5.5 times).In addition; The antigen-specific IgG of experimental mice vaginal secretion and IgA level also are significantly increased; Explain and also be enhanced its mucosa-immune level (wherein the mouse vagina excretory antigen-specific IgG of intramuscular injection immunization ways and IgA level rise to 13270.1302 and 2931.04532 by 1903.28406 and 243.08172 respectively, raise 7.0 times and 12.1 times respectively; The mouse vagina excretory antigen-specific IgG and the IgA level of collunarium immunization ways rise to 4830.55718 and 5447.3058 by 567.79924 and 1967.86764 respectively, raise 8.5 times and 2.8 times respectively).
Description of drawings
Fig. 1 is a kind of adjuvant mCCL28 improves antigen-specific antibodies in the mice serum of immunity back through the intramuscular injection immunization ways a horizontal synoptic diagram.
Figure 1A is an antigen-specific IgA antibody horizontal in the serum; Figure 1B is an antigen-specific IgG antibody horizontal in the serum.PCN140+VECTOR is a control group, and pCN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Fig. 2 is a kind of adjuvant mCCL28 improves immunity back mouse vagina mucous membrane surface antigen-specific antibodies through the intramuscular injection immunization ways a horizontal synoptic diagram.
Fig. 2 A is an antigen-specific IgA antibody horizontal in the vaginadouche appearance; Fig. 2 B is an antigen-specific IgG antibody horizontal in the vaginadouche appearance.PCN140+VECTOR is a control group, and pCN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Fig. 3 is a kind of adjuvant mCCL28 improves antigen-specific secretion INF-γ in the mouse spleen of immunity back through the intramuscular injection immunization ways a lymphocyte number synoptic diagram.
PCN140+VECTOR is a control group, and pCN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Fig. 4 is a kind of adjuvant mCCL28 improves antigen-specific antibodies in the mice serum of immunity back through the collunarium immunization ways a horizontal synoptic diagram.
Fig. 4 A is an antigen-specific IgA antibody horizontal in the serum; Fig. 4 B is an antigen-specific IgG antibody horizontal in the serum.CN140+VECTOR is a control group, and CN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Fig. 5 is a kind of adjuvant mCCL28 improves immunity back mouse vagina mucous membrane surface antigen-specific antibodies through the collunarium immunization ways a horizontal synoptic diagram.
Fig. 5 A is an antigen-specific IgA antibody horizontal in the vaginadouche appearance; Fig. 5 B is an antigen-specific IgG antibody horizontal in the vaginadouche appearance.CN140+VECTOR is a control group, and CN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Fig. 6 is a kind of adjuvant mCCL28 improves antigen-specific secretion INF-γ in the mouse spleen of immunity back through the collunarium immunization ways a lymphocyte number synoptic diagram.
CN140+VECTOR is a control group, and CN140+mCCL28 is an experimental group.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.The p value is shown in the histogram top.
Embodiment
Embodiment 1:
The preparation method of a kind of vaccine adjuvant CCL28 the steps include:
1, obtain spleen cell or PMBC (PBMC) from the separation of immunization acceptor, and its total mRNA of extracting (test kit is available from invitrogen company);
2, through RT-PCR technology (Molecular Cloning:A Laboratory Manual, the third edition), be strand cDNA (related reagent is available from invitrogen company) with gained mRNA rt;
3, the PCR primer of designs C CL28 specific amplification is right, and through primer to introducing suitable restriction enzyme site respectively at the gene two ends, obtain the CCL28 gene fragment through PCR (Molecular Cloning:A Laboratory Manual, the third edition) amplification then; Described primer is to being (primer is introduced restriction enzyme site and indicated sequence by underscore, and the restriction enzyme site title marks in the bracket behind primer):
Mouse:
The positive-sense strand primer: 5 '-GAT
GAATTCATGCAGCAAGCAGGGCTCACACTC-3 ' (EcoRI)
The antisense strand primer: 5 '-TTA
CTCGAGCTAACGAGAGGCTTCGTGCCTGTG-3 ' (XhoI)
The people:
The positive-sense strand primer: 5 '-AT
GAATTCATGCAGCAGAGAGGACTCGC-3 ' (EcoRI)
The antisense strand primer: 5 '-CG
CTCGAGCTAATAAGGAGTTTTATGGC-3 ' (XhoI)
4, to CCL28 gene fragment and pcDNA3.1 (+) (invitrogen company), carry out double digestion (NEB company) respectively, enzyme is cut the sticky end fragment that obtains and is connected through T4 ligase enzyme (NEB company), obtains recombinant expression plasmid;
5, will connect product transformed into escherichia coli DH5 α (TaKaRa company), with intestinal bacteria a large amount of amplification cultivation in the LB substratum, extract recombinant plasmid (plasmid extraction kit is available from Omega company) from cultured intestinal bacteria at last then;
6, behind the recombinant plasmid sequencing (Shanghai Sani company), the recombinant plasmid that sequence is correct is genotype adjuvant CCL28-pcDNA3.1 of the present invention.Wherein a kind of isolating gene (mouse source genotype adjuvant CCL28), its sequence is a nucleotide sequence shown in the SEQ ID 1, a kind of isolating gene (human source gene type adjuvant CCL28), its sequence is a nucleotide sequence shown in the SEQ ID 3.
7, sequence is correct CCL28 full length sequence or partial sequence; Behind pcr amplification; Subclone is transformed into intestinal bacteria Rosetta bacterial strain (Novagen company), behind prokaryotic expression and purifying to prokaryotic expression carrier pET-28a (Novagen company); (method is identical with the patent " polypeptide of recombination fusion protein CLD, encoding sequence and preparation method and application " that the applicant has applied for, and concrete steps are referring to this patent to obtain protein type or polypeptide type CCL28 adjuvant.The patent No.: 201110030466.2).Wherein a kind of expression and purified proteins matter (mouse source protein type adjuvant CCL28), its sequence is the aminoacid sequence shown in the SEQ ID 2, a kind of expression and purified proteins matter (people's source protein type adjuvant CCL28), its sequence is an aminoacid sequence shown in the SEQ ID 4.
The preparation method of the aminoacid sequence of recombination fusion protein CLD (number of patent application: 201110030466.2), the steps include:
The expression and purification of recombination fusion protein:
It is (commercially available that A, expression vector transform bacterial strain E.coli Rosetta bacterial strain; Novagen company), picking list bacterium colony is 0.6-1.0 to the test tube overnight cultures according to 1: 100 ratio enlarged culturing to OD value; Add IPTG to final concentration be 1mM; Induce centrifugal collection thalline after 4-5 hour, ultrasonication under the condition of ice bath, centrifugal collection inclusion body.Inclusion body is with containing the solution washing twice of 1M Guanidinium hydrochloride, then with the guanidine hydrochloride solution dissolving that contains 5mM DTT, and room temperature (20-25 ℃, below identical) 100rpm concussion 4 hours, 4 ℃, centrifugal 30 minutes of 12000rpm, supernatant cross 0.45 μ m membrane filtration.Supernatant is diluted in and contains 10% (volume, below identical) glycerine, 20mM Tris, and in the 500mM NaCl renaturation buffer, 1mM reduced glutathion/0.2mM Sleep-promoting factor B is as redox couple, under 4 ℃ of conditions, renaturation 24 hours.
B, recombinant protein are behind nickel chelating His-albumen affinity chromatography resin purification, and (0.2MNaCl) imidazoles is removed in dialysis for 5% volume glycerine, 20mM Tris, and after the balance, product concentrates with the ultrafiltration pipe with dialyzate.Each renaturation fusion rotein is concentrated into finite concentration, and purity detecting adopts SDS-PAGE, shows that the purity of protein of acquisition can reach more than 90%.
Embodiment 2:
The application of a kind of vaccine adjuvant CCL28 in intramuscular injection type vaccine the steps include:
Mouse source CCL28 (mCCL28) strengthens humoral immunization, cellular immunization and the mucosa-immune level of mouse to CN54-gp140 with the altogether immune BALB/c mouse of plasmid antigen pCN54-gp140
1. vaccine is formed
Antigen: through codon optimized pCN54-gp140 plasmid (be called for short pCN140, CN54 is HIV-107B '/C hypotype) (Cranage, Fraser et al.2011)
Adjuvant: mCCL28-pcDNA3.1 (being called for short mCCL28)
Contrast: pcDNA3.1 (+) empty carrier (being called for short VECTOR, invitrogen company)
Every each immunizing dose of mouse is: antigen 50 μ g, adjuvant perhaps contrast 100 μ g, are diluted to TV 80 μ l, spiral mixing with saline water.
2. experimental design
1) experiment is divided into groups
Experimental group: pCN140 (50 μ g)+mCCL28 (100 μ g)
Control group: pCN140 (50 μ g)+VECTOR (100 μ g)
2) immunization strategy
Ages in SPF level 6 to 8 week, female BALB/c mouse (available from Beijing China Fukang biotech inc) are divided into two groups at random, 5 every group, raise in SPF level experimental animal room.Mouse immune is an immunity cycle with 3 weeks, in 0,3,6 weeks mouse is carried out immunity respectively.Immunity adopts intramuscular injection to add the mode that electric shock is strengthened.Vaccine that mixes and adjuvant are injected in two back leg quadricepss muscle of thigh (40 μ l/ leg) about mouse, and with 100V, the positive antipole of the pulsed voltage of 50ms respectively shocks by electricity three times, promote the antigen and the adjuvant composition of plasmid form to be transfected into muscle cell.
3) sample collecting
Blood sample and vaginadouche appearance: respectively at exempting from week collection with three before the immunity.Blood sample is gathered through the eye socket rear vein beard, and gather and be placed on 4 ℃, 4-6 hour, centrifuging and taking serum ,-80 ℃ of preservations, subsequent use.Vaginadouche appearance obtains with the PBS washing vagina that 100 μ l contain proteolytic enzyme inhibitor (Roche company).After the collection, the centrifuging and taking supernatant ,-80 ℃ of preservations, subsequent use.
Spleen cell and mesenteric lymph nodes cell: be collected in three and exempt from a week.Mouse is got its spleen and mesenteric lymph nodes after disconnected neck is put to death, grind through 70 μ m nylon wire filters (BD Falcon company) and filter, and obtains single cell suspension, after the lymphocyte separation medium sorting, obtains the lymphocyte suspension again.The lymphocyte that obtains just can be used for subsequent experimental.
3. result and conclusion
1) CN140 specific IgG, IgA titre in serum and the vaginadouche appearance
CN140 antigen-specific IgG, IgA titre all adopt terminal point titre ELISA to measure in the sample; Concrete grammar is following: 96 hole elisa plate (NUNC MaxiSorp; Thermo Scientific company) the CN140 albumen 50 μ l/ holes with 5 μ g/ml encapsulate and spend the night in room temperature (20-25 ℃, identical up and down).The plank that encapsulates in 37 ℃ of sealings after one hour, by being fit to extent of dilution adding elisa plate, and is done gradient dilution with 1: 3 with the mouse sample through PBS+0.05%Tween washing and 1%BSA, places 37 ℃ to hatch then one hour.After the PBS+0.05%Tween washing, the detection two that adds the HRP mark is anti-.When measuring the IgG titre, add goat anti-mouse IgG-HRP (dilution in 1: 5000, Abcam company), hatched one hour for 37 ℃; When measuring the IgA titration, successively add goat anti-mouse IgA-Biotin (dilution in 1: 5000, Southern Biotechnology company), hatched one hour and Streptavidin-HRP (1: 5000, R&D company), hatched 30 minutes for 37 ℃ for 37 ℃.The elisa plate of hatching adds 1M H through TMB (Sigma company) colour developing 5 minutes
2SO
4Termination reaction reads the OD value of 450nm and 570nm (with reference to wavelength) respectively.At last, with negative sample OD
450MV+3 times standard deviation be cutoff value, calculate each sample antibody endpoint titre.Experimental result is seen explanation accompanying drawing 1 and Fig. 2.Figure 1A is an antigen-specific IgA titre in the serum; Figure 1B is an antigen-specific IgG titre in the serum; Fig. 2 A is an antigen-specific IgA titre in the vaginadouche appearance; Fig. 2 B is an antigen-specific IgG titre in the vaginadouche appearance.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.
Can find out from experimental result; Compare with control group; Through mCCL28 and the coimmune mouse of pCN140, specific IgG of CN140 and IgA titre all have remarkable rising in its serum and the vaginadouche appearance, explain that mCCL28 can strengthen mouse to antigenic HI and mucosal immune response.
2) spleen cell INF-γ ELISPOT
The mouse INF-γ ELISPOT test kit that this experiment is used is bio tech ltd available from reaching section, and experimental procedure is carried out according to product description.Be summarized as follows: mouse spleen lymphocyte adds in the ELISPOT plate 1 * 10 behind viable count
6/ hole, each mouse sample is established three repetitions (experimental port), and establishes negative and positive parallel control.Experimental port adds stimulator CN140 albumen to 20 μ g/ml, and the positive control hole adds concanavalinA (Sigma company), and negative control adds substratum.Each pore volume is transferred to 100 μ l with substratum, and mixing is in 37 ℃, 5%CO
2Cultivated 72 hours in the incubator.Hypotonic then smudge cells also cleans the ELISPOT plate, adds vitamin H respectively and detects antibody and Streptavidin-HRP and hatched 1 hour in 37 ℃.AEC develops the color in 37 ℃ and carries out, and about 25 minutes, clean termination reaction with tap water then, after room temperature is dried, read plate, counting, analytical data (Biosys company).Experimental result is seen explanation accompanying drawing 3.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.
Can find out from experimental result; Compare with control group; Through mCCL28 and the coimmune mouse of pCN140, the T lymphocyte of CN140 specificity in its spleen, secretion INF-γ has significantly and increases, and explain that mCCL28 can strengthen mouse and react to antigenic cellullar immunologic response.
Embodiment 3:
The application of a kind of vaccine adjuvant CCL28 in collunarium immunologic pattern vaccine the steps include:
Mouse source CCL28 (mCCL28) strengthens humoral immunization, cellular immunization and the mucosa-immune level of mouse to CN54-gp140 with the altogether immune BALB/c mouse of proteantigen CN54-gp140
1. vaccine is formed
Antigen: CN54-gp140 albumen (be called for short CN140, expressed and purifying by Polymun Scientific company, CN54 is HIV-107B '/C hypotype)
Adjuvant: mCCL28-pcDNA3.1 (being called for short mCCL28)
Contrast: pcDNA3.1 (+) empty carrier (being called for short VECTOR, invitrogen company)
Every each immunizing dose of mouse is: antigen 5 μ g, adjuvant perhaps contrast 15 μ g.
The antigen preparation: 5 μ g CN54-gp140 albumen are dissolved in the saline water that contains 1%Chitosan the spiral mixing.
The adjuvant preparation: use commercial adjuvant PEI (in vivo-jet PEI, Polyplus transfection company), 15 μ g adjuvants or control vector are mixed (N/P=7, TV are 30 μ l) with PEI, and detailed step is referring to product description.
2. experimental design
1) experiment is divided into groups
Experimental group: CN140 (5 μ g)+mCCL28 (15 μ g)
Control group: CN140 (5 μ g)+VECTOR (15 μ g)
2) immunization strategy
Ages in SPF level 6 to 8 week, female BALB/c mouse (available from Beijing China Fukang biotech inc) are divided into two groups at random, 5 every group, raise in SPF level experimental animal room.Mouse immune is an immunity cycle with 3 weeks, in 0,3,6 weeks mouse is carried out immunity respectively, and protein immunization carried out prior to plasmid in immune three days.The collunarium mode is all adopted in protein immunization and plasmid immunity: mouse dropwise splashes into the mouse nostril with sample after anesthesia.
3) sample collecting
Sample collection method is with application example 1.
3. result and conclusion
1) CN140 specific IgG, IgA titre in serum and the vaginadouche appearance
CN140 antigen-specific IgG, IgA titre all adopt terminal point titre ELISA to measure in the sample, and method is identical with application example 2.Experimental result is seen explanation accompanying drawing 4 and Fig. 5.Fig. 4 A is an antigen-specific IgA titre in the serum; Fig. 4 B is an antigen-specific IgG titre in the serum; Fig. 5 A is an antigen-specific IgA titre in the vaginadouche appearance; Fig. 5 B is an antigen-specific IgG titre in the vaginadouche appearance.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.
Can find out from experimental result; Compare with control group; Through mCCL28 and the coimmune mouse of CN140, specific IgG of CN140 and IgA titre all have remarkable rising in its serum and the vaginadouche appearance, explain that mCCL28 can strengthen mouse to antigenic HI and mucosal immune response.
2) spleen cell INF-γ ELISPOT
The mouse INF-γ ELISPOT test kit that this experiment is used is bio tech ltd available from reaching section, and experimental procedure is carried out according to product description.Method is identical with application example 2.Experimental result is seen explanation accompanying drawing 6.Data statistic analysis adopts two tail t checks, and the p value thinks that less than 0.05 there were significant differences between group, and the p value thinks between group utmost point significant difference is arranged less than 0.01.
Can find out from experimental result; Compare with control group; Through mCCL28 and the coimmune mouse of CN140, the T lymphocyte of CN140 specificity in its spleen, secretion INF-γ has significantly and increases, and explain that mCCL28 can strengthen mouse and react to antigenic cellullar immunologic response.
4. conclusion
CCL28 can significantly improve body to antigenic humoral immunization, cellular immunization and mucosa-immune level as immunological adjuvant.
Claims (7)
1. isolating gene, its sequence is the nucleotide sequence shown in the SEQ ID 1.
2. express and purified proteins matter for one kind, its sequence is the aminoacid sequence shown in the SEQ ID 2.
3. isolating gene, its sequence is the nucleotide sequence shown in the SEQ ID 3.
4. express and purified proteins matter for one kind, its sequence is the aminoacid sequence shown in the SEQ ID 4.
5. the preparation method of the described a kind of vaccine adjuvant CCL28 of claim 1 to 4 the steps include:
A, separate from the immunization acceptor and to obtain spleen cell or PMBC, and its total mRNA of extracting;
B, technological through RT-PCR is strand cDNA with gained mRNA rt;
The PCR primer of C, designs C CL28 specific amplification is right, and through primer to introducing suitable restriction enzyme site respectively at the gene two ends, obtain the CCL28 gene fragment through pcr amplification then; Described primer is to being:
Mouse:
The positive-sense strand primer: 5 '-GAT
GAATTCATGCAGCAAGCAGGGCTCACACTC-3 '
The antisense strand primer: 5 '-TTA
CTCGAGCTAACGAGAGGCTTCGTGCCTGTG-3 '
The people:
The positive-sense strand primer: 5 '-AT
GAATTCATGCAGCAGAGAGGACTCGC-3 '
The antisense strand primer: 5 '-CG
CTCGAGCTAATAAGGAGTTTTATGGC-3 '
D, to CCL28 gene fragment and pcDNA3.1 (+), carry out double digestion respectively, enzyme is cut the sticky end fragment that obtains and is connected through the T4 ligase enzyme, obtains recombinant expression plasmid;
E, will connect product transformed into escherichia coli DH5 α, with intestinal bacteria a large amount of amplification cultivation in the LB substratum, extract recombinant plasmid from cultured intestinal bacteria at last then;
Behind F, the recombinant plasmid sequencing, the recombinant plasmid that sequence is correct is genotype adjuvant CCL28-pcDNA3.1 of the present invention.
6. the application of a kind of vaccine adjuvant CCL28 in intramuscular injection type vaccine described in the claim 1 to 5.
7. the application of a kind of vaccine adjuvant CCL28 in collunarium immunologic pattern vaccine described in the claim 1 to 5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106692965A (en) * | 2017-02-15 | 2017-05-24 | 无锡市第五人民医院 | HSV-2 DNA vaccine used by mucous membranes, as well as preparation method and application thereof |
| CN108014332A (en) * | 2016-10-28 | 2018-05-11 | 哈尔滨润科旺生物技术开发有限公司 | A kind of live vaccine adjuvant |
| CN108567978A (en) * | 2018-04-23 | 2018-09-25 | 山东农业大学 | The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant |
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2012
- 2012-01-13 CN CN2012100095750A patent/CN102559693A/en active Pending
Non-Patent Citations (4)
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| CASTELLETTI E ET AL: "The mucosae-associated epithelian chemokine (MEC/CCL28) modulates immunity in HIV infection", 《PLOS ONE》 * |
| KUTZLER MA ET AL: "Plasmids encoding the mucosal chemokines CCL27 and CCL28 are effective adjuvants in eliciting antigen-specific immunity in vivo", 《GENE THERAPY》 * |
| STRAUSBERG RL ET AL: "Homo sapiens chemokine (C-C motif) ligand 28, mRNA (cDNA clone MGC:71902 IMAGE: 30326117), complete cds", 《GENBANK ACCESSION: BC062668》 * |
| STRAUSBERG RL ET AL: "Mus musculus chemokine (C-C motif) ligand 28, mRNA (cDNA IMAGE: 4240191), complete cds", 《GENBANK ACCESSION:BC055846》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108014332A (en) * | 2016-10-28 | 2018-05-11 | 哈尔滨润科旺生物技术开发有限公司 | A kind of live vaccine adjuvant |
| CN106692965A (en) * | 2017-02-15 | 2017-05-24 | 无锡市第五人民医院 | HSV-2 DNA vaccine used by mucous membranes, as well as preparation method and application thereof |
| CN108567978A (en) * | 2018-04-23 | 2018-09-25 | 山东农业大学 | The vaccine of carbon quantum dot adjuvant and carbon containing quantum dot adjuvant |
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Application publication date: 20120711 |