CN1772299A - The pIL-4 gene adjuvant for pig vaccine and its prepn process - Google Patents
The pIL-4 gene adjuvant for pig vaccine and its prepn process Download PDFInfo
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Abstract
The present invention discloses one kind of adjuvant for pig vaccine and its preparation process and constituted pig vaccine. The gene adjuvant for pig vaccine is adjuvant including pig interleukin-4 gene (pIL-4), or recombinant plasmid pcDNA-pIL-4 of animal cell expression plasmid pcDNA3.1 and pig interleukin-6 gene (pIL-4), or the combination of pcDNA-pIL-4 and No. 206 adjuvant. The pig vaccine is composition of pig's cysticercus-resisting vaccine composition; pig's deactivated foot-and-mouth disease virus vaccine and the gene adjuvant. The gene adjuvant of the present invention is prepared through gene cloning, recombination, recombinant plasmid proliferation, extraction and purification and other processes.
Description
Technical field
The present invention relates to adjuvant and its preparation method that pig vaccine uses, and use vaccine with the pig that adjuvant of the present invention constitutes.
Background technology
Since the twenties in last century, many materials are attempted as immunological adjuvant, but have only the aluminium glue adjuvant to obtain the permission of human vaccine, and are used for pig vaccine in a large number.It is as antigenic storage vault and carrier, thereby slowly released antigen prolongs the humoral immune reaction of inducing body, but shortcoming is only to induce the Th2 humoral immune reaction, and antibody is based on the IgG1 type, and no TCL reacts.Though oil adjuvant and Freund's complete adjuvant can obviously improve the immunne response ability, often occur untoward reaction in actual applications, as injection site swelling, pain, fever, or allergy etc. takes place, only for being used for the laboratory animal test.206 adjuvants are at present commercial efficient animal vaccine oil adjuvants, have been widely used in the animal vaccine, and it is reliable, effective and safe that practice confirms, but needs import, and price is higher.
China is the world big country of raising pigs, and the feeding live pig amount is occupied bigger proportion more than 1,100,000,000 since 2002 in animal husbandry is produced, and pig industry also is the main production pillar and the new growth engines of China part provinces and regions simultaneously.But in the face of the great eqpidemic disease of current pig industry take place and popular more sophisticated situation---old complaint does not eliminate, new disease rises again, epidemic situation is continuous, this just requires us must use new scientific theory and production technology to develop pig with the prevention of safety, efficient vaccine with control the generation of various eqpidemic diseases and popular, and the adjuvant development is the key component of vaccine research.
Summary of the invention
First purpose of the present invention provides a kind of new gene adjuvant that uses for pig vaccine, and this adjuvant can make to hang down and reply or no response and reply infull vaccine and produce effective immunoreation.
Second purpose of the present invention provides a kind of new adjuvant with the aforesaid gene adjuvant of the present invention and existing certain adjuvant compatibility.
The 3rd purpose of the present invention provides the preparation method of gene adjuvant of the present invention.
The 4th purpose of the present invention provides two classes and adopts the pig of adjuvant of the present invention to use vaccine.
The gene adjuvant that pig vaccine of the present invention uses is meant the adjuvant that includes porcine interleukin 4 genes (pIL-4).
The adjuvant of example of the present invention is the recombiant plasmid pcDNA-pIL-4 of animal cell expression plasmid pcDNA3.1 and porcine interleukin 4 genes (pIL-4).
The new adjuvant of gene adjuvant of the present invention and existing certain adjuvant compatibility is the compatibility of pcDNA-pIL-4 gene adjuvant and 206 adjuvants.
The preparation method of the recombiant plasmid pcDNA-pIL-4 of porcine interleukin 4 genes in the adjuvant of the present invention is: with pig IL-4 secretory cell after stimulating inducing culture, adopt RT-PCR technology amplification clone to obtain complete genome sequence, read frame both sides design expression type primer according to the characteristic of pcDNA3.1 carrier at pig IL-4 gene again and clone its complete genome sequence, and introduce corresponding restriction enzyme site, amplification is back with corresponding enzyme difference while enzyme action genes of interest and carrier, connect reorganization with the T4 ligase, make up annular recombinant expression plasmid pcDNA-pIL-4, recombinant plasmid expression vector transformed into escherichia coli with gained, carry out amplification cultivation, and then extract and the recombinant expression plasmid DNA of described these genes of interest of purification with the alkaline bleach liquor cleavage method is a large amount of.
Two class pig vaccines of the present invention are pig vaccine combination and Schweineseuche viral inactivation vaccine compositionss with anti-cysticercosis, and this two classes vaccine combination is respectively:
Pig is to be selected from cysticercus cellulosae somatic antigen and TSO18 recombinant antigen vaccine by containing with the vaccine combination of anti-cysticercosis, with gene adjuvant of the present invention or gene adjuvant and 206 adjuvants mix or emulsifying forms;
Schweineseuche viral inactivation vaccine compositions be by the Schweineseuche viral inactivation vaccine with gene adjuvant of the present invention or gene adjuvant and 206 adjuvants mix or emulsifying forms.
Cytokine is the regulatory factor that a class of interior immunocyte of body or the generation of non-immunocyte has extensive biologic activity, can activate and regulate immunologically competent cell in vivo, to the generation of immunne response and adjusting have important function (Sun Weimin etc. write. cytokine research methodology, front page in 1999).Before dna vaccination produces, just have the people cytokine as adjuvant and vaccine coupling, but since cytokine in vivo in cell or the body fluid content extremely low, and the half-life is too short, old friend worker's extraction involves great expense, and fails extensive use in traditional vaccine.Be subjected to dna vaccination with the inspiration of plasmid as antigen vectors, the form of structure cytokine recombinant expression plasmid is expressed and brought into play immunoregulation effect in animal body is gene adjuvant.Inject simultaneously with cytokine gene adjuvant and vaccine, can make low replying or no response and reply infull vaccine and produce effective immunoreation.This is a theoretical foundation of setting up adjuvant of the present invention.
(Interleukin 4, IL-4) are the cytokines with various biological function, are a kind of important immunocompetence regulatory factors for interleukin-4.IL-4 is the cytokine that can strengthen the B cell response, and it can suppress cell-mediated immunoreation by stoping the T cell to produce the Th1 type cytokines, promotes the generation of IgE and IgG1 antibody-like.In addition, IL-4 can also promote the function of macrophage submission antigen and killing tumor cell, mainly is a kind of Th2 type cytokines gene adjuvant.
The superiority of gene adjuvant of the present invention has: 1) host's expression in vivo required cytokine, near the natural molecule structure, active unaffected on conformation; 2) once give to hang down scale for a long time on a small quantity and reach, need not multiple dosing; 3) preparation is simple, and quality is easy to control, and easily large-scale production is with low cost, is easy to storage and transport; 4) safety is good.Cytokine is the immune modulatory molecules that itself exists in the body, and human body is had no side effect, and also is subjected to the control of immunity of organism regulating networks simultaneously; 5) be easy to make up and transform.Utilize Protocols in Molecular Biology can freely select the cytokine kind, realize desired immune response strength and type at gene level.Generally speaking, inoculate certain type cytokine (Th1 or Th2 class) plasmid and can promote the immunoreation of respective type.
Recombinant expression plasmid pcDNA-pIL-4 of the present invention has an advantages of higher stability external, has tangible immunoenhancement result after expressing in vivo, can be as the adjuvant of multiple vaccine, especially for the vaccine adjuvant that prevents and/or treats common schweineseuche disease.
Show that through relevant experiment gene adjuvant of the present invention has can make low replying or no response and reply infull vaccine and produce effective immunoreactive effect, adopts the vaccine of adjuvant of the present invention more not use the vaccine of adjuvant that more intensive immunization is arranged.And during the new adjuvant that adopts that gene of the present invention and 206 adjuvant compatibilities form, can have the advantage of the different adjuvants of two classes concurrently, can reduce the preparation cost of adjuvant simultaneously greatly.And method of the present invention provides the effective measures that prepare gene adjuvant of the present invention or compatibility adjuvant.
Description of drawings
Fig. 1: the pcDNA-pIL-4 expression plasmid is to the reinforced effects of the immune antibody of cysticercus cellulosae somatic antigen.Among Fig. 1: CAg represents cysticercus cellulosae antigen; IL-4 represents the pcDNA-pIL-4 recombinant expression plasmid; V represents the pcDNA empty plasmid; 206 represent 206 adjuvants; Control representative inoculation does not contain antigenic vaccine diluent; The OD value that on behalf of the ELISA method, the longitudinal axis detect, transverse axis is represented the natural law after the immunity.
Fig. 2: the pcDNA-pIL-4 expression plasmid is to the reinforced effects of the immune antibody of cysticercosis cellulosae TS018 recombinant antigen.Among Fig. 2: TSO18 represents the cysticercosis cellulosae recombinant antigen; IL-4 represents the pcDNA-pIL-4 recombinant expression plasmid; 206 represent 206 adjuvants; Control representative inoculation does not contain antigenic vaccine diluent; The OD value that on behalf of the ELISA method, the longitudinal axis detect, transverse axis is represented the natural law after the immunity.
Fig. 3: the pcDNA-pIL-4 expression plasmid is to the reinforced effects of the immune antibody titre of Schweineseuche inactivated vaccine.Among Fig. 3: FAg represents the Schweineseuche inactivated vaccine; IL-4 represents the pcDNA-pIL-4 recombinant expression plasmid; V represents the pcDNA empty plasmid; Control representative inoculation does not contain antigenic vaccine diluent.The antibody titer value that longitudinal axis representative blocking-up ELISA method detects, transverse axis is represented the natural law after the immunity.
The specific embodiment
Details are as follows for the preparation method of adjuvant of the present invention:
(1) from pig peripheral blood or lymph node, separates mononuclearcell, after derivant stimulates cultivation, RT-PCR clones the purpose cytokine gene, through sequence homology, heredity develop relation with and molecular structure and function prediction analysis after, as the candidate gene of gene adjuvant;
(2) adopt the genetic engineering recombinant technique, select the suitable expression type primer that contains restriction enzyme site that the purpose cytokine gene is cloned into the animal cell expression plasmid vector;
(3) recombinant plasmid vector with step (2) gained transforms the prokaryotic hosts bacterium, selects positive colony, and order-checking is carried out amplification cultivation after identifying again; With
(4) recombinant plasmid dna of a large amount of extractions and purification genes of interest.
Wherein the plasmid vector in the step (2) is that carrier commonly used in the genetic engineering nucleic acid vaccine is the pcDNA3.1 plasmid vector, is used for the amplification of genes of interest, and makes stable in animal body, the lasting expression of cytokine, to keep its adjuvant effect.This class carrier is that those of ordinary skills know, and the restriction enzyme site of interpolation and clone's method are conventional meanses of the prior art, and restriction enzyme site is the specific site of its multiple clone site normally.
Prokaryotic hosts bacterium in the step (3) also is commonly used in the genetic engineering field, can increase in a large number recombiant plasmid, for example escherichia coli.The cultivation of the conversion of host cell, the screening of positive colony, sequencing, host strain and the extraction purification of recombinant expression plasmid all are well known to those skilled in the art, for example be described in " Molecular Cloning:A Laboratory Manual " (New York of people such as Sambrook, cold spring harbor laboratory, calendar year 2001).
Below be a specific embodiment of the present invention:
From Landrace peripheral blood or lymph node, separate mononuclearcell with lymphocyte separation medium, after 37 ℃ of derivants (PHA, LPS or the two associating) stimulate cultivation, the total RNA or the mRNA of cultured cell have been stimulated in the different time sections extraction, the design Auele Specific Primer (the pIL-4 forward primer is: 5`-GCT CGA TTC ATGGGT GTA AC-3`, and downstream primer is: 5`-GAG ATT CAA CAC TTT GAG TAT TT-3`; ), be that template RT-PCR clones purpose cytokine gene pIL-4 complete sequence with it, respectively through sequence homology, heredity develop relation with and molecular structure and function prediction analysis after, as the candidate gene of gene adjuvant.
At pIL-4, it is (the same to design the expression type primer that contains restriction enzyme site respectively, be IL-4 forward primer: 5`-CGGGATCCATGCCACAAGTGCGACATCACCTT-3`, downstream primer: 5`-CGCTCGAGTTAACACTTTGAGTATTTCTCCT-3`, restriction enzyme site is BamH I and Xho I), by the genetic engineering recombinant technique, orientation is cloned into it among animal cell expression plasmid vector pcDNA3.1, makes up recombinant expression carrier pcDNA-pIL-4; Recombinant plasmid expression vector is transformed the prokaryotic hosts bacterium, select positive colony, order-checking is carried out amplification cultivation after identifying again.Cultivate the gene order of products therefrom and see nucleotide sequence and nucleotides sequence e-file.
Select the height copy positive strain of cytokine recombiant plasmid respectively, be inoculated into 1000ml and contain in the LB culture medium of ammonia benzyl, 220rpm cultivates after 12～14 hours for 37 ℃ in shaking table, extracts recombiant plasmid in a large number with the SDS alkaline lysis.The thick plasmid that extracts after the ice-cold LiCl of 5M separates, its supernatant equal-volume isopropanol precipitating, reuse 70% washing with alcohol precipitates, the centrifugal supernatant of removing; Will precipitation with the TE buffer dissolving that contains RNase, room temperature treatment is after 30 minutes, reuse phenol: chloroform extracting 2 times, 2 times of dehydrated alcohol precipitations are centrifugal; Behind 1ml aquesterilisa dissolution precipitation, add 0.5ml PEG-MgCl
2Solution (30mM MgCl
2The middle 40%PEG 8000 that adds), fully placed 15 minutes behind the mixing, centrifugal 20 minutes of 13000rpm, precipitation is with 70% washing with alcohol 2 times, reuse TE buffer or sterilized water dissolution precipitation, promptly obtain so-called adjuvant, through measuring its content and purity respectively, standby 4 ℃ of preservations with the nucleic acid-protein detector.
Below for the middle pIL-4 gene and the derivation aminoacid sequence thereof of gene adjuvant of the present invention:
atg?ggt?gtc?acc?tcc?caa?ctg?atc?cca?acc?ctg?gtc?tgc?tta?ctg?45
Met?Gly?Val?Thr?Ser?Gln?Leu?Ile?Pro?Thr?Leu?Val?Cys?Leu?Leu
-24 -20 -15 -10
gct?tgt?acc?agc?aac?ttc?gtt?cac?gga?cac?aag?tgc?gac?atc?acc?90
Ala?Cys?Thr?Ser?Asn?Phe?Val?His?Gly?His?Lys?Cys?Asp?Ile?Thr
-5 -1 1 5
tta?caa?gag?atc?atc?aaa?acc?ttg?aac?att?ctc?aca?gcg?aga?aag?135
Leu?Gln?Glu?Ile?Ile?Lys?Thr?Leu?Asn?Ile?Leu?Thr?Ala?Arg?Lys
10 15 20
aac?tcg?tgc?atg?gag?ctg?ccc?gtg?acg?gac?gtc?ttt?gct?gcc?cca?180
Asn?Ser?Cys?MET?Glu?Leu?Pro?Val?Thr?Asp?Val?Phe?Ala?Ala?Pro
25 30 35
gag?aac?acg?acg?gag?aag?gaa?acc?ttc?tgc?cgg?gcc?tcg?act?gtg?225
Glu?Asn?Thr?Thr?Glu?Lys?Glu?Thr?Phe?Cys?Arg?Ala?Ser?Thr?Val
40 45 50
ctt?cgg?cac?atc?tac?aga?cac?cac?acg?tgc?atg?aag?agc?ctc?ctg?270
Leu?Arg?His?Ile?Tyr?Arg?His?His?Thr?Cys?MET?Lys?Ser?Leu?Leu
55 60 65
agc?gga?ctt?gac?agg?aac?ctg?agc?agc?atg?gca?aac?atg?acc?tgt?315
Ser?Gly?Leu?Asp?Arg?Asn?Leu?Ser?Ser?MET?Ala?Asn?MET?Thr?Cys
70 75 80
tct?gtg?cat?gaa?gcc?aag?aag?agc?act?ttg?aaa?gac?ttc?ttg?gaa?360
Ser?Val?His?Glu?Ala?Lys?Lys?Ser?Thr?Leu?Lys?Asp?Phe?Leu?Glu
85 90 95
agg?cta?aag?acg?att?atg?aag?gag?aaa?tac?tca?aag?tgt?tga 402
Arg?Leu?Lys?Thr?Ile?MET?Lys?Glu?Lys?Tyr?Ser?Lys?Cys?***
100 105
The related experiment that adjuvant of the present invention is stated as follows to the influence of vaccine immunity effect:
1, pcDNA-pIL-4 recombinant expression plasmid gene adjuvant is to the antigenic immunological enhancement of cysticercosis cellulosae:
(1) with recombinant expression plasmid pcDNA-pIL-4 and cysticercus cellulosae somatic antigen (200 μ g/ only) compatibility, the preparation vaccine immune mouse, with 206 adjuvants (200 μ l/ are only) and pcDNA3.1 empty carrier (100 μ g/ are only) is the adjuvant contrast, dosage of inoculation 400 μ l, and 200 μ l are respectively injected in hind leg thigh inboard; After the immunity first time, each booster immunization of 25d and 63d once.Different time is cut tail blood sampling separation of serum after immunity, the content of antibody in the mensuration serum and immune spleen cell secretion IL-4.Evidence pcDNA-pIL-4 recombinant expression plasmid can obviously strengthen the humoral immunity level of mouse anti cysticercus cellulosae somatic antigen, is higher than or is equivalent to the immunostimulant level of 206 adjuvants, and apparently higher than empty plasmid immunity matched group, (see figure 1).
(2) with recombinant expression plasmid pcDNA-pIL-4 and 206 adjuvants (250 μ l/ head) compatibility and cysticercosis cellulosae TSO18 recombinant antigen (200 μ g/ only) combination; preparation molecular vaccine immunity man pig (500 μ l/ head); exempted from back 7 days at head; carrying out two exempts from; two exempt from back 7 days challenge infections (25000 taeniasis suis worm's ovums); antibody is checked in blood sampling subsequently, and massacres animal in back 90 days in infection and check the cysticercus sneak case, determines immune protective effect.The result shows that aspect inductive antibody horizontal, pcDNA-pIL-4 adjuvant group is significantly higher than immune matched group; Aspect worm reduction rate, also be significantly higher than immune matched group, referring to accompanying drawing 2.
2, pcDNA-pIL-4 recombinant expression plasmid gene adjuvant is to the effect of Schweineseuche inactivated vaccine
With recombinant expression plasmid pcDNA-pIL-4 (100 μ g) separately respectively with Schweineseuche inactivated vaccine (200 μ g/ only) compatibility, preparation vaccine immune mouse (400 μ l/ only), with 206 adjuvants (200 μ l/ only) and pcDNA3.1 empty carrier (100 μ g/ only) is the adjuvant contrast, and 200 μ l are respectively injected in hind leg thigh inboard; After the immunity first time, each booster immunization of 25d and 63d once.Different time is cut tail blood sampling separation of serum after immunity, detects the foot-and-mouth disease antibody titre with liquid phase blocking-up ELISA method.Find that porcine cytokine recombiant plasmid gene adjuvant all has stronger adjuvant effect or immunoregulation effect to Schweineseuche antigen, its antibody titer can reach 300 above (see figure 3)s in 90 days behind initial immunity.
More than experiment shows that adjuvant properties of the present invention is in particular in:
(1) can significantly strengthen the immune effect of cysticercosis cellulosae somatic antigen and recombinant antigen.
A. the laboratory animal mice: these cytokine recombiant plasmid whiles and cysticercus cellulosae somatic antigen formulated in combination vaccine intramuscular inoculation immune mouse, it can be at the mice expression in vivo, regulate body and produce stronger immunne response, it strengthens the cysticercus somatic antigen and induces the ability of humoral response to be higher than 206 standard adjuvants, but the intensity that its enhance immunity is replied is relevant with the kind of cytokine with type.
B. the target animals pig: with IL-4 recombinant expression plasmid and the man pig of cysticercosis cellulosae TS018 recombinant antigen formulated in combination vaccine intramuscular inoculation immunity, these cytokines of Biao Daing can be regulated body and be produced stronger immunne response in vivo, and its IL-4 recombiant plasmid induces antibody horizontal apparently higher than other matched groups; Aspect the challenge infection protection, adjuvant of the present invention significantly strengthens the challenge infection of host to the taeniasis suis worm's ovum.
(2) can significantly strengthen the immune effect of Schweineseuche inactivated vaccine.With IL-4 while and Schweineseuche inactivated vaccine intramuscular inoculation immune mouse, recombiant plasmid can be regulated body in this cytokine of mice expression in vivo and produce stronger immunne response, it strengthens the Schweineseuche inactivated vaccine and induces the ability of humoral response to be significantly higher than the standard vaccine contrast, its antibody titer can reach more than 300 in the time of back 90 days in immunity, and empty plasmid standard vaccine matched group only is about 60.
(3) to have a using dosage little for this gene adjuvant, the characteristic that toxicity is low.Use the recombiant plasmid immunostimulation mice and the tame pig of 100 μ g, 300 μ g dosage respectively, can reach, also do not find toxic and side effects mice and tame pig than the better immune effect of 206 adjuvant standard doses.
(4) this porcine cytokine recombinant expression plasmid also has the activity of intersection to the immunostimulation of mice, so animal spectrum and spectrotype that such gene adjuvant adapts to are wider, and the laboratory animal mice can be used as the animal model of this gene adjuvant effect assessment.
Relevant experiment also shows, when gene adjuvant of the present invention and 206 adjuvant compatibilities can form a kind of new adjuvant, both can reduce the use amount of 206 adjuvants, can have the advantage of the different adjuvants of two classes again concurrently.Can determine by test as for its concrete proportion compatibility.
Claims (6)
1, a kind of gene adjuvant of pig vaccine use is characterized in that this gene adjuvant includes porcine interleukin 4 genes (pIL-4).
2, the gene adjuvant of pig vaccine use according to claim 1 is characterized in that this adjuvant is the recombiant plasmid pcDNA-pIL-4 of animal cell expression plasmid pcDNA3.1 and porcine interleukin 4 genes (pIL-4).
3, the adjuvant that uses of a kind of pig vaccine is characterized in that adjuvant is the gene adjuvant of recombiant plasmid pcDNA-pIL-4 of animal cell expression plasmid pcDNA3.1 and porcine interleukin 4 genes (pIL-4) and the compatibility of 206 adjuvants.
4, the preparation method of the recombiant plasmid pcDNA-pIL-4 of porcine interleukin 4 genes in the adjuvant that uses according to claim 2 or 3 described pig vaccines, it is characterized in that pig IL-4 secretory cell after stimulating inducing culture, adopt RT-PCR technology amplification clone to obtain complete genome sequence, read frame both sides design expression type primer according to the characteristic of pcDNA3.1 carrier at pig IL-4 gene again and clone its complete genome sequence, and introduce corresponding restriction enzyme site, amplification is back with corresponding enzyme difference while enzyme action genes of interest and carrier, connect reorganization with the T4 ligase, make up annular recombinant expression plasmid pcDNA-pIL-4, recombinant plasmid expression vector transformed into escherichia coli with gained, carry out amplification cultivation, and then extract and the recombinant expression plasmid DNA of described these genes of interest of purification with the alkaline bleach liquor cleavage method is a large amount of.
5, a boar is characterized in that with the vaccine combination of anti-cysticercosis compositions is selected from cysticercus cellulosae somatic antigen and TSO18 recombinant antigen vaccine by containing, and mixes with claim 1 or 2 or 3 described adjuvants or emulsifying forms.
6, a kind of Schweineseuche viral inactivation vaccine compositions is characterized in that the Schweineseuche viral inactivation vaccine mixes with claim 1 or 2 or 3 described adjuvants or emulsifying forms.
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| CN103012577A (en) * | 2012-12-28 | 2013-04-03 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine interleukin 4, and encoding gene and expression method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103012577A (en) * | 2012-12-28 | 2013-04-03 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine interleukin 4, and encoding gene and expression method thereof |
| CN103012577B (en) * | 2012-12-28 | 2014-09-10 | 江苏众红生物工程创药研究院有限公司 | Recombinant porcine interleukin 4, and encoding gene and expression method thereof |
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