CN102558342B - Pig 4-1BB receptor, gene for encoding pig 4-1BB receptor and application thereof - Google Patents
Pig 4-1BB receptor, gene for encoding pig 4-1BB receptor and application thereof Download PDFInfo
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Abstract
The invention provides a pig 4-1BB receptor molecule, which is the flowing protein (1) or (2): the protein (1) is composed of amino acid sequences shown in SEQ ID NO. 3 or 4, and the protein (2) is derived from the protein (1) by substitution, deletion or insertion of one or more amino acids in the amino acid sequences shown in SEQ ID NO. 3 or 4, and has the same activity. . The invention also provides a gene for encoding the pig 4-1BB receptor, which has nucleotide sequences shown in SEQ ID NO. 1 or 2. The costimulatory receptor 4-1BB provided by the invention is specifically and highly expressed in peripheral blood T cells of pig, can enhance activation, proliferation and secretion activity of cell factor when transgenic T cells are suffered from antigenic stimulation, thus enhance acquired immunity response of host and immunity effect of vaccine.
Description
Technical field
The invention belongs to animal genetic engineering field, be specifically related to pig 4-1BB acceptor, encode its gene and application thereof.
Background technology
China is a pork production and consumption big country, but current swine fever, breeding and respiratory syndrome (blue otopathy, PRRSV) etc. epidemic disease is serious popular is China's greatest difficulty that industry faces of raising pigs, and therefore control and prevention of disease becomes stability and development pig industry significant problem urgently to be resolved hurrily.But swine disease generally presents the polyinfection of many cause of diseases intersections in recent years, the resistance against diseases of pig and the prevention effect of vaccine obviously reduce.Therefore to carry out control and prevention of disease work, need to find the New Policy of vaccine research and development, or seek to substitute or auxiliary approach, as cultivated, there is the new product boar of disease resistance.But existing achievement in research is devoted to utilize specified disease resistant gene to cultivate the domestic animal new variety of single disease-resistant characteristic more.And this strategy realistic situation gap obvious and that current China pig industry epidemic disease is multiple is too large.Therefore, cultivate the new product boar with broad spectrum resistance and become numerous scientific research personnel's ideal.
Adaptive immune response reaction is being brought into play vital effect in animal opposing pathogenic agent invasive procedure.The activation of T cell is the most important thing of this reaction especially.Studies have shown that in recent years, effective activation of T cell needs two signals model.The one, MHC-antigen peptide is combined the first signal producing with TCR, and the 2nd, common costimulatory receptor is mainly the acceptors such as 4-1BB and the second signal of its ligand binding generation.Under normal body microenvironment, often comparatively small amt of antigen presenting cell submission antigen peptide out, the combination of itself and TCR is not enough to activating T cell, easily causes t cell responses incapability.And the costimulatory signal path that T cell co-stimulatory acceptor 4-1BB etc. provide can make up weak TCR signal, thus activated T cell.Moreover, when TCR and antigen peptide affine not enough, be often difficult to activating T cell, and enough costimulatory signals also can play the effect that strengthens TCR signal path, thus activated T cell.In a word, enough costimulatory signals, both can overcome the less problem of TCR occupation rate, can also make up the deficiency of TCR avidity.Therefore the signal of costimulatory receptor mediation can strengthen adaptive immune system function altogether, thereby possesses the potential that becomes broad spectrum antidisease gene candidate.
, since 1989 are found, its vital role being risen on costimulatory signal path is increasingly bright and clear for 4-1BB molecule (also name CD137).The signal Main Function transmitting that combines with its part 4-1BBL is to strengthen the activity of cd8 t cell and the antiviral activity of memory t cell, has also strengthened amplification and the effective removing to pathogen of T cell function signal simultaneously.And 4-1BB molecule again produces and does the used time and also mediate the necessary signal of transmission antigen at memory t cell.In addition, 4-1BB also plays the part of the dual role of the removing of mediation tumour and preventing autoimmune disease in acquired immunity regulation and control simultaneously.
The foundation of 4-1BB acceptor to t cell immune response, strengthen and maintain and all have vital role, this is indicating that they have good prospect as target gene in transgenic breeding research and application.But current most costimulatory molecules correlative study is all based on gene knockout or utilizes the monoclonal antibody of anti-4-1BB to remove or the mode that strengthens costimulatory signal is carried out.These strategies are clearly not applicable for breeding research.Moreover the 4-1BB acceptor of pig only has the gene order information of prediction to come forth at present, the encoding sequence that it is definite and protein function thereof all do not have report before this.
Summary of the invention
The object of the present invention is to provide a boar 4-1BB acceptor, encode its gene and application thereof.
For achieving the above object, first the present invention provides pig 4-1BB acceptor molecule, and it is the protein being comprised of the aminoacid sequence shown in SEQ ID NO.3 or 4.
The amino acid length of these two pig 4-1BB acceptor molecules is respectively 255 (variants 1) and 241 (variants 2), homology is between the two up to 94.5%, and acceptor molecule 2 is only lacking 14 continuous amino acid than acceptor molecule 1 near carboxyl terminal.Shown in SEQ ID NO.3 is the aminoacid sequence of pig 4-1BB variant 1, and shown in SEQ ID NO.4 is the aminoacid sequence of pig 4-1BB variant 2.
Should be appreciated that those skilled in the art can, according to aminoacid sequence disclosed by the invention, not affect under its active prerequisite, replace, lack and/or increase one or several amino acid, obtain the mutant nucleotide sequence of described albumen.Therefore, pig 4-1BB acceptor of the present invention also comprises by being substituted, lacking in the aminoacid sequence shown in SEQ ID NO.3 or 4 or adding one or several amino acid and have the protein by the protein derived shown in SEQ ID NO.3 or 4 of same isoreactivity.For example, in the extracellular region of two variants of 4-1BB, the Serine of the 79th is replaced with to Threonine, or by the glutamine disappearance of the 107th, or increase by 3 proline(Pro) below at 198.
Preferably, the homology of the aminoacid sequence shown in the aminoacid sequence of derived protein and SEQ ID No.3 or 4 can be more than 70%, preferably more than 80%, more preferably more than 90%.
The present invention also provides the gene of the above-mentioned albumen of coding.Preferably, the nucleotide sequence of the gene of coding pig 4-1BB acceptor provided by the invention is as shown in SEQ ID No.1 or 2.
Should be understood that the codon of considering that the degeneracy of codon and the preferences of different plant species codon, those skilled in the art can be used applicable specific species to express as required.
The carrier that contains pig 4-1BB acceptor gene, clone and Host Strains that the present invention also provides all belong to protection scope of the present invention.
Pig 4-1BB acceptor described in the present invention also provides is in the application improving in pig broad spectrum resistance.As developed biotechnological formulation by preparing anti-pig 4-1BB monoclonal antibody, after oral or injection system administration, the combination of this monoclonal antibody and 4-1BB acceptor can strengthen the required second signal of t cell activation, and then strengthens t cell immune response, improves the resistance against diseases of pig.
The present invention also provides above-mentioned pig 4-1BB acceptor gene in the application of cultivating in broad spectrum resistance pig.Concrete grammar is:
1) pig 4-1BB acceptor gene is cloned on carrier for expression of eukaryon, or obtains pig 4-1BB acceptor gene mRNA by the method for in-vitro transcription;
2) utilize electroporation that the recombinant vectors of preparation or mRNA are imported to pig embryonic cell;
3) obtain 4-1BB expression level and raise, thus the transgenic pig that broad spectrum disease resistance improves.
The transgenosis strategy that the present invention is based on common costimulatory receptor 4-1BB is a novel strategy of study on animal breeding.To be total to costimulatory receptor 4-1BB specifically high expression level in T cell, can strengthen activation, propagation and the cytokine secretion activity of T cell when being subject to antigenic stimulation, and then strengthen host acquired immunity and reply, strengthen immune effect of vaccine.So after those skilled in the art will envision that transgenic animal individuality is cultivated successfully, the immunologic function when pathogen infection and resistance against diseases thereof also will obviously improve.Based on the strategy of high expression level 4-1BB, compared with cultivating the domestic animal new variety of single disease-resistant characteristic, can more effectively strengthen the resistance against diseases of host when being subject to many cause of diseases intersection polyinfection, and improve vaccine effect.
Accompanying drawing explanation
Fig. 1 is that carrier pMax-4-1BBHA forms mode chart, wherein pCMV: cytomegalovirus (CMV) promotor, can regulate and control the promotor that goal gene is expressed in eukaryotic cell; 4-1BB-HA: the gene coding region of the fusion rotein of pig 4-1BB and HA label, HA label is a fragment gene sequence of a coding HA (influenza virus haemagglutinin antigenic determinant) small peptide, can be used to detect the expression of institute's fusion rotein; SV40: be one section of polyadenylic acid residue of vacuolating virus of monkey 40mRNA end, there is the effect that stops the stability of transcribing, strengthen RNA.
After Fig. 2 is pig peripheral blood mononuclearcell (PBMC) transfection pMax-4-1BBHA plasmid, detect its exogenous protein expression situation by Western blot method.
Fig. 3 is being subject to after antigen (PRRSV) stimulation 24h after pig peripheral blood mononuclearcell (PBMC) transfection pMax-4-1BBHA plasmid, and the transcriptional level of its t cell activation marker CD25 molecule obviously improves.Contrast: without pig PBMC CD25 transcriptional level after PRRSV infects of transfection; Idle running is dyed: the pig PBMC that idle running is dyed is CD25 transcriptional level after PRRSV infects; 4-1BB: turn pig PBMC CD25 transcriptional level after PRRSV infects then through pMax-4-1BBHA plasmid).
Fig. 4 is being subject to after antigen (PRRSV) stimulation 24h after pig peripheral blood mononuclearcell (PBMC) transfection pMax-4-1BBHA plasmid, and in its PBMC cell, IFN-γ transcriptional level obviously improves.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
The clone of embodiment 14-1BB gene
The present invention has higher similarity as theoretical basis take people and pig gene order comparison result, according to people's 4-1BB primers, transfers the doubtful sequence of 4-1BB of pig take the cDNA of Wuzhi Mountain pig as template.Concrete operation step is:
(1) by comparing with the mankind's 4-1BB gene order, in the genome of pig, find the gene fragment that similarity is higher, and design accordingly primer (in Table 1:P1 and P2).
(2) extraction of pig peripheral blood mononuclearcell (PBMC) cDNA:
From the aseptic 20ml heparin sodium anticoagulation of taking of precaval vein of pig, after the dilution of PBS equal-volume, mixing, it is slowly added on to isopyknic pig lymphocyte parting liquid (Tianjin Hao Xiang, China) upper, the centrifugal 1800rpm of horizontal rotor, 20min.Draw afterwards the buffy coat under blood plasma, obtain PBMC.Utilize Invitrogen company Trizol reagent (
lSReagent) extract total RNA of PBMC, then use the cDNA of the synthetic pig of reverse transcription test kit (First Strand Synthesis Kit for RT-PCR) of ABI company.
(3), take the cDNA of pig as template, utilize the increase doubtful sequence of its 4-1BB of the phusion exo+ polymerase test kit of NEB company.
Amplification reaction system: distilled water, 34.5 μ l; 5 × HF buffer, 10 μ l; 10mM dNTPmix, 1 μ l; 25 μ M P1,1 μ l; 25 μ M P2,1 μ l; Phusion polysaccharase, 0.5 μ l; CDNA, 2 μ l.
Response procedures: denaturation: 98 ℃, 1min;
Circulation (35): 98 ℃, 10s; 55 ℃, 20s; 72 ℃, 1min;
Supplement and extend: 72 ℃, 10min.
It is upper that amplification products therefrom is connected to commercialization cloning vector (pEASY-Blunt Simple), delivers to the order-checking of Shanghai Mei Ji biological medicine Science and Technology Ltd..Through order-checking, find, the nucleotide coding frame of pig 4-1BB has two kinds, a long 768bp (variant 1), another long 726bp (variant 2).Both homologys are up to 94.5%, and compared with variant 1, variant 2 is lacking 42 continuous Nucleotide near 3 ' end.The aminoacid sequence of variant 1 is as shown in SEQ ID No.3, and the aminoacid sequence of variant 2 is as shown in SEQ ID No.4.
(4) according to the measured sequence information design 5 ' RACE of step (3) and the required primer of 3 ' RACE (table 1:5 ' GSP1 and 5 ' GSP2; 3 ' GSP1 and 3 ' GSP2), utilize the test kit (5 '-Full RACE Kit and 3 ' Full RACE Core Set Ver.2.0) of TAKARA company, obtain its 5 ' and 3 ' end transcribe non-translational region (UTR) sequence information.The cDNA full length sequence of variant 1 is as shown in SEQ ID No.1, and the cDNA full length sequence of variant 1 is as shown in SEQ IDNo.2.To carry the cloning vector called after pEASY-4-1BB-1 of variant 1, carry the cloning vector called after pEASY-4-1BB-2 of variant 2.
Table 1 pig 4-1BB gene clone primer used
Embodiment 2 impacts of pig 4-1BB gene up-regulated expression on pig t cell immune response
Utilize strategy provided by the invention, by the plasmid transfection pig peripheral blood mononuclearcell (PBMC that contains pig 4-1BB gene, mainly comprise T lymphocyte), on cell levels, detect its be subject to antigen (PRRSV) stimulate time cell activation and cytokine secretion activity.
(1) structure of carrier for expression of eukaryon pMax-4-1BBHA.Design upstream primer: 5 ' (Kpn 1)
gGTACCATGGGAAATGGCTACTACAA3 ' and downstream primer: 5 ' (Sac1)
cGAGCTCTCA tAG tTCACACTCGCCTTCCT3 ' (square frame part is HA sequence label), take the cloning vector pEASY-4-1BB-2 containing 4-1BB gene as template, the open reading frame fragment of amplification pig 4-1BB variant 2.
Reaction system and program are with the step 3 in embodiment 1.Kpn1 and Sac1 restriction enzyme site are contained respectively in the fragment two ends of cloning, and downstream introduced HA sequence label, are convenient to detect expressing fusion protein situation with Western-blot.Finally, the fragment of amplification gained is connected to through Kpn1 and Sac1 and is total on the skeleton carrier pMax-GFP (purchased from Lonza Amaxa) after enzyme is cut.Plasmid after structure is shown in Fig. 1.
(2) a large amount of extractions of pMax-4-1BBHA plasmid:
The E.Z.N.A.TM Endo-Free Plsamid Maxi Kit specification sheets of producing with reference to OMEGA company carries out.After plasmid extraction completes, by the concentration of spectrophotometric determination plasmid.
(3) extraction of pig peripheral blood mononuclearcell (PBMC):
From the aseptic 20ml heparin sodium anticoagulation of taking of precaval vein of pig, after the dilution of PBS equal-volume, mixing, it is slowly added on to isopyknic pig lymphocyte parting liquid (Tianjin Hao Xiang, China) upper, the centrifugal 1800rpm of horizontal rotor, 20min.Draw afterwards the buffy coat under blood plasma, obtain PBMC.
(4) transfection of pig peripheral blood PBMC:
After cell counting, by 5 × 10
6pMBC is dissolved in (AMAXA in the mouse lymphotactin transfection damping fluid that 100 μ l return to room temperature, mouse T cell transfection kit), add 4 μ gpMax-4-1BBHA, set up negative group simultaneously, do not add plasmid, after mixing gently, transfer in 2mm consideration convey cup (AMAXA).Consideration convey cup is put into consideration convey instrument (AMAXA), carry out transfection by the intrinsic program X001 program in consideration convey instrument (AMAXA LONZA), then rapidly cell is transferred in complete 1640 substratum of 37 ℃ of preheatings of 2ml, and be placed in 37 ℃, 5%CO
2cell culture incubator in cultivate.
(5) use Western-blot method to detect the expression of foreign gene.Because of transfection carrier pMax-4-1BBHA used, the 4-1BB gene that it carries merges with HA label, therefore the hybridization of the commercialization antibody of the anti-HA label in available mouse source and fusion rotein, then use that the anti-mouse two of HRP (horseradish peroxidase) mark is anti-to be detected hybridisation events.
(6) after pig peripheral blood mononuclearcell (PBMC) transfection be subject to antigen (PRRSV) stimulate time, it activates the detection of situation: with carrier pMax-4-1BBHA By Transfecting Porcine after PBMC8 hour, infect these cells with 0.1MOI PRRSV.Infect after 24 hours, utilize relative fluorescence quantitative PCR (SYBGreen dye method) to analyze the transcriptional level of its t cell activation Marker CD25 molecule, and GAPDH gene is set is reference gene.The primer sees the following form 2.
Primer used when the transcriptional level of the activation Marker (CD25 molecule) of relative fluorescence quantitative PCR for table 2 (SYBGreen dye method) to pig and IFN-γ is analyzed
(7) after pig peripheral blood mononuclearcell (PBMC) transfection be subject to antigen (PRRSV) stimulate time, the detection of its IFN-γ transcriptional level: with carrier pMax-4-1BBHA By Transfecting Porcine PBMC after 8 hours, infect these cells with 0.1MOI PRRSV.Infect after 24 hours, utilize relative fluorescence quantitative PCR (SYBGreen dye method) to analyze IFN-γ transcriptional level in PBMC cell, and GAPDH gene is set is reference gene.The primer is in Table 2.
Result as shown in drawings, after carrier pMax-4-1BBHA By Transfecting Porcine PBMC cell, detects that with anti-HA monoclonal antibody fusion rotein can successful expression (Fig. 2) in cell.The PBMC cell of high expression level 4-1BB variant 2 is after the presenting cell that is infected PRRSV stimulates, the transcriptional level of its activation marker molecule CD25 obviously raises (Fig. 3), and the transcriptional level (Fig. 4) of its cytokine of the PBMC of activation IFN-γ also obviously improves.
Take cloning vector pEASY-4-1BB-1 as template, by above-mentioned identical method, build the carrier for expression of eukaryon of 4-1BB variant 1 open reading frame fragment, and by this carrier By Transfecting Porcine peripheral blood mononuclear cell (PBMC), the PBMC cell of high expression level 4-1BB variant 1 is after the presenting cell that is infected PRRSV stimulates, the transcriptional level of its activation marker molecule CD25 obviously raises, and the transcriptional level of its cytokine of the PBMC of activation IFN-γ also obviously improves.The above results shows, two variants of pig 4-1BB acceptor molecule all have activity, can improve immunologic function and the resistance against diseases thereof of pig when pathogen infection.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (7)
1. pig 4-1BB acceptor molecule, it is the protein being comprised of the aminoacid sequence shown in SEQ ID NO.3 or 4.
2. the gene of coding pig 4-1BB acceptor claimed in claim 1.
3. gene according to claim 2, is characterized in that, nucleotide sequence is as shown in SEQ ID NO.1 or 2.
4. contain the carrier of gene described in claim 2 or 3.
5. contain the host cell of carrier described in claim 4.
6. the application of pig 4-1BB acceptor claimed in claim 1 in the monoclonal antibody formulation of preparation 4-1BB.
7. application according to claim 6, is characterized in that, take pig 4-1BB acceptor molecule claimed in claim 1 as immunogen, and the monoclonal antibody of preparation 4-1BB, then be prepared into preparation.
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| CN1440812A (en) * | 2002-02-27 | 2003-09-10 | 上海中信国健药业有限公司 | Method of strengthening immune response with CD137 conjugated excitomotor |
| CN102206678A (en) * | 2011-04-12 | 2011-10-05 | 中国农业大学 | Method for transfecting pig T lymphocytes in vitro by applying nuclear transfection method |
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| CN1440812A (en) * | 2002-02-27 | 2003-09-10 | 上海中信国健药业有限公司 | Method of strengthening immune response with CD137 conjugated excitomotor |
| CN102206678A (en) * | 2011-04-12 | 2011-10-05 | 中国农业大学 | Method for transfecting pig T lymphocytes in vitro by applying nuclear transfection method |
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