CN103014034A - Method for preparing creatinekinase isoenzyme and application - Google Patents
Method for preparing creatinekinase isoenzyme and application Download PDFInfo
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- CN103014034A CN103014034A CN2012105674745A CN201210567474A CN103014034A CN 103014034 A CN103014034 A CN 103014034A CN 2012105674745 A CN2012105674745 A CN 2012105674745A CN 201210567474 A CN201210567474 A CN 201210567474A CN 103014034 A CN103014034 A CN 103014034A
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Abstract
The invention discloses a method for preparing creatinekinase isoenzyme and the application. The method includes the following steps: modifying a whole-genome synthesized humanized creatinekinase isoenzyme encoding gene sequence, and removing rare codons of escherichia coli; inserting the modified creatinekinase isoenzyme encoding gene sequence into an expression vector, and adopting bacterial strains of escherichia coli for transformation and expression; inoculating the transformed and expressed bacterial strains with an LB culture medium and performing IPTG (isopropyl-Beta-d-thiogalactoside) induction; and breaking the obtained thalli, performing centrifugal separation, and passing by the Ni-NTA columns for purification. The operation for preparing creatinekinase isoenzyme is very simple, the preparation cost is low, the product yield is high, the purity is high, and high biological activity is obtained; the prepared creatinekinase isoenzyme serving as antigenic substances can be used for animal immunization, related antibodies can be screened; and meanwhile, the method lays the foundation of quick establishment for a calibrator preparing platform imperative to the domestic clinical chemistry diagnosis as well as development and preparation for diagnostic reagent standard substances and references.
Description
Technical field
The invention belongs to genetically engineered and protein expression field, relate to a kind of method that efficiently prepares Creatine kinase MB with biotechnology, product can be used as antigenic substance and carries out animal immune, the screening associated antibodies is diagnosed manufacturing of required calibration object, reference material and reference material for clinical chemistry simultaneously and is laid the foundation.
Background technology
Creatine kinase (Creatinekinase, CK) extensively is present in the various tissues, and is relevant with the regeneration of Triphosaden (ATP), and the function of this enzyme is to keep intracellular Triphosaden concentration at physiological level.Its katalysis is reversible, is about to high-energy phosphate bond and is transferred to adenosine diphosphate (ADP) (ADP) upward or from Triphosaden high-energy phosphate bond is transferred to creatine from phosphocreatine, forms phosphocreatine.
CK is comprised of M and two subunits of B, is combined into CK-BB, CK-MM, and three kinds of isozymes of CK-MB also have another kind of isozyme in cell mitochondrial, be referred to as CK-Mt.CK-BB mainly is present in the organs such as brain, prostate gland, and CK-MM mainly is present in bone and cardiac muscle, and CK-MB then mainly is present in cardiac muscle, and major part is CK-MM in the normal human blood, a small amount of CK-MB, and CK-BB is few.
The isozyme of creatine kinase is of great significance in clinical diagnosis, when various pathologies comprise that amyotrophy and myocardial infarction occur, the creatine kinase level improves rapidly in people's the serum, thinks that at present the specific activity of mensuration creatine kinase is had an electro-cardiogram more reliable in the diagnosis of myocardial infarction.During myocardial infarction, creatine kinase raises in onset 6 hours, reaches the peak in 24 hours, recovers normal in 3-4 day.Wherein the specificity of the isozyme CK-MB of creatine kinase diagnosis is the highest.Creatine kinase has important physiological function because of it and clinical value has caused that people pay attention to widely and deep research.
China is vast in territory, and is populous, and reagent for clinical diagnosis market is huge, has a extensive future.With regard to domestic present situation, the diagnostic reagent industry of China still is in early days of growth, i.e. at the input initial stage of research and development of products and production, the growth stage is inreal arrival also, and China's diagnostic reagent market scale is about 1/14 of world market, and larger growing space is arranged.Develop cheap and good-quality diagnostic reagent product very necessary and the tool significance beyond doubt.
External product normally pushes market to reagent, reference material (calibration object, quality control product), instrument as a system (closed system) at present, very large advantage is arranged, and have corner on the market in detection repeatability, accuracy.Experience and strength that domestic main diagnostic reagent manufacturer does not nearly all produce corresponding quality control product, the homemade goods of approving on the market is very few.
Summary of the invention
For the problem that prior art exists, the object of the present invention is to provide a kind of simple to operate, preparation cost is cheap, the method for preparing Creatine kinase MB that success ratio is high.
For achieving the above object, a kind of method for preparing Creatine kinase MB of the present invention, the method comprises:
1) transforms synthetic two the subunit's coding gene sequences of humanization creatine kinase of also full gene, remove the intestinal bacteria rare codon;
2) with step 1) in two subunit's coding gene sequences of improved creatine kinase insert in the expression vector respectively, and transform to express with coli strain;
3) will transform the expression inoculation in the LB substratum, IPTG induces;
4) the broken bacterium of gained thalline, the Ni-NTA column purification is crossed in centrifugation;
Wherein, the synthetic humanization creatine kinase CKM of the subunit coding gene sequence of improved full gene is step 1):
1 ATGGGCCCAT TCGGTAACAC CCACAACAAG TTCAAGCTGA ATTACAAGCC TGAGGAGGAG
61 TACCCCGACC TCAGCAAACA TAACAACCAC ATGGCCAAGG TACTGACCCT TGAACTCTAC
121 AAGAAGCTGC GGGACAAGGA GACTCCATCT GGCTTCACTG TAGACGATGT CATCCAGACA
181 GGAGTGGACA ACCCAGGTCA CCCCTTCATC ATGACCGTGG GCTGCGTGGC TGGTGATGAG
241 GAGTCCTACG AAGTTTTCAA GGAACTCTTT GACCCCATCA TCTCGGATCG CCACGGGGGC
301 TACAAACCCA CTGACAAGCA CAAGACTGAC CTCAACCATG AAAACCTCAA GGGTGGAGAC
361 GACCTGGACC CTAACTACGT GCTCAGCAGC CGCGTCCGCA CTGGCCGCAG CATCAAGGGC
421 TACACGTTGC CCCCACACTG CTCCCGTGGC GAGCGCCGGG CGGTGGAGAA GCTCTCTGTG
481 GAAGCTCTCA ACAGCCTGAC GGGCGAGTTC AAAGGGAAGT ACTACCCTCT GAAGAGCATG
541 ACGGAGAAGG AGCAGCAGCA GCTCATCGAT GACCACTTCC TGTTCGACAA GCCCGTGTCC
601 CCGCTGCTGC TGGCCTCAGG CATGGCCCGC GACTGGCCCG ACGCCCGTGG CATCTGGCAC
661 AATGACAACA AGAGCTTCCT GGTGTGGGTG AACGAGGAGG ATCACCTCCG GGTCATCTCA
721 ATGGAGAAGG GGGGCAACAT GAAGGAGGTT TTCCGCCGCT TCTGCGTAGG GCTGCAGAAG
781 ATTGAGGAGA TCTTTAAGAA AGCTGGCCAC CCCTTCATGT GGAACCAGCA CCTGGGCTAC
841 GTGCTCACCT GCCCATCCAA CCTGGGCACT GGGCTGCGTG GAGGCGTGCA TGTGAAGCTG
901 GCGCACCTGA GCAAGCACCC CAAGTTCGAG GAGATCCTCA CCCGCCTGCG TCTGCAGAAG
961 AGGGGTACAG GTGGCGTGGA CACAGCTGCC GTGGGCTCAG TATTTGACGT GTCCAACGCT
1021 GATCGGCTGG GCTCGTCCGA AGTAGAACAG GTGCAGCTGG TGGTGGATGG TGTGAAGCTC
1081 ATGGTGGAAA TGGAGAAGAA GTTGGAGAAA GGCCAGTCCA TTTAG
Step 1) the synthetic humanization creatine kinase CKB of the subunit coding gene sequence of improved full gene is in:
1 ATGGGTCCAT TTAGCAATTC TCACAACGCA CTGAAACTGC
GCTTTCCGGC TGAAGATGAG
61 TTCCCGGACC TGTCCGCACA TAACAACCAC ATGGCTAAAG
TTCTGACTCC GGAACTGTAC
121 GCAGAACTGC GCGCAAAATC TACCCCGAGC GGCTTTACTC
TGGACGACGT AATCCAGACC
181 GGCGTGGATA ACCCGGGTCA CCCATATATC ATGACCGTTG
GTTGTGTTGC CGGCGATGAA
241 GAAAGCTACG AAGTCTTCAA AGACCTGTTC GATCCGATTA
TCGAAGATCG CCACGGCGGT
301 TACAAACCAA GCGATGAACA CAAAACCGAT CTGAATCCAG
ACAACCTGCA GGGTGGCGAC
361 GATCTGGATC CGAACTACGT ACTGTCTAGC CGTGTGCGCA
CCGGCCGTTC TATCCGCGGT
421 TTCTGTCTGC CACCGCACTG TTCCCGTGGT GAACGTCGTG
CCATCGAAAA ACTGGCTGTG
481 GAAGCTCTGT CCAGCCTGGA TGGTGACCTG GCAGGTCGTT
ATTATGCGCT GAAGAGCATG
541 ACCGAAGCCG AACAACAACA GCTGATCGAT GATCACTTCC
TGTTCGACAA GCCTGTTTCT
601 CCGCTGCTGC TGGCTTCTGG TATGGCGCGC GACTGGCCGG
ACGCACGTGG CATTTGGCAT
661 AACGACAACA AAACGTTCCT GGTGTGGGTC AACGAAGAAG
ACCACCTGCG TGTAATTTCT
721 ATGCAGAAAG GTGGCAACAT GAAAGAAGTT TTCACCCGTT
TTTGCACCGG CCTGACCCAG
781 ATTGAAACTC TGTTCAAATC TAAAGACTAT GAATTTATGT
GGAACCCGCA CCTGGGTTAT
841 ATTCTGACCT GTCCGTCCAA CCTGGGTACT GGCCTGCGTG
CGGGTGTGCA CATTAAGCTG
901 CCGAACCTGG GTAAACATGA AAAGTTCAGC GAGGTCCTGA
AACGCCTGCG TCTGCAGAAG
961 CGTGGTACGG GCGGTGTTGA TACTGCAGCC GTTGGTGGTG
TGTTCGACGT TAGCAACGCG
1021 GATCGTCTGG GCTTCTCTGA AGTTGAACTG GTTCAGATGG
TTGTTGATGG CGTGAAACTG
1081 CTGATTGAGA TGGAACAGCG TCTGGAACAG GGTCAGGCTA
TTGATGACCT GATGCCGGCA
1141 CAAAAATAG
Further, expression vector is step 2): pRSF Duet, pET express series.
Further, coli strain is step 2): BL21 (DE3), Rosetta (DE3).
Further, described BL21 (DE3) bacterial strain is used for efficient cloning by expression in the gene of the expression vector that contains the phage t7 promotor.
Further, lambda particles phage DE3 contains the T7 phage rna polymerase in the district, and this district is integrated in and forms described BL21 (DE3) on the karyomit(e) of BL21.
Further, broken bacterium method step 4) is broken, the ultrasonic or N,O-Diacetylmuramidase enzymolysis of high pressure.
A kind of application of method in calibration object, reference material and reference material preparation for preparing as claimed in claim 1 Creatine kinase MB CKMB.
Further, the sterling of described preparation Creatine kinase MB CKMB is added into people source or zoogenous serum, is mixed with standard substance or the calibration object of prescribed concentration.
A kind ofly utilize product that the described method for preparing Creatine kinase MB CKMB of claim 1 makes as the antigenic substance of immunity usefulness, screen associated antibodies.
Beneficial effect of the present invention is: it is very simple that the present invention prepares the Creatine kinase MB operation, preparation cost is low, finished product yield is high, purity is high and have a high biological activity, can be used as antigenic substance and carry out animal immune, the screening associated antibodies, for the calibration object of setting up as early as possible domestic clinical chemistry diagnosis urgent need prepares platform, development diagnostic reagent reference material and reference material lay the foundation.
Description of drawings
Fig. 1 obtains the approximately Insert Fragment of 1.2kb for the CKM-CKB pRSF that makes up cuts with NcoR I and EcoR I enzyme, cuts with NdeI and XhoI enzyme, obtains approximately that the Insert Fragment of 1.2kb shows insertion vector of goal gene, successfully constructs;
Fig. 2 is the SDS-PAGE electrophorogram of the CKMB finished product of purifying, and product purity can reach more than 90%.
Embodiment
Below, with reference to the accompanying drawings, the present invention is more fully illustrated, shown in the drawings of exemplary embodiment of the present invention.Yet the present invention can be presented as multiple multi-form, and should not be construed as the exemplary embodiment that is confined to narrate here.But, these embodiment are provided, thereby make the present invention comprehensively with complete, and scope of the present invention is fully conveyed to those of ordinary skill in the art.
The present invention has set up a kind of method for preparing Creatine kinase MB simple to operate, rapid, lower-cost.The principle of this method mainly is: two subunits of the creatine kinase encoding gene after will optimizing inserts the expression vector that builds, and induces its high efficient expression in intestinal bacteria, and the method is specially:
1. the structure of Creatine kinase MB expression vector:
Transform humanization Creatine kinase MB coding gene sequence, remove the intestinal bacteria rare codon, from the synthetic improved CKM of the full gene of Beijing invitrogen company and CKB encoding sequence;
Utilize http://www.faculty.ucr.edu/～mmaduro/codonusage/usage.htm web page analysis, in original CKM and CKB coding gene sequence, codon usage frequency all accounts for 12% 10% with interior in the intestinal bacteria; Improved gene order is removed the intestinal bacteria rare codon fully, and e. coli codon is activated fully.
Utilize NcoR I and EcoR I site that CKM is cloned in the PRSFDuet carrier by genetic engineering means, be configured to CKM PRSF, recycling NdeI and XhoI site are cloned into CKB in the CKM PRSF carrier, and conversion intestinal bacteria TOP10 bacterial strain, several transformed clones of picking extract plasmid, identify whether insertion vector (as shown in Figure 1) of goal gene by digestion with restriction enzyme, choose the plasmid that inserts goal gene and carry out determined dna sequence, the result shows that two subunits of creatine kinase successfully are cloned in the pRSF Duet carrier, and we are with its called after CKM-CKBpRSF.
The synthetic humanization creatine kinase CKM of the subunit coding gene sequence of improved full gene is:
1 ATGGGCCCAT TCGGTAACAC CCACAACAAG TTCAAGCTGA ATTACAAGCC TGAGGAGGAG
61 TACCCCGACC TCAGCAAACA TAACAACCAC ATGGCCAAGG TACTGACCCT TGAACTCTAC
121 AAGAAGCTGC GGGACAAGGA GACTCCATCT GGCTTCACTG TAGACGATGT CATCCAGACA
181 GGAGTGGACA ACCCAGGTCA CCCCTTCATC ATGACCGTGG GCTGCGTGGC TGGTGATGAG
241 GAGTCCTACG AAGTTTTCAA GGAACTCTTT GACCCCATCA TCTCGGATCG CCACGGGGGC
301 TACAAACCCA CTGACAAGCA CAAGACTGAC CTCAACCATG AAAACCTCAA GGGTGGAGAC
361 GACCTGGACC CTAACTACGT GCTCAGCAGC CGCGTCCGCA CTGGCCGCAG CATCAAGGGC
421 TACACGTTGC CCCCACACTG CTCCCGTGGC GAGCGCCGGG CGGTGGAGAA GCTCTCTGTG
481 GAAGCTCTCA ACAGCCTGAC GGGCGAGTTC AAAGGGAAGT ACTACCCTCT GAAGAGCATG
541 ACGGAGAAGG AGCAGCAGCA GCTCATCGAT GACCACTTCC TGTTCGACAA GCCCGTGTCC
601 CCGCTGCTGC TGGCCTCAGG CATGGCCCGC GACTGGCCCG ACGCCCGTGG CATCTGGCAC
661 AATGACAACA AGAGCTTCCT GGTGTGGGTG AACGAGGAGG ATCACCTCCG GGTCATCTCA
721 ATGGAGAAGG GGGGCAACAT GAAGGAGGTT TTCCGCCGCT TCTGCGTAGG GCTGCAGAAG
781 ATTGAGGAGA TCTTTAAGAA AGCTGGCCAC CCCTTCATGT GGAACCAGCA CCTGGGCTAC
841 GTGCTCACCT GCCCATCCAA CCTGGGCACT GGGCTGCGTG GAGGCGTGCA TGTGAAGCTG
901 GCGCACCTGA GCAAGCACCC CAAGTTCGAG GAGATCCTCA CCCGCCTGCG TCTGCAGAAG
961 AGGGGTACAG GTGGCGTGGA CACAGCTGCC GTGGGCTCAG TATTTGACGT GTCCAACGCT
1021 GATCGGCTGG GCTCGTCCGA AGTAGAACAG GTGCAGCTGG TGGTGGATGG TGTGAAGCTC
1081 ATGGTGGAAA TGGAGAAGAA GTTGGAGAAA GGCCAGTCCA TTTAG
The synthetic humanization creatine kinase CKB of the subunit coding gene sequence of improved full gene is:
1 ATGGGTCCAT TTAGCAATTC TCACAACGCA CTGAAACTGC
GCTTTCCGGC TGAAGATGAG
61 TTCCCGGACC TGTCCGCACA TAACAACCAC ATGGCTAAAG
TTCTGACTCC GGAACTGTAC
121 GCAGAACTGC GCGCAAAATC TACCCCGAGC GGCTTTACTC
TGGACGACGT AATCCAGACC
181 GGCGTGGATA ACCCGGGTCA CCCATATATC ATGACCGTTG
GTTGTGTTGC CGGCGATGAA
241 GAAAGCTACG AAGTCTTCAA AGACCTGTTC GATCCGATTA
TCGAAGATCG CCACGGCGGT
301 TACAAACCAA GCGATGAACA CAAAACCGAT CTGAATCCAG
ACAACCTGCA GGGTGGCGAC
361 GATCTGGATC CGAACTACGT ACTGTCTAGC CGTGTGCGCA
CCGGCCGTTC TATCCGCGGT
421 TTCTGTCTGC CACCGCACTG TTCCCGTGGT GAACGTCGTG
CCATCGAAAA ACTGGCTGTG
481 GAAGCTCTGT CCAGCCTGGA TGGTGACCTG GCAGGTCGTT
ATTATGCGCT GAAGAGCATG
541 ACCGAAGCCG AACAACAACA GCTGATCGAT GATCACTTCC
TGTTCGACAA GCCTGTTTCT
601 CCGCTGCTGC TGGCTTCTGG TATGGCGCGC GACTGGCCGG
ACGCACGTGG CATTTGGCAT
661 AACGACAACA AAACGTTCCT GGTGTGGGTC AACGAAGAAG
ACCACCTGCG TGTAATTTCT
721 ATGCAGAAAG GTGGCAACAT GAAAGAAGTT TTCACCCGTT
TTTGCACCGG CCTGACCCAG
781 ATTGAAACTC TGTTCAAATC TAAAGACTAT GAATTTATGT
GGAACCCGCA CCTGGGTTAT
841 ATTCTGACCT GTCCGTCCAA CCTGGGTACT GGCCTGCGTG
CGGGTGTGCA CATTAAGCTG
901 CCGAACCTGG GTAAACATGA AAAGTTCAGC GAGGTCCTGA
AACGCCTGCG TCTGCAGAAG
961 CGTGGTACGG GCGGTGTTGA TACTGCAGCC GTTGGTGGTG
TGTTCGACGT TAGCAACGCG
1021 GATCGTCTGG GCTTCTCTGA AGTTGAACTG GTTCAGATGG
TTGTTGATGG CGTGAAACTG
1081 CTGATTGAGA TGGAACAGCG TCTGGAACAG GGTCAGGCTA
TTGATGACCT GATGCCGGCA
1141 CAAAAATAG
2. the expression of Creatine kinase MB:
1) take BL21 (DE3) as Host Strains, CKM-CKB pRSF is transformed, picking list bacterium colony spends the night in 3ml LB culture medium culturing;
2) with 1: 500 ratio bacterium liquid is forwarded to 400ml LB substratum, constant temperature is at 37 ℃, and 230rpm is cultured to OD600=0.4-0.6;
3) add IPTG and induce, it is 0.5mM that IPTG induces final concentration, keeps expressing after 8 hours under 25 ℃ of conditions receiving bacterium.
BL21 (DE3) bacterial strain is used for efficient cloning by expression in the gene of the expression vector that contains the phage t7 promotor (such as pET series).Bacillus coli communis does not have the t7 RNA polymerase, so can not express the gene on those carriers.Lambda particles phage DE3 contains the T7 phage rna polymerase in the district, and this district is integrated on the karyomit(e) of BL21, just is BL21 (DE3).
3. the purifying of Creatine kinase MB:
1) get 400ml nutrient solution 3000rpm centrifugation and collect thalline after 10 minutes, thalline is resuspended in (50mM Na-PO in the 10ml protein extraction damping fluid
4, pH8.0,500mM NaCl, 10mM Imidazle), 15MPa is high to crush bacterium, 12,000rpm, 4 ℃ of centrifugal 10min, supernatant liquor and Ni-NTA were hatched 1 hour on ice;
2) cross the Ni-NTA affinity column, with protein extraction buffer washing, use at last the 250mMImidazle wash-out, collect elutriant.
3) the albumen elutriant is crossed the G50 post and is removed Imidazle, can obtain like this CKM-CKB (as shown in Figure 2) of 85% above purity.
4. Creatine kinase MB quality and determination of activity:
The sterling of preparation Creatine kinase MB is with Luo Shi E601 ElectrochemiluminescDetermination Determination, and quality can reach 10000ug/L, steps auspicious BS2000 and measures actively, and enzyme work can reach 5000U/L, and frozen 6 months of-20 ℃ of glycerine, and enzyme is lived and substantially remained unchanged.This sterling can be added into people source or zoogenous serum, is mixed with standard substance or the calibration object of prescribed concentration.
The above-mentioned method for preparing Creatine kinase MB can be used as antigenic substance and carries out animal immune, the screening associated antibodies, and in calibration object, reference material and reference material preparation, use, not to be diagnosed as purpose, do not belong to the diagnostic method of disease.
Unless specifically defined, it is known term in the relevant technologies field that the present invention describes used term.The chemical symbol of standard and dummy suffix notation can with its full name Alternate.
Unless special indicating, the present invention uses but clearly sets forth or simple technology and the method for setting forth refers to the normally used technology of the art and method, can carry out according to technology well known in the art and method.The use of test kit is to carry out according to the specification sheets that manufacturers or supplier provide.
Claims (9)
1. a method for preparing Creatine kinase MB is characterized in that, the method comprises:
1) transforms the synthetic humanization Creatine kinase MB coding gene sequence of full gene, remove the intestinal bacteria rare codon;
2) with step 1) in improved Creatine kinase MB coding gene sequence insert in the expression vector, and transform with coli strain and to express;
3) will transform the expression inoculation in the LB substratum, IPTG induces;
4) the broken bacterium of gained thalline, the Ni-NTA column purification is crossed in centrifugation;
Wherein, the synthetic humanization creatine kinase CKM of the subunit coding gene sequence of improved full gene is step 1):
1 ATGGGCCCAT TCGGTAACAC CCACAACAAG TTCAAGCTGA ATTACAAGCC TGAGGAGGAG
61 TACCCCGACC TCAGCAAACA TAACAACCAC ATGGCCAAGG TACTGACCCT TGAACTCTAC
121 AAGAAGCTGC GGGACAAGGA GACTCCATCT GGCTTCACTG TAGACGATGT CATCCAGACA
181 GGAGTGGACA ACCCAGGTCA CCCCTTCATC ATGACCGTGG GCTGCGTGGC TGGTGATGAG
241 GAGTCCTACG AAGTTTTCAA GGAACTCTTT GACCCCATCA TCTCGGATCG CCACGGGGGC
301 TACAAACCCA CTGACAAGCA CAAGACTGAC CTCAACCATG AAAACCTCAA GGGTGGAGAC
361 GACCTGGACC CTAACTACGT GCTCAGCAGC CGCGTCCGCA CTGGCCGCAG CATCAAGGGC
421 TACACGTTGC CCCCACACTG CTCCCGTGGC GAGCGCCGGG CGGTGGAGAA GCTCTCTGTG
481 GAAGCTCTCA ACAGCCTGAC GGGCGAGTTC AAAGGGAAGT ACTACCCTCT GAAGAGCATG
541 ACGGAGAAGG AGCAGCAGCA GCTCATCGAT GACCACTTCC TGTTCGACAA GCCCGTGTCC
601 CCGCTGCTGC TGGCCTCAGG CATGGCCCGC GACTGGCCCG ACGCCCGTGG CATCTGGCAC
661 AATGACAACA AGAGCTTCCT GGTGTGGGTG AACGAGGAGG ATCACCTCCG GGTCATCTCA
721 ATGGAGAAGG GGGGCAACAT GAAGGAGGTT TTCCGCCGCT TCTGCGTAGG GCTGCAGAAG
781 ATTGAGGAGA TCTTTAAGAA AGCTGGCCAC CCCTTCATGT GGAACCAGCA CCTGGGCTAC
841 GTGCTCACCT GCCCATCCAA CCTGGGCACT GGGCTGCGTG GAGGCGTGCA TGTGAAGCTG
901 GCGCACCTGA GCAAGCACCC CAAGTTCGAG GAGATCCTCA CCCGCCTGCG TCTGCAGAAG
961 AGGGGTACAG GTGGCGTGGA CACAGCTGCC GTGGGCTCAG TATTTGACGT GTCCAACGCT
1021 GATCGGCTGG GCTCGTCCGA AGTAGAACAG GTGCAGCTGG TGGTGGATGG TGTGAAGCTC
1081 ATGGTGGAAA TGGAGAAGAA GTTGGAGAAA GGCCAGTCCA TTTAG
Step 1) the synthetic humanization creatine kinase CKB of the subunit coding gene sequence of improved full gene is in:
1 ATGGGTCCAT TTAGCAATTC TCACAACGCA CTGAAACTGC
GCTTTCCGGC TGAAGATGAG
61 TTCCCGGACC TGTCCGCACA TAACAACCAC ATGGCTAAAG
TTCTGACTCC GGAACTGTAC
121 GCAGAACTGC GCGCAAAATC TACCCCGAGC GGCTTTACTC
TGGACGACGT AATCCAGACC
181 GGCGTGGATA ACCCGGGTCA CCCATATATC ATGACCGTTG
GTTGTGTTGC CGGCGATGAA
241 GAAAGCTACG AAGTCTTCAA AGACCTGTTC GATCCGATTA
TCGAAGATCG CCACGGCGGT
301 TACAAACCAA GCGATGAACA CAAAACCGAT CTGAATCCAG
ACAACCTGCA GGGTGGCGAC
361 GATCTGGATC CGAACTACGT ACTGTCTAGC CGTGTGCGCA
CCGGCCGTTC TATCCGCGGT
421 TTCTGTCTGC CACCGCACTG TTCCCGTGGT GAACGTCGTG
CCATCGAAAA ACTGGCTGTG
481 GAAGCTCTGT CCAGCCTGGA TGGTGACCTG GCAGGTCGTT
ATTATGCGCT GAAGAGCATG
541 ACCGAAGCCG AACAACAACA GCTGATCGAT GATCACTTCC
TGTTCGACAA GCCTGTTTCT
601 CCGCTGCTGC TGGCTTCTGG TATGGCGCGC GACTGGCCGG
ACGCACGTGG CATTTGGCAT
661 AACGACAACA AAACGTTCCT GGTGTGGGTC AACGAAGAAG
ACCACCTGCG TGTAATTTCT
721 ATGCAGAAAG GTGGCAACAT GAAAGAAGTT TTCACCCGTT
TTTGCACCGG CCTGACCCAG
781 ATTGAAACTC TGTTCAAATC TAAAGACTAT GAATTTATGT
GGAACCCGCA CCTGGGTTAT
841 ATTCTGACCT GTCCGTCCAA CCTGGGTACT GGCCTGCGTG
CGGGTGTGCA CATTAAGCTG
901 CCGAACCTGG GTAAACATGA AAAGTTCAGC GAGGTCCTGA
AACGCCTGCG TCTGCAGAAG
961 CGTGGTACGG GCGGTGTTGA TACTGCAGCC GTTGGTGGTG
TGTTCGACGT TAGCAACGCG
1021 GATCGTCTGG GCTTCTCTGA AGTTGAACTG GTTCAGATGG
TTGTTGATGG CGTGAAACTG
1081 CTGATTGAGA TGGAACAGCG TCTGGAACAG GGTCAGGCTA
TTGATGACCT GATGCCGGCA
1141 CAAAAATAG 。
2. prepare as claimed in claim 1 the method for Creatine kinase MB, it is characterized in that step 2) described in expression vector be: pRSF Duet, pET express series.
3. prepare as claimed in claim 1 the method for Creatine kinase MB, it is characterized in that step 2) described in coli strain be: BL21 (DE3), Rosetta (DE3).
4. prepare as claimed in claim 3 the method for Creatine kinase MB, it is characterized in that, described BL21 (DE3) bacterial strain is used for efficient cloning by expression in the gene of the expression vector that contains the phage t7 promotor.
5. prepare as claimed in claim 4 the method for Creatine kinase MB, it is characterized in that, lambda particles phage DE3 contains the T7 phage rna polymerase in the district, and this district is integrated in and forms described BL21 (DE3) on the karyomit(e) of BL21.
6. prepare as claimed in claim 1 the method for Creatine kinase MB, it is characterized in that step 4) described in broken bacterium method be broken, the ultrasonic or N,O-Diacetylmuramidase enzymolysis of high pressure.
7. the application of method in calibration object, reference material and reference material preparation for preparing as claimed in claim 1 Creatine kinase MB.
8. application as claimed in claim 7 is characterized in that, the sterling of described preparation Creatine kinase MB is added into people source or zoogenous serum, is mixed with standard substance or the calibration object of prescribed concentration.
9. one kind is utilized product that the described method for preparing Creatine kinase MB of claim 1 makes as the antigenic substance of immunity usefulness, screens associated antibodies.
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| CN110904068A (en) * | 2018-09-17 | 2020-03-24 | 杭州博茵生物技术有限公司 | CK-MB type creatine kinase isozyme, preparation method and application |
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Non-Patent Citations (2)
| Title |
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| YOSHIKO SUNAHARA，ET AL: "Production of recombinant human creatine kinase (r-hCK) isozymes by tandem repeat expression of M and B genes and characterization of r-hCK-MB", 《CLINICAL CHEMISTRY》 * |
| 张建 等: "人线粒体肌酸激酶广泛型亚型的原核表达、抗体制备及其临床应用意义", 《第二军医大学学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110904068A (en) * | 2018-09-17 | 2020-03-24 | 杭州博茵生物技术有限公司 | CK-MB type creatine kinase isozyme, preparation method and application |
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