CN1459505A - Chinese Han nationality people stroma metallopankrin-9 gene reconstitution, expression, purification and its medical application - Google Patents
Chinese Han nationality people stroma metallopankrin-9 gene reconstitution, expression, purification and its medical application Download PDFInfo
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Abstract
A matrix metal proteinase gene MMP-9 in Han nationality people of China, its clone, recombination, expression and purification methods, its special primer, and the application of the MMP-9 protein and its analog and derivative in diagnosing and treating liver cancer are disclosed. Said MMP-9 gene is prepared from the liver cell cDNA of said Chinese through PCR method.
Description
Technical field
The present invention relates to express after the reconstruct of human body gene in the genetically engineered field, purification process, particularly reorganization, expression and the purifying of people's matrix metalloproteinase-9 (MMP-9) gene; And MMP-9 albumen itself, analogue, the application of derivative aspect liver cancer medical diagnosis, treatment.
Technical background
Malignant tumour shift be one continuously, the multistage process of complicated, multifactor participation.Wherein extracellular matrix (ECM) is the first road barrier that tumour cell faces in transfer process.Tumour cell can produce a large amount of protease hydrolysis matrix, makes tumour cell be easy to shift.ECM degrading enzyme system comprises six big classes at least, and its matrix metalloproteinase (MMPs) is the most important, and it almost can degradation of polysaccharide whole ECM compositions in addition.In the Invasion and Metastasis process of tumour, the MMPs overexpression.
The clinical application of MMP-9:
The inventor herein has detected 170 routine liver cancer (primary hepatocarcinoma 115 example and secondary liver cancer 55 examples) patient's peripheral blood and 40 routine hepatic benign lesions (liver innocent tumour 11 example and liver cirrhosis 29 examples) patient's peripheral blood, 30 routine healthy human peripheral blood MMP-9.The result shows: serum MMP-9 content the liver cancer group (523.25 ± 410.96ng/ml) and the hepatic benign lesions group (200.06 ± 163.59ng/ml), (123.39 ± 58.51ng/ml) have significant difference (p=0.0001, p=0.0001 value) (table one) between organizing to healthy people; Liver cancer, optimum hepatopathy and normal people's positive rate is respectively 73.5%, 35% and 3.3% (cut-off value 240.41ng/ml), judges liver cancer susceptibility 73.5%, specific degree 96.7% (table two).Show that MMP-9 concentration is the reference index of assisting diagnosing liver cancer in the detection by quantitative liver cancer patient peripheral blood.
Table one MMP-9 expresses in hepatic diseases
Case grouping N Mean (ng/ml) Std Deviation Std Error Minim Maxim p
Liver cancer 170 523.25 410.96 31.52 26.20 2160.0
Primary hepatocarcinoma 115 490.39 397.50 37.07 26.20 2160.0
Secondary liver cancer 55 591.97 433.45 58.45 93.00 1812.0 0.080
Hepatic benign lesions 40 200.06 163.59 25.87 22.30 677.40 0.0001
Benign tumor of liver 11 270.40 228.70 68.96 26.50 677.40
Liver cirrhosis 29 173.38 126.15 23.42 22.30 564.20 0.094
The normal people 30 123.39 58.51 10.68 21.30 257.60 0.0001
The positive rate that table two MMP-9 expresses
Case grouping N MMP-9 expresses P (2-sided)
Negative positive
Liver cancer 170 45 (26.5%) 125 (73.5%)
Hepatic benign lesions 40 26 (65.0%) 14 (35.0%)＜0.01
The normal people 30 29 (96.7%) 1 (3.3%)＜0.01
The inventor has also confirmed recurrence, the transfer closely related (table three) of serum MMP-9 level and liver cancer by research.Therefore the high-caliber Patients with Primary of peripheral blood MMP-9 can be considered to give the systematize treatment before and after topical therapeutics such as intervention or operation, prevents the generation of its transfer, in the hope of improving curative ratio.
Table three liver cancer MMP-9 level with whether shift
Whether shift N MMP-9 (ng/ml) Mean Std Deviation Std Error p
Do not have and shift 45 391.23 286.96 33.36
Transferase 12 8 530.86 363.27 63.55 0.044 is arranged
Summary of the invention
In order stably to obtain the MMP-9 albumen of capacity, be used to study the proteic anti-efficiently MMP-9 antibody of MMP-9 for preparation simultaneously, the present inventor is by the method for gene recombination, obtained reorganization Chinese han population MMP-9 cDNA, in order to strengthen its antigenicity, the contriver has further made up a kind of PMMP-9 gene again, and is stable simultaneously, expressed people's MMP-9 albumen of recombinating efficiently.
The method that the purpose of this invention is to provide above-mentioned Chinese han population MMP-9 gene recombination, expression and MMP-9 protein purification.Disclose simultaneously that the plasma content of MMP-9 and tissue content and liver cancer recurrence shift, the internal relation of early prediction.
Technical scheme of the present invention is: clone, reorganization, expression and purification people MMP-9 and the proteic method of PMMP-9 may further comprise the steps: the gene of (1), clone's Chinese han population MMP-9, with the gene of reverse transcription PCR method amplification MMP-9; (2), the MMP-9 gene with above-mentioned amplification is connected structure people MMP-9 cloned plasmids with cloning vector; (3), above-mentioned people MMP-9 cloned plasmids subclone is advanced expression vector, the plasmid DNA transfection intestinal bacteria after will connecting again; (4), expressing human MMP-9
A, antibiotic LB plate is contained in the above-mentioned intestinal bacteria shop that has a Chinese han population MMP-9 expression carrier, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, put into dress LB culture medium culturing bottle, shake in the case at 30 ℃ and cultivate, survey bacterium liquid A650 OD value and be and stopped cultivation at 1.0 o'clock;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
D, results bacterium; (5), purifying people MMP-9
A, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal after, again with the phosphate buffered saline buffer of the pH8.0 precipitation that suspends;
B, cracking bacterium, the centrifuging and taking supernatant liquor;
C, preliminary purification people MMP-9;
D, carry out purifying with ion-exchange and gel permeation chromatography.(6), the structure of reorganization Chinese han population PMMP-9 gene:
A, be that template is made pcr amplification with new primer with the MMP-9 plasmid DNA
Obtain the derivative PMMP-9 of MMP-9;
B, PCR product P MMP-9 is cloned on the cloning vector plasmids;
C, cut PMMP-9 gene subclone to expression vector through enzyme; (7), reorganization proteic expression of Chinese han population PMMP-9 and purifying
A, antibiotic LB plate is contained in the above-mentioned intestinal bacteria shop that has a recombinant human PMMP-9 expression carrier, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, put into dress LB culture medium culturing bottle, shake in the case at 30 ℃ and cultivate, survey bacterium liquid A650 OD value and be and stopped cultivation at 1.0 o'clock;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
D, results bacterium;
E, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal after, again with the phosphate buffered saline buffer of the pH8.0 precipitation that suspends;
F, cracking bacterium, the centrifuging and taking supernatant liquor;
G, preliminary purification recombinant human PMMP-9;
H, carry out purifying with ion-exchange and gel permeation chromatography.The gene order of primer special of the present invention is
Primer 35 ' CTACTCTGCCTGCACCACCGAC
Primer 45 ' CTTGGTCCGGGCAGAAGCCCCA
Description of drawings
Below in conjunction with accompanying drawing embodiments of the invention are described further.
Fig. 1 is the electrophorogram of the Chinese han population MMP-9 of purifying
Fig. 2 is the electrophorogram of the reorganization Chinese han population PMMP-9 of purifying
Embodiment
Embodiment 1:
One, the acquisition of Chinese han population MMP-9 gene cDNA
1, gets liver cell, adopt U.S. Trizol RNA extraction agent box recommend method to extract total RNA, total RNA is walked sex change glue, confirm the reliable in quality of total RNA;
2, the design primer is as follows:
Primer 15 ' CATGAGCCTCTGGCAGCCCCT 3 '
Primer 25 ' GACGGGAGCCCTAGTCCTCAG 3 '
3, be masterplate with above-mentioned total RNA, the cDNA of the synthetic people MMP-9 of reverse transcription, pcr amplification, PCR reaction system reference literature (Scarf S.J., In PCR Protocol:A Guide toMethod and Application, and ed.New York, Academic Press, USA 1990) carry out, reaction conditions is:
94 ℃ of sex change 50 seconds,
Annealed 1 minute for 55 ℃,
72 ℃ were extended 1 minute and 30 seconds,
Totally 35 circulations, last 72 ℃ of insulations 10 minutes
4, the PCR product is after the preliminary conclusive evidence of 1% agarose electrophoresis, and phenol/chloroform extracting is reclaimed;
Two, make up Chinese han population MMP-9 cloned plasmids
1,, mend the PCR product flat with the Klenow polymerase according to Klenow polymerase recommend method.
2, will mend the pBluscript KS carrier that flat PCR product and EcoRV enzyme cut (any cloning vector is all passable, as T-easy, pGEX etc.) flush end and connect, the ligation system is as follows:
PBluscript KS (the EcoRV enzyme is cut) 1ul
5x connects damping fluid (what damping fluid) 2ul
T4 dna ligase 1ul
EcoRV(1u/1ul) 0.5ul
PCR product 4ul
H2O 1.5ul
Above mixture connects 12 hours for 16 ℃;
3, will connect product Transformed E .Coli JM109 competent cell, coating penbritin LB plate, the picking positive colony prepares plasmid DNA with alkaline lysis, and plasmid DNA is identified recombinant plasmid pBLU-HMMP-9 with HindIII and EcoRI double digestion;
Three, the structure of expression plasmid pET-MMP-9
1, the gene fragment of people MMP-9 (HMMP-9) in the above-mentioned pBLU-MMP-9 cloning vector is used under NdeI and the XhoI double digestion,, reclaimed the gene fragment of the CMMP-9 of about 2Kb through 1% agarose electrophoresis;
2, the pET expression vector (expression vector is various, as pBluescriptKS, pPUC, pGEM etc.) that above-mentioned this fragment and same enzyme are cut connects, and the ligation system is as follows:
PET (NdeI, the XhoI enzyme is cut) 1ul
5x connects damping fluid 2ul
T4 dna ligase 1ul
HMMP-9 5ul
H2O 1ul
Above mixture connects 12 hours for 16 ℃
3, will connect product Transformed E .coli BL21 competent cell, coating kantlex LB plate, picking positive colony;
4, prepare plasmid DNA with alkaline lysis, plasmid DNA is identified recombinant plasmid pET-HMMP-9 with NdeI and XhoI double digestion;
Four, the expression of MMP-9 in intestinal bacteria
The recombinant plasmid pET-HMMP-9 clone inoculation 10ml LB test tube in E.coliJM109 that 1, will filter out, 16 ℃ of 300r/min shaking tables are cultivated, and survey bacterium liquid A650OD value and be to stop cultivation at 1.0 o'clock;
2, collect bacterium, centrifugal 5000r/min20 minute, remove supernatant liquor, with the molten precipitation of phosphate buffered saline buffer (pH7.2), ultrasonication thalline, centrifugal 10000r/min20 minute, drying precipitated.
3, above-mentioned 10ml bacterium liquid is put into the culturing bottle that the 1000mlLB substratum is housed, shake at 37 ℃ and to cultivate 300r/min in the case, survey bacterium liquid A650 OD value, when the OD value is 1.0, adding IPTG (isopropyl-) in bacterium liquid induces, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
4, collect bacterium,, 4 ℃, centrifugal 20 minutes, remove supernatant liquor, collecting precipitation with 3000 rev/mins.With the miscible precipitation of the phosphate buffered saline buffer of pH8.0, and add PMSF (phenylmethylsulfonyl fluoride) immediately to 1mM, be put in-20 ℃ frozen;
Five, the separation and purification of MMP-9
1, bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal 5000 rev/mins, 4 ℃, 5 minutes, remove supernatant liquor, suspend with phosphate buffered saline buffer again and precipitate;
2, make the bacterium cracking with sonioation method (also can use the N,O-Diacetylmuramidase cracking process), under 4 ℃ of conditions 12, the centrifugal 10min of 000r/min, precipitation be resuspended in 30ml basis damping fluid (8M urea, 10mM Tris, pH7.5).12, the centrifugal 10min of 000r/min gets supernatant liquor;
3, supernatant liquor is through the DEAE ion exchange chromatography: respectively with basal liquid add 100mM, 200mM, 400mM NaCl carries out wash-out.Each component is analyzed through 12.5%SDS-PAGE.The component that contains desirable proteins is carried out Ni-NTG-His post affinity chromatography, adds 15mM, 30mM, 60mM, 120mM, 220mM imidazoles wash-out with basal liquid respectively.Each component row 12.5%SDS-PAGE analyzes.
As shown in Figure 1, gained MMP-9 solution S DS-PAGE electrophoresis presents a band.In the above-described embodiments, the structure of expression plasmid can also respectively have primer with corresponding two restriction endonuclease sites of expression vector with 5 of a pair of upstream and downstream ' end, and DNA is a template with people MMP-9 cloned plasmids, does pcr amplification; The PCR product is carried out purifying with ordinary method; With two corresponding restriction enzymes PCR product and expression vector are carried out enzyme and cut PCR product and expression vector after cutting with the ordinary method purifying enzyme; Be connected with expression vector with the PCR product of T4 ligase enzyme purifying.
Embodiment 2:
One, the acquisition of reorganization Chinese han population PMMP-9 gene
1, the design primer is as follows:
Primer 35 ' CTACTCTGCCTGCACCACCGAC 3 '
Primer 45 ' CTTGGTCCGGGCAGAAGCCCCA 3 '
2, selecting above-mentioned Chinese han population MMP-9 cDNA is template; Obtain the gene of reorganization Chinese han population PMMP-9, pcr amplification, PCR reaction system reference literature (Scarf S.J., In PCR Protocol:A Guide to Method and Application, and ed.New York, Academic Press, USA 1990) carry out, reaction conditions is:
94 ℃ of sex change 50 seconds,
Annealed 1 minute for 55 ℃,
72 ℃ were extended 1 minute and 30 seconds,
Totally 35 circulations, last 72 ℃ of insulations 10 minutes
3, the PCR product is after the preliminary conclusive evidence of 1% agarose electrophoresis, and phenol/chloroform extracting is reclaimed;
Two, make up reorganization Chinese han population PMMP-9 cloned plasmids
1,, mend the PCR product flat with the Klenow polymerase according to Klenow polymerase recommend method.
2, will mend the pBluscript KS carrier that flat PCR product and EcoRV enzyme cut (any cloning vector all can) flush end and be connected, the ligation system is as follows:
PBluscript KS (the EcoRV enzyme is cut) 1ul
5x connects damping fluid (what damping fluid) 2ul
T4 dna ligase 1ul
EcoRV(1u/1ul) 0.5ul
PCR product 4ul
H2O 1.5ul
Above mixture connects 12 hours for 16 ℃;
3, will connect product Transformed E .Coli JM109 competent cell, coating penbritin LB plate, the picking positive colony prepares plasmid DNA with alkaline lysis, and plasmid DNA is identified recombinant plasmid pBLU-HPMMP-9 with HindIII and EcoRI double digestion;
Three, the structure of expression plasmid pET-PMMP-9
1, the gene fragment of recombinant human PMMP-9 in the above-mentioned pBLU-PMMP-9 cloning vector is used under NdeI and the XhoI double digestion,, reclaimed the gene fragment of the PMMP-9 of about 450bp through 1% agarose electrophoresis;
2, the pET expression vector (expression vector is various, as pBluescriptKS, pPUC, pGEM etc.) that above-mentioned this fragment and same enzyme are cut connects, and the ligation system is as follows:
PET (NdeI, the XhoI enzyme is cut) 1ul
5x connects damping fluid 2ul
T4 dna ligase 1ul
HPMMP-9 5ul
H2O 1ul
Above mixture connects 12 hours for 16 ℃
3, will connect product Transformed E .coli JM109 competent cell, coating penbritin LB plate, picking positive colony;
4, prepare plasmid DNA with alkaline lysis, plasmid DNA is identified recombinant plasmid pET-HPMMP-9 with NdeI and XhoI double digestion;
Four, the expression of PMMP-9 in intestinal bacteria
The recombinant plasmid pET-HPMMP-9 clone inoculation 10ml LB test tube in E.coliJM109 that 1, will filter out, 16 ℃ of 300r/min shaking tables are cultivated, and survey bacterium liquid A650OD value and be to stop cultivation at 1.0 o'clock;
2, collect bacterium, centrifugal 5000r/min20 minute, remove supernatant liquor, with the molten precipitation of phosphate buffered saline buffer (pH7.2), ultrasonication thalline, centrifugal 10000r/min20 minute, drying precipitated.
3, above-mentioned 10ml bacterium liquid is put into the culturing bottle that the 1000mlLB substratum is housed, shake cultivation survey in 16 hours bacterium liquid A650 OD value in the case at 37 ℃, when the OD value is 1.0, adding IPTG (isopropyl-) in bacterium liquid induces, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
4, collect bacterium,, 4 ℃, centrifugal 20 minutes, remove supernatant liquor, collecting precipitation with 3000 rev/mins.With the miscible precipitation of the phosphate buffered saline buffer of pH8.0, and add PMSF (phenylmethylsulfonyl fluoride) immediately to 1mM, be put in-20 ℃ frozen;
Five, the separation and purification of PMMP-9
1, bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal 5000 rev/mins, 4 ℃, 5 minutes, remove supernatant liquor, suspend with phosphate buffered saline buffer again and precipitate;
2, make the bacterium cracking with sonioation method (also can use the N,O-Diacetylmuramidase cracking process), 4 ℃ of conditions following 12000 rev/mins centrifugal, 30 minutes, get supernatant liquor;
3, supernatant liquor is through DEAE ion exchange chromatography and Ni-NTG-His post affinity chromatography:
Respectively with basal liquid add 100mM, 200mM, 400mM NaCl carries out wash-out.Each component is analyzed through 12.5% SDS-PAGE.The component that contains desirable proteins is carried out Ni-NTG-His post affinity chromatography, adds 15mM, 30mM, 60mM, 120mM, 220mM imidazoles wash-out with basal liquid respectively.Each component row 12.5%SDS-PAGE analyzes.
Claims (8)
1, the method for a kind of expression and purification Chinese han population MMP-9 cDNA gene and reorganization PMMP-9 is characterized in that: may further comprise the steps:
(1), the gene of clone Chinese han population MMP-9, with the gene of reverse transcription PCR method amplification MMP-9;
(2), the MMP-9 gene with above-mentioned amplification is connected structure people MMP-9 cloned plasmids with cloning vector;
(3), above-mentioned people MMP-9 cloned plasmids subclone is advanced expression vector, the plasmid DNA transfection intestinal bacteria after will connecting again;
(4), express Chinese han population MMP-9
A, antibiotic LB plate is contained in the above-mentioned intestinal bacteria shop that has a Chinese han population MMP-9 expression carrier, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, put into dress LB culture medium culturing bottle, shake in the case at 30 ℃ and cultivate, survey bacterium liquid A650 OD value and be and stopped cultivation at 1.0 o'clock;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
D, results bacterium;
(5), purifying people MMP-9
A, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal after, again with the phosphate buffered saline buffer of the pH8.0 precipitation that suspends;
B, cracking bacterium, the centrifuging and taking supernatant liquor;
C, preliminary purification people MMP-9;
D, carry out purifying with ion-exchange and gel permeation chromatography.
(6), the structure of reorganization Chinese han population PMMP-9 gene
A, be template obtains MMP-9 as pcr amplification with new primer derivative PMMP-9 with the MMP-9 plasmid DNA;
B, PCR product P MMP-9 is cloned on the cloning vector plasmids;
C, cut PMMP-9 gene subclone to expression vector through enzyme;
(7), reorganization proteic expression of Chinese han population PMMP-9 and purifying
A, antibiotic LB plate is contained in the above-mentioned intestinal bacteria shop that has reorganization Chinese han population PMMP-9 expression carrier, plate is placed 37 ℃ of incubator incubated overnight;
Select a bacterium colony on b, the slave plate, put into dress LB culture medium culturing bottle, shake in the case at 30 ℃ and cultivate, survey bacterium liquid A650 OD value and be and stopped cultivation at 1.0 o'clock;
C, add IPTG in bacterium liquid, making its final concentration is 0.5mM, continues to cultivate 6-8 hour;
D, results bacterium;
E, above-mentioned bacterial precipitation is washed once with the phosphate buffered saline buffer of pH8.0, centrifugal after, again with the phosphate buffered saline buffer of the pH8.0 precipitation that suspends;
F, cracking bacterium, the centrifuging and taking supernatant liquor;
G, preliminary purification recombinant human PMMP-9;
H, carry out purifying with ion-exchange and Ni-NTG-His post affinity chromatography.
2, the method for expression and purification recombinant human MMP-9 gene according to claim 1 and PMMP-9 is characterized in that: gene and the used primer of PMMP-9 of described reverse transcription PCR method amplification MMP-9 are CMMP-9 primer 15 ' CATGAGCCTCTGGCAGCCCCT 3 ' CMMP-9 primer 25 ' GACGGGAGCCCTAGTCCTCAG 3 ' PCMMP-9 primer 35 ' CTACTCTGCCTGCACCACCGAC 3 ' PCMMP-9 primer 45 ' CTTGGTCCGGGCAGAAGCCCCA 3 '
3, the method for expression and purification recombinant human MMP-9 according to claim 1 and 2 and PMMP-9 gene is characterized in that: the described method that people MMP-9 and PMMP-9 cloned plasmids subclone are advanced expression vector is: expression vector is arbitrary expression vector; Respectively have primer with corresponding two restriction endonuclease sites of expression vector with 5 of a pair of upstream and downstream ' end, DNA is a template with people MMP-9 cloned plasmids, does pcr amplification; The PCR product is carried out purifying with ordinary method; With two corresponding restriction enzymes PCR product and expression vector are carried out enzyme and cut PCR product and expression vector after cutting with the ordinary method purifying enzyme; Be connected with expression vector with the PCR product of T4 ligase enzyme purifying.
4, the method for expression and purification recombinant human MMP-9 according to claim 1 and 2 and PMMP-9 gene is characterized in that: the described method that people MMP-9 and PMMP-9 cloned plasmids subclone are advanced expression vector is: expression vector is arbitrary expression vector; Select identical restriction endonuclease sites on cloning vector and the expression vector for use.With two restriction enzymes cloning vector and expression vector enzyme are cut, cut out MMP-9 and PMMP-9 gene order in the cloning vector, cut the multiple clone site sequence in the expression vector; With ordinary method purifying MMP-9 and PMMP-9 gene order and the expression vector that cuts multiple clone site sequence in the expression vector; People MMP-9 with purifying is connected with expression vector with the PMMP-9 gene order with the T4 ligase enzyme.
5, the method for expression and purification recombinant human MMP-9 gene according to claim 1 and 2 is characterized in that: in the process of described expression total length people MMP-9 and PMMP-9, the microbiotic that adds in the LB substratum is a penbritin; Results bacterium method be with culture with 3000 rev/mins centrifugal 20 minutes, remove supernatant liquor, collecting precipitation.
6, the method for expression and purification recombinant human MMP-9 according to claim 1 and 2 and PMMP-9 gene, it is characterized in that: in the step of described purifying people MMP-9 and PMMP-9, after washing bacterial precipitation with phosphate buffered saline buffer, centrifugal speed is 5000 rev/mins, continues 5 minutes; Bacterium cracked method is sonioation method or N,O-Diacetylmuramidase cracking process, and centrifugal after the bacterium cracking is 12000 rev/mins, continues 30 minutes, gets supernatant liquor; The method of preliminary purification is ion exchange chromatography or sieve method.
7, the primer special in expression and purification recombinant human MMP-9 and the PMMP-9 gene, its gene order is: CMMP-9 primer 15 ' CATGAGCCTCTGGCAGCCCCT 3 ' CMMP-9 primer 25 ' GACGGGAGCCCTAGTCCTCAG 3 ' PCMMP-9 primer 35 ' CTACTCTGCCTGCACCACCGAC 3 ' PCMMP-9 primer 45 ' CTTGGTCCGGGCAGAAGCCCCA 3 '
8, the dependency of MMP-9 blood plasma and tissue content and hepatocarcinoma early diagnosis and relapse and metastasis process:
1). MMP-9 concentration is the reference index of assisting diagnosing liver cancer in the detection by quantitative liver cancer patient peripheral blood, and its threshold value is＞300ng/ml
2). the level of MMP-9 can be as the index preferably of predicting liver cancer relapse and metastasis in the blood around detecting before the treatment; Its value is＞800ng/ml to surpass this value indication liver cancer recurrence and transfer.
3). the high-caliber liver cancer patient of peripheral blood MMP-9 can be considered to give the systematize treatment before and after topical therapeutics such as intervention or operation before the treatment, prevents the generation of its transfer, in the hope of improving curative ratio.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335616C (en) * | 2003-12-29 | 2007-09-05 | 中国医学科学院肿瘤医院肿瘤研究所 | Process for obtaining solid tumor related free protein in blood |
| CN101056979B (en) * | 2004-09-03 | 2013-06-05 | 阿梅特拉斯法玛有限公司 | Anti-histone H1 monoclonal antibody and hybridoma capable of producing the same |
| CN104395345A (en) * | 2012-02-29 | 2015-03-04 | 吉联亚生物科技有限公司 | Antibodies to matrix metalloproteinase 9 |
| CN113249361A (en) * | 2021-03-29 | 2021-08-13 | 南京欧凯生物科技有限公司 | Matrix metalloprotease and its preparing process |
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2002
- 2002-05-21 CN CN 02117369 patent/CN1459505A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100335616C (en) * | 2003-12-29 | 2007-09-05 | 中国医学科学院肿瘤医院肿瘤研究所 | Process for obtaining solid tumor related free protein in blood |
| CN101056979B (en) * | 2004-09-03 | 2013-06-05 | 阿梅特拉斯法玛有限公司 | Anti-histone H1 monoclonal antibody and hybridoma capable of producing the same |
| CN104395345A (en) * | 2012-02-29 | 2015-03-04 | 吉联亚生物科技有限公司 | Antibodies to matrix metalloproteinase 9 |
| CN113249361A (en) * | 2021-03-29 | 2021-08-13 | 南京欧凯生物科技有限公司 | Matrix metalloprotease and its preparing process |
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