CN103923897B - Caspase recombinates proenzyme and encoding gene thereof and application - Google Patents
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Abstract
The present invention discloses caspase and recombinates proenzyme and encoding gene thereof and application. Described caspase proenzyme amino acid sequence of recombinating is made up of the protein subunit sequence of caspase forming tool protease activity and the connection peptides sequence that connects described protein subunit sequence; At least one sequence in described connection peptides sequence is the amino acid recognition sequence comprising HRV 3CP crack protein matter. Protein provided by the present invention has by HRV 3CP specific recognition and the characteristic activated, and makes the apoptosis of infection virus and terminates infecting. The infection that the present invention is the duplication of the virus suppressing coding HRV 3CP and the virus of propagation and treatment and prevention coding HRV 3CP provides a kind of new approach, has very wide application prospect.
Description
Technical field
The present invention relates to caspase to recombinate proenzyme and encoding gene thereof and application.
Background technology
HRV 3CP is a kind of lyase participated in virus assembly, it it is the specific enzymes class integrating serine protease and L-Cysteine HCL Anhydrous characteristic, the recognition site of HRV 3CP is LeuGluValLeuPheGlnGlyPro, cleavage site is between Gln-Gly, it is called Q-G site, and exists without Homology in its host cell. Virus infection host cell, after viral RNA enters the tenuigenin of host cell, viral RNA instructs as mRNA immediately and synthesizes a polyprotein, this polyprotein is finally cracked into independent structural protein, structural protein and viral RNA under the effect of HRV 3CP and is self-assembled into virion further. Therefore, HRV 3CP plays an important role in a large amount of amplifications in host cell body of the maturation of each albumen of virus and progeny virus.
Aspartic acid specificity cysteine protease (cysteine-containingaspartate-specificproteases, caspase) is and an apoptosis-related proteinoid enzyme, plays key effect in the process of apoptosis. The member of caspase is more, has identified 14 kinds of different caspase the mankind. Various caspase is rich in halfcystine, after they are activated, it is possible to cut at the special asparagicacid residue position of target protein. In normal cell, these enzymes exist with the proenzyme state of dormancy, turn into having the maturing enzyme of catalytic activity under specific condition through process. Based on substrate specificity and cell function, caspase can be divided into 3 groups: I inflammation group: Caspase-1 ,-4 ,-5 ,-11 ,-12 ,-13 ,-14. II withers dies and opens beginning group: caspase-6 ,-8 ,-9 ,-10. III apoptosis effect group: caspase-2 ,-3 ,-7.
Apoptosis be the protease cascade reaction process that a complicated caspase family guides, although it is different for the caspase participated in the apoptotic process that different cells or different signal transduction path bring out, but caspase-3 is the only way which must be passed of apoptotic protease cascade reaction, is also the key enzyme and executive that wither and die. Caspase-3 exists with single pepsinogen precursor forms usually, after the caspase-3 zymogen precursor of non-activity receives the dead signal withering and dying and start class caspase transmission, become P17 (Ser29-Asp175) and P12 (Ser176-Asp277) two sub-bases through proteolysis at Asp28 and Asp175 place. Large and small sub-base discharges formation heterodimer. Two heterodimers form organized enzyme after being assembled into �� 2 �� 2 tetramer, by two the independent catalytic site His121 and Gln161-Ala162-Cys163Arg164-Gly165 (QACRG) exercising function contained on it, substrate is cut off in Asp site, thus perform apoptosis function, also carry out oneself's activation of zymogen precursor by this kind of mode simultaneously. Research shows, caspase-3 can cause the cell generation Chromatin condensation of all types and form DNA fragmentation. In addition, caspase-3 can suppress the biologic activity of interleukin-18, and caspase-3 plays crucial effect in apoptosis and inflammatory reaction.
Summary of the invention
It is an object of the invention to provide a kind of protein, this protein has following effect: the infection suppressing the virus of the duplication of the virus of coding HRV 3CP in host cell and/or propagation and prevention and/or treatment coding HRV 3CP.
The aminoacid sequence of described protein is by the caspase(aspartic acid specificity cysteine protease forming tool protease activity) protein subunit sequence and the connection peptides sequence that connects described protein subunit sequence form; At least one sequence in described connection peptides sequence is the amino acid recognition sequence comprising HRV 3CP crack protein matter;
The protein subunit of the caspase of described tool protease activity should be at least 2.
In above-mentioned protein, described connection peptides sequence can be the 148th of sequence 1 to the 155th bit sequence or sequence 2 the 148th to the 153rd bit sequence;
In above-mentioned protein, described caspase specifically can be caspase-3;
In above-mentioned protein, when described caspase is caspase-3, described protein subunit is Dare's base and little sub-base; The aminoacid sequence of described Dare base specifically can be the 1st of sequence 1 to the 147th bit sequence;
And/or, the aminoacid sequence of described little sub-base specifically can be the 156th of sequence 1 to the 257th bit sequence.
Above-mentioned protein specifically can be the protein of following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 2.
The present invention protects the encoding gene of above-mentioned arbitrary described protein.
Described gene is following 1) 4) in arbitrary described gene:
1) DNA molecular shown in sequence 3;
2) DNA molecular shown in sequence 4;
3) under strict conditions with 1) or 2) the DNA sequence dna hybridization that limits and coding have the protein DNA molecule of caspase proenzyme function;
4) with 1) or 2) or 3) DNA sequence dna that limits there is more than 90% homology and coding have caspase proenzyme function protein DNA molecular.
The present invention protects the expression cassette containing above-mentioned arbitrary described gene, recombinant vectors, recombinant cell lines or recombinant virus.
Described recombinant vectors specifically can be carrier pcDNA3.1 (+) multiple clone site place (as between BamH I and Xho I restriction enzyme site) insert above-mentioned arbitrary as described in the recombinant vectors of gene.
The present invention protects above-mentioned arbitrary described protein, gene, expression cassette, recombinant vectors, recombinant cell lines and/or recombinant virus to have following A in preparation) and B) at least one effect product (medicine) in application:
A) duplication of virus in host cell and/or the propagation of coding HRV 3CP is suppressed;
B) infection of the virus of prevention and/or treatment coding HRV 3CP.
Present invention also offers and there is above-mentioned A) and B) in the product (medicine) of at least one effect, its activeconstituents is above-mentioned arbitrary described protein, gene, expression cassette, recombinant expression vector, recombinant cell lines and/or recombinant virus.
The virus of above-mentioned coding HRV 3CP can be picornavirus, it is specially Coxsackie virus, in an embodiment of the present invention, described Coxsackie virus is American Type Culture preservation center (AmericanTissueCultureColection(ATCC), network address: http://www.atcc.org/) the virus being numbered VR-30, the aminoacid sequence of the HRV 3CP of this encoding viral is as shown in sequence 6.
Experiment proves, contain reconstitution cell A and B of caspase-3 restructuring proenzyme and the reconstitution cell CK of proenzyme of not recombinating containing caspase-3 with the virus infection of coding HRV 3CP after, compared with CK, duplication viral in reconstitution cell A and B and propagation weaken, the toxic action of cell is weakened by virus, and cytoactive is obviously recovered. Protein provided by the present invention has by HRV 3CP specific recognition and the characteristic activated, and makes the apoptosis of infection virus and terminates infecting. The infection that the present invention is the duplication of the virus suppressing coding HRV 3CP and the virus of propagation and treatment and prevention coding HRV 3CP provides a kind of new approach, has very wide application prospect.
Accompanying drawing explanation
Fig. 1 is the Activity determination result of caspase-3 in reconstitution cell.
Fig. 2 is the caspase-3 activity change of the reconstitution cell that infects of Coxsackie virus (CVB virus) under different incubation time.
Fig. 3 be real-time fluorescence quantitative PCR detection after CVB virus infection in 6,7 and 8 little reconstitution cells constantly CVB virus conservative gene at the relative expression quantity of rna level.
Fig. 4 is the activity of reconstitution cell after CVB virus infection in embodiment 6.
Fig. 5 is the activity change of reconstitution cell under different incubation time after CVB virus infection in embodiment 7.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The information of biomaterial used in following embodiment is as follows:
Carrier pcDNA3.1 (+): Invitrogen, products catalogue is numbered V790-20;
Coxsackie virus (hereinafter referred to as CVB virus): American Type Culture preservation center (AmericanTissueCultureColection(ATCC), network address: http://www.atcc.org/) be numbered VR-30, the HRV 3CP sequence of this encoding viral is as shown in sequence 6;
293T/17 cell: American Type Culture preservation center (AmericanTissueCultureColection(ATCC), network address: http://www.atcc.org/) be numbered CRL-11268.
Embodiment 1, caspase-3 restructuring proenzyme and encoding gene thereof
By the Liang Geya base Dare base P17 (Ser of composition caspase-329Asp175) and little sub-base P12 (Ser176Asp277) with connection peptides LEVLFQGP or VLFQGP connect (this connection peptides can by HRV 3CP specific recognition and cut, after making cutting formed caspase-3 have activity and can active cell wither die), obtaining the recombinant protein A as shown in sequence 1 and the recombinant protein B as shown in sequence 2 respectively, recombinant protein A and B are caspase-3 restructuring proenzyme.
DNA molecular A shown in composition sequence table sequence 3, this DNA molecule encode recombinant protein A;
DNA molecular B shown in composition sequence table sequence 4, this DNA molecule encode recombinant protein B.
The structure of embodiment 2, recombinant expression vector
The DNA molecular A of embodiment 1 or DNA molecular B is connected into eukaryotic expression vector pcDNA3.1 (+) multiple clone site on BamH I and Xho I restriction enzyme site between, obtain recombinant vectors pcDNA3.1 (+)-A and pcDNA3.1 (+)-B. Through order-checking confirm, described recombinant vectors pcDNA3.1 (+)-A be pcDNA3.1 (+) multiple clone site on BamH I and Xho I restriction enzyme site between insert the DNA molecular A shown in sequence 3; Described recombinant vectors pcDNA3.1 (+)-B be pcDNA3.1 (+) multiple clone site on BamH I and Xho I restriction enzyme site between insert the DNA molecular B shown in sequence 4.
The acquisition of embodiment 3, reconstitution cell
Embodiment 2 is obtained recombinant vectors pcDNA3.1 (+)-A and pcDNA3.1 (+)-B and empty carrier pcDNA3.1 (+) transfection 293T/17 cell respectively, obtain respectively containing recombinant vectors pcDNA3.1 (+) the reconstitution cell A of-A and containing recombinant vectors pcDNA3.1 (+) the reconstitution cell B of-B and containing empty carrier pcDNA3.1 (+) reconstitution cell CK(compare), concrete transfection method is as follows:
1) cultivation of transfection cell: 24h before transfection, with the substratum RPMI1640(Hyclone company containing 10% foetal calf serum, products catalogue is numbered SH30809.01B), at 37 DEG C, 5%CO2Cultivate 293T/17 cell under condition, when adherent rate reaches 80%, carry out transfection;
2) transfection: get 96 orifice plates, every hole add 0.1 �� g recombinant vectors pcDNA3.1 (+) A or pcDNA3.1 (+)-B or pcDNA3.1 (+), useSerum free medium is diluted to 9.8 �� L; Every hole adds 0.2 �� LThermoScientificTurboFectTranfectionReagent transfection reagent (ThermoScientific company, products catalogue is numbered #R0531), and vortex mixes even, and room temperature leaves standstill 15min; Obtaining 10 �� L solution joins in the cell culture fluid that step 1) obtains mixed even, 37 DEG C, 5%CO2The nutrient solution of reconstitution cell A, B and CK is obtained, for the virus infection of embodiment 47 after cultivating 24h under condition.
In embodiment 4, reconstitution cell, caspase-3 restructuring proenzyme can by virus HRV 3CP specific activation
1, build the recombinant expression vector pCDNA3.1-3c(containing HRV 3CP encoding sequence namely carrier pcDNA3.1 (+) BamH I and Xho I restriction enzyme site between the DNA fragmentation shown in insertion sequence table sequence 5, this fragment coding HRV 3CP). by this recombinant expression vector pCDNA3.1-3c and recombinant vectors pcDNA3.1 (+) A, pcDNA3.1 (+)-B or pcDNA3.1 (+) cotransfection 293T/17 cell (method is identical with embodiment 3), add in every hole of 96 orifice plates during transfection 0.05 �� g recombinant vectors pcDNA3.1 (+) A, pcDNA3.1 (+)-B or pcDNA3.1 (+), and 0.05 recombinant vectors pCDNA3.1-3c of �� g, 30h is cultivated after transfection, obtain containing recombinant vectors pcDNA3.1 (+) the reconstitution cell A1 of-A and pCDNA3.1-3c, containing recombinant vectors pcDNA3.1 (+) the reconstitution cell B1 of-B and pCDNA3.1-3c and containing recombinant vectors pcDNA3.1 (+) and the reconstitution cell CK1 of pCDNA3.1-3c, the 293T/17 cell (WT) of the untransfected cultivated under measuring same condition respectively and reconstitution cell A1, the activity of caspase-3 in B1 and CK1.
Result: as shown in Figure 1, the caspase-3 of reconstitution cell A1 and B1 is active obviously higher than WT and CK1, illustrates that recombinant plasmid pcDNA3.1 (+)-A and pcDNA3.1 (+)-B can encode caspase-3 and recombinate the HRV 3CP specific activation of proenzyme loaded body pCDNA3.1-3c coding.
2, by the virus-Coxsackie virus (hereinafter referred to as CVB virus) of coding HRV 3CP, reconstitution cell A, B and CK that embodiment 3 obtains are infected respectively, and detect the activity infecting caspase-3 in rear reconstitution cell.
Result: as shown in Figure 2, reconstitution cell A and B after CVB virus infection, the caspase-3 activity in cell relatively reconstitution cell CK significantly raise, and cultivating in 17 hours the prolongation with infection time, caspase-3 activity increases. Result shows: have expressed caspase-3 restructuring proenzyme in reconstitution cell A and B, and caspase-3 restructuring proenzyme can by the HRV 3CP specific activation of CVB virus.
In step 2 as follows by virus method that reconstitution cell is infected: the culture supernatant sucking-off of reconstitution cell A, B or CK of 24h will be cultivated after embodiment 3 transfection, (MOI=10000:1 is mixed with CVB viral suspension, when namely infecting, virus is 10000:1 with the ratio of cell quantity), 4 DEG C of standing 1h, the virus not combined is washed with PBS, add the fresh RPMI1640(Hyclone company containing 2% foetal calf serum again, products catalogue is numbered SH30809.01B), cultivate 1,2,5,7,8 at being placed in 37 DEG C respectively and little carry out caspase-3 determination of activity constantly.
Caspas-3 activity determination method in step 1 and 2 is specific as follows: use test kit3/7Assay (Promega) carries out caspase-3 determination of activity according to operation instruction, specifically it is operating as: the caspase-GloR3/7Reagent detection reagent in test kit is equilibrated to room temperature, this detection reagent of 100ul is added in the cell culture fluid to be measured of every hole, the 300 centrifugal 30s of 500rpm, system is mixed, room temperature lucifuge places 40min, take out 100ul in check-out console, the fluorescence radiation trace routine in single passage multiple labeling detector (PerkinElmer, EnVisionTM2103MulilabelReader) is utilized to carry out luminous intensity mensuration.
In embodiment 5, reconstitution cell, caspase-3 recombinates the duplication of proenzyme attenuated virus in cell
By CVB virus, reconstitution cell A, B and CK that embodiment 3 obtains is infected respectively, and the CVB viral genome copy number infected in rear 6h, 7h and 8h reconstitution cell is carried out real-time fluorescence quantitative PCR. Result as shown in Figure 3, infects after 6h at CVB containing recombinate reconstitution cell A and B of proenzyme of caspase-3, in cell CVB genome copy number relatively reconstitution cell CK(compare) significantly reduce. Result shows, the cell containing caspase-3 restructuring proenzyme is after CVB infects, and the caspase-3 in cell recombinates proenzyme by the HRV 3CP specific activation of virus, and active cell withers and dies, and suppresses the duplication of virus in cell, thus the infection of attenuated virus.
The method that reconstitution cell is infected by the present embodiment virus is as follows: will cultivate the culture supernatant sucking-off of reconstitution cell A, B or CK of 24h after embodiment 3 transfection, (MOI=1000:1 is mixed with CVB viral suspension, when namely infecting, virus is 1000:1 with the ratio of cell quantity), 4 DEG C of standing 1h, the virus not combined is washed with PBS, add the fresh RPMI1640 containing 2% foetal calf serum again, it is placed at 37 DEG C and cultivates 6, the 7 and 8 little total serum IgE extracting reconstitution cell constantly respectively.
The method that the present embodiment real-time fluorescence quantitative PCR is analyzed is as follows: the total serum IgE of reconstitution cell is carried out reverse transcription, obtain cDNA, taking this cDNA as template, real-time fluorescence quantitative PCR amplification is carried out with special primer CVBF and CVBR of CVB conservative gene, taking GAPDH as internal reference, primer is GAPDH-F and GAPDH-R. Real-time fluorescence quantitative PCR carries out on BIO-RadIQ5 real-time fluorescence quantitative PCR instrument, and 3 repetitions are established in a parallel test. Utilize the method that LivakKJ and SchmittgenTD (2001) reports, namely 2-����CTCalculate relative expression quantity.
����CT=(CT.Target-CT.Actin)Timex-(CT.Target-CT.Actin)Time0
Timex represents any time point, Time0Represent that the target gene of 1 times amount after GAPDH corrects is expressed.
The sequence (5 ' 3 ') of above-mentioned primer is as follows:
CVBF:5 '-TCACAGAATACGGCTTCC-3 ';
CVBR:5 '-TTCACCTTGCTCATCATTG-3 ';
GAPDH-F:5 '-TGAGTATGTCGTGGAGTC-3 ';
GAPDH-R:5 '-CAATCTTGAGTGAGTTGTCAT-3 '.
Embodiment 6, containing caspase-3 recombinate proenzyme reconstitution cell can attenuated virus to the toxic action of cell
By CVB virus, reconstitution cell A, B and CK that embodiment 3 obtains is infected respectively, infect latter 12 hours by culture supernatant sucking-off, join on the 293T/17 cell of normal cultivation, continue to cultivate 12 hours, carry out cytoactive detection. Result as shown in Figure 4, compared with reconstitution cell CK, containing caspase-3 recombinate proenzyme reconstitution cell A and B can the infection of attenuated virus, then the cytoactive after infecting is significantly higher than control group.
The method that reconstitution cell is infected by the present embodiment virus is as follows: will cultivate the culture supernatant sucking-off of reconstitution cell A, B or CK of 24h after embodiment 3 transfection, (MOI=100:1 is mixed with CVB viral suspension, when namely infecting, virus is 100:1 with the ratio of cell quantity), 4 DEG C of standing 1h, the virus not combined is washed with PBS, add the fresh RPMI1640 containing 2% foetal calf serum again, by culture supernatant sucking-off after cultivating respectively at being placed in 37 DEG C 12 hours, join on the 293T/17 cell of normal cultivation, continue to cultivate 12 hours, carry out cytoactive detection.
The measuring method of the present embodiment cytoactive is as follows: use test kitLuminescent (Promega) carries out cytoactive detection, carries out according to operation instruction, is specifically operating as: willDetection reagent equilibrates to room temperature, adds 100ul detection reagent, contents were mixed 2 minutes on an orbit determination vibrator, inducing cell lysis to containing in every hole of cell culture fluid to be measured; Room temperature lucifuge places 10min, take out 100ul in check-out console, utilize single passage multiple labeling detector (PerkinElmer, EnVisionTM2103MulilabelReader) the fluorescence radiation trace routine in carries out luminous intensity mensuration, according to formula: versus cell activity=experimental group cytoactive detection value/control group (cell of uninfecting virus with CMC model) cytoactive detection value �� 100%.
The reconstitution cell of embodiment 7, proenzyme of recombinating containing caspase-3 is by the recovery of cytoactive after virus infection
By CVB virus, reconstitution cell A, B and CK that embodiment 3 obtains is infected respectively, and the reconstitution cell infecting latter 12nd, 24,36 and 48 hour is carried out cytoactive detection, wherein, the 24th hour after infection, cells and supernatant is changed into fresh substratum and continues to cultivate. Result as shown in Figure 5, compared with reconstitution cell CK, is changing after nutrient solution the 12 24 hour containing recombinate reconstitution cell A and B of proenzyme of caspase-3, and cytoactive has the trend of recovery. Result shows, the CVB virus in cell is had Scavenging activity by caspase-3 restructuring proenzyme.
The method that reconstitution cell is infected by the present embodiment virus is as follows: will cultivate the reconstitution cell A of 24h after embodiment 3 transfection, the culture supernatant sucking-off of B or CK, (MOI=10:1 is mixed with CVB viral suspension, when namely infecting, virus is 10:1 with the ratio of cell quantity), 4 DEG C of standing 1h, the virus not combined is washed with PBS, add the fresh RPMI1640 containing 2% foetal calf serum again, 12 are cultivated respectively at being placed in 37 DEG C, 24, 36 and 48 hours, measure cytoactive, wherein, the 24th hour after infection, cells and supernatant changes into the fresh RPMI1640 containing 2% foetal calf serum continue to cultivate.
The measuring method of the present embodiment cytoactive is identical with embodiment 6.
Claims (8)
1. a protein, its aminoacid sequence is be made up of the protein subunit sequence of the caspase forming tool protease activity and the connection peptides sequence that connects described protein subunit sequence; At least one sequence in the connection peptides sequence of the described protein subunit sequence of described connection is the amino acid recognition sequence comprising HRV 3CP crack protein matter;
Described connection peptides sequence is that the 148th of sequence 1 to the 155th bit sequence or sequence 2 the 148th is to the 153rd bit sequence; Described caspase is caspase-3;
Described protein subunit is Dare's base and little sub-base;
The aminoacid sequence of described Dare base is that the 1st of sequence 1 is to the 147th bit sequence;
And/or, the aminoacid sequence of described little sub-base is that the 156th of sequence 1 is to the 257th bit sequence.
2. protein according to claim 1, it is characterised in that: described protein is the protein of following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
B protein that () is made up of the aminoacid sequence shown in sequence in sequence table 2.
3. the encoding gene of protein described in claim 1 or 2.
4. gene according to claim 3, it is characterised in that: described gene is following 1)-2) and in arbitrary described gene:
1) DNA molecular shown in sequence 3;
2) DNA molecular shown in sequence 4.
5. expression cassette containing gene described in claim 3 or 4, recombinant vectors, recombinant cell lines or recombinant virus.
6. recombinant vectors according to claim 5, it is characterised in that: described recombinant vectors for carrier pcDNA3.1 (+) multiple clone site place insertion claim 3 or 4 described in the recombinant vectors that obtains of gene.
7. in claim 1-6, arbitrary described protein, gene, expression cassette, recombinant vectors, recombinant cell lines and/or recombinant virus have following A in preparation) and B) at least one effect product in application:
A) duplication of virus in host cell and/or the propagation of coding HRV 3CP is suppressed;
B) infection of the virus of prevention and/or treatment coding HRV 3CP.
8. there is A in claim 7) and B) in the product of at least one effect, it is characterised in that: the activeconstituents of described product is arbitrary described protein, gene, expression cassette, recombinant expression vector, recombinant cell lines and/or recombinant virus in claim 1-6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1353761A (en) * | 1999-04-13 | 2002-06-12 | 依达研究发展有限公司 | Preparation of biologically active molecules |
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| CN1353761A (en) * | 1999-04-13 | 2002-06-12 | 依达研究发展有限公司 | Preparation of biologically active molecules |
Non-Patent Citations (2)
| Title |
|---|
| Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32);MICHAEL BITZER et al;《JOURNAL OF VIROLOGY》;19990131;第704页右栏第二段 * |
| The 3C Protease Activity of Enterovirus 71 Induces Human Neural Cell Apoptosis;Mei-Ling Li et al;《Virology》;20021231;386-395 * |
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