CN1143116A - New human erythropoietin - Google Patents

Abstract

The geneclone and expression in cell of mammal of human erythropoietin features that the total RNA extracted is reversely transcribed to synthesize the first chain of cDNA and then cloned via polymerase chain reaction to obtain DNA segment of coded human erythropoietin, and the said DNA segement is connected with expression carrier PCR III to obtain recombination plasmid pE/C, which, together with plasmid pDHFR, is transfected to ovarium cell of Chinese hamstser of dihydrofolate reductase deficiency to obtain the cell strain expressing human erythropoietin. The cell strain of the present invention has the features of stable passage and high expression amount.

Description

A kind of new erythropoietin

The present invention relates to a kind of erythropoietin technical field, a kind of gene clone of erythropoietin specifically and the method for in mammalian cell, expressing thereof.

(Erythropoietin EPO) is a kind of important hemopoieticgrowth factor to erythropoietin, and in vivo, it can stimulate propagation, the differentiation of red corpuscle precursor cell in the marrow specifically, increases sophisticated red corpuscle from the burst size of marrow to peripheral blood.Kidney is the main generation position of erythropoietin in the human body of growing up, and the generation minimizing of the erythropoietin that causes because of renal failure can cause anaemia.Erythropoietin has been used for the treatment of clinically anaemia that renal failure causes, patient AIDS because of anaemia due to the zidovudine treatment and tumour patient because of the anaemia due to the chemotherapy, in operation and bone marrow transplantation, erythropoietin also is well used.Behind epo treatment, pcv and hemoglobin level obviously raise in the patient body, and the dependency of transfusing blood is obviously reduced, and therefore, the clinical application of erythropoietin prospect is very wide.

The natural human erythropoietin mainly is to extract from anaemia patients'blood or urine, and is very limited because of its source, can't satisfy the heavy demand of clinical and scientific research.The development of recombinant DNA technology is for the mass production erythropoietin provides a new approach.Since 1985, existing many scholars have successfully expressed erythropoietin (Fu-kuen, Lin etc., Proc.Natl. Acad.Sci.USA, 82:7580-7584,1985 in mammalian cell; Kenneth Jacobs etc., Nature, 313:806-810,1985; Jerry S.Powell etc., Proc.Natl.Acad.Sci.USA, 83:6465-6469,1986; Huang Liwen etc., Chinese science B collects, 24:178-184,1994).These genetically engineered erythropoietins of expressing in mammalian cell have in the body identical with the natural human erythropoietin, external activity.Following method is mainly followed in the gene clone and the expression in mammalian cell thereof of genetically engineered erythropoietin at present.

1. the acquisition of the dna fragmentation of coding erythropoietin: mainly contain the dna fragmentation that dual mode obtains the coding erythropoietin.A kind of is to extract tire liver chromosomal DNA, is primer then with the specific oligonucleotide, goes out the gene fragment of erythropoietin through PCR amplification, carries out external splicing then.Another kind is to extract people's tire liver mRNA, through the synthetic cDNA library of reverse transcription.Or screening people tire liver gene group library, the gene of the erythropoietin that obtains encoding downcuts human epo gene and is connected with expression vector, imports mammalian cell, extraction mRNA, and another mistake is transcribed into the cDNA library.Screening cDNA library, the dna fragmentation of the erythropoietin that obtains encoding.

2. the dna fragmentation of coding erythropoietin is connected with expression vector.Expression vector commonly used has pDSVL, pSV2, and these two kinds of plasmids have Tetrahydrofolate dehydrogenase (dhfr) gene, also can use pDll, do not contain the dhfr gene.

3. the structure of cell strain: recombinant plasmid is imported mammalian cell, obtain needed cell strain through screening.Host cell commonly used is COS cell and Chinese hamster ovary celI.

According to the document of having delivered, the genomic constitution of Chinese's erythropoietin is identical with the document of abroad delivering with the aminoacid sequence of structure and erythropoietin.193 amino acid of EPO gene coding region codified, wherein preceding 27 amino acid are formed the targeting signal peptide, and all the other 166 amino acid are formed maturation protein, and its molecular weight is 18399D.The recombinant epo molecule is because of degree of glycosylation and sugared type composition difference, and its molecular weight does not wait from 26KD to 34KD.

The invention provides a kind of new erythropoietin and production method thereof, this production method comprises the structure of recombinant DNA carrier, with recombinant vectors DNA transfection mammalian cell, and culturing cell, purifying obtains a kind of new erythropoietin from supernatant.

The present invention also provides a kind of new dna molecular of the erythropoietin of encoding.The 418th bit base of this dna molecular is C, and the document of having delivered is G.

The present invention also provides a kind of amino acid composition sequence of new erythropoietin.This sequence is made up of 193 amino acid, and wherein preceding 27 amino acid are formed signal peptide, and back 166 amino acid are formed maturation protein, and the 113rd amino acids of maturation protein is Arg, rather than Gly.Through the sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, apparent molecular weight is 36～45KD, is a glycoprotein.The derivative of erythropoietin of the present invention is also included among the present invention.

The present invention also provides a kind of recombinant DNA molecules.The dna fragmentation of coding erythropoietin is connected with expression vector pCRIII, obtains recombinant plasmid pE/C.

The present invention also provides the mammalian cell that is used for the expressing human erythropoietin.With plasmid pE/C and pDHFR cotransfection Chinese hamster ovary cell, obtain the engineering cell strain of express recombinant erythropoietin, its expression amount surpasses 10000U/ml10 ⁶Cell is 2～3 times of existing cell strain expression amount.

The present invention also provides and has contained a kind of new erythropoietin and the medicine of derivative thereof, and erythropoietin of the present invention and derivative thereof are as all or part of active constituent of medicine in this medicine.

The present invention also provides a kind of new erythropoietin has been used for the method for disease treatment, contains this new people's cell and generates plain medicine and can be used for treating the renal failure anaemia, and anaemia due to the chemotherapy of tumors is also as the bone marrow transplantation toughener.Realize that technical essential of the present invention is:

1. from fresh Chinese's tire liver, extract total RNA,, amplify the dna fragmentation of coding Chinese erythropoietin again through polymerase chain reaction (PCR) through first chain of the synthetic cDNA of reverse transcription.

2. the DNA product with amplification is connected with expression vector pCRIII, connects the competent cell that product is used for transformed into escherichia coli DH5 α, through screening, evaluation, obtains recombinant molecule pE/C.

3. adopt United States Biochemical, the sequence of the dna fragmentation of coding Chinese erythropoietin among the Inc. Sequenase kit measurement plasmid pE/C.Sequencing result shows that coding region the 418th bit base is C, and the 113rd amino acids of the maturation protein of deriving thus is Arg.

4. recombinant plasmid pE/C and plasmid pDHFR (available from Invitrogen) are adopted the Chinese hamster ovary cell (CHO dhfr-) of lipofection cotransfection Tetrahydrofolate dehydrogenase defective type, pressurization screening in the ever-increasing methotrexate of concentration (MTX) is after resulting cell strain is confirmed in enzyme immunoassay.

The invention will be further described below by embodiment

1. the structure of recombinant plasmid pE/C

Extract total RNA from 1 gram Chinese tire liver, 1.2% agarose electrophoresis shows that 18S and 28S RNA are complete.Getting the total RNA of 5ug is template, first chain with the synthetic cDNA of avian myeloblastosis virus reverse transcriptase (AMV-RT), this synthetic product is got 1ul as template by dilution in 1: 10, with specific oligonucleotide as 3 '-end and 5 '-the end primer, as archaeal dna polymerase, amplify the dna fragmentation of coding Chinese erythropoietin with the Tag enzyme through polymerase chain reaction (PCR).The PCR reaction conditions is: 95 ℃ of sex change 10 minutes, and cycle stage 95 ℃ of sex change 45 seconds, 56 ℃ of annealing 50 seconds, 72 ℃ were extended 2 minutes, extended 10 minutes at 72 ℃ after 35 circulations.The PCR product shows that through 1% agarose electrophoresis amplified production is about 630bp, and is consistent with the expection size.Reclaim the 630bp fragment from agarose, be connected at 12 ℃ with carrier pCRIII and spend the night, ligase enzyme is the T4 dna ligase.Connect product and be used for transformed into escherichia coli DH5 α competent cell.Picking list colony inoculation in the 5ml substratum, 37 ℃ of overnight incubation.Prepare plasmid DNA in a small amount, through E _CORI digestion show that recombinant plasmid contains the 630bp fragment, and direction of insertion is correct.The plasmid called after pE/C that obtains, its structure is seen accompanying drawing 1, its complete sequence sees Table 1..

The full DNA sequence Sequence Range:1 to 6079 of table 1. plasmid pE/C

* * * * *

GACGGATCGG?GAGATCTCCC?GATCCCCTAT?GGTCGACTCT?CAGTACAATC

100

* * * * *

TGCTCTGATG?CCGCATAGTT?AAGCCAGTAT?CTGCTCCCTG?CTTGTGTGTT

* * * * *

GGAGGTCGCT?GAGTAGTGCG?CGAGCAAAAT?TTAAGCTACA?ACAAGGCAAG

200

* * * * *

GCTTGACCGA?CAATTGCATG?AAGAATCTGC?TTAGGGTTAG?GCGTTTTGCG

* * * * *

CTGCTTCGCG?ATGTACGGGC?CAGATATACG?CGTTGACATT?GATTATTGAC

300

* * * * *

TAGTTATTAA?TAGTAATCAA?TTACGGGGTC?ATTAGTTCAT?AGCCCATATA

* * * * *

TGGAGTTCCG?CGTTACATAA?CTTACGGTAA?ATGGCCCGCC?TGGCTGACCG

400

* * * * *

CCCAACGACC?CCCGCCCATT?GACGTCAATA?ATCACGTATG?TTCCCATAGT

* * * * *

AACGCCAATA?GGGACTTTCC?ATTGACGTCA?ATGGGTGGAC?TATTTACGGT

500

* * * * *

AAACTGCCCA?CTTGGCAGTA?CATCAAGTGT?ATCATATGCC?AAGTACGCCC

* * * * *

CCTATTGACG?TCAATGACGG?TAAATGGCCC?GCCTGGCATT?ATGCCCAGTA

600

* * * * *

CATGACCTTA?TGGGACTTTC?CTACTTGGCA?GTACATCTAC?GTATTAGTCA

* * * * *

TCGCTATTAC?CATGGTGATG?CGGTTTTGGC?AGTACATCAA?TGGGCGTGGA

700

* * * * *

TAGCGGTTTG?ACTCACGGGG?ATTTCCAAGT?CTCCACCCCA?TTGACGTCAA

* * * * *

TGGGAGTTTG?TTTTGGCAGC?AAAATCAACG?GGACTFTCCA?AAATGTCGTA

800

* * * * *ACAACTCCGC?CCCATTGACG?CAAATGGGCG?GTAGGCGTGT?ACGGTGGGAG

* * * * *GTGTATATAA?GCAGAGCTCT?CTGGCTAACT?AGAGAACCCG?CTGCTTAACT

900

* * * * *GGCTTATCGA?AATTAATACG?ACTCACTATA?GGGAGACCCA?AGCTTGAGAT

* * * * *GGGGGTGCAC?GAATGTCCTG?CCTGGCTGTG?GCTTCTCCTG?TCCCTGCTGT

1000

* * * * *CGCTCCCTCT?GGGCCTCCCA?GTCCTGGGCG?CCCCACCACG?CCTCATCTGT

* * * * * GACAGCCGAG?TCCTGGAGAG?GTACCTCTTG?GAGGCCAAGG?AGGCCGAGAA

1100

* * * * *TATCACGACG?GGCTGTGCTG?AACACTGCAG?CTTGAATGAG?AATATCACTG

* * * * *TCCCAGACAC?CAAAGTTAAT?TTCTATGCCT?GGAAGAGGAT?GGAGGTCGGG

1200

* * * * *CAGCAGGCCG?TAGAAGTCTG?GCAGGGCCTG?GCCCTGCTGT?CGGAAGCTGT

* * * * *CCTGCGGGGC?CAGGCCCTGT?TGGTCAACTC?TTCCCAGCCG?TGGGAGCCCC

1300

* * * * *TGCAGCTGCA?TGTGGATAAA?GCCGTCAGTG?GCCTTCGCAG?CCTCACCACT

* * * * *CTGCTTCGGG?CTCTGCGAGC?CCAGAAGGAA?GCCATCTCCC?CTCCAGATGC

1400

* * * * * GGCCTCAGCT?GCTCCACTCC?GAACAATCAC?TGCTGACACT?TTCCGCAAAC

* * * * *TCTTCCGAGT?CTACTCCAAT?TTCCTCCGGG?GAAAGCTGAA?GCTGTACACA

1500

* * * * *GGGGAGGCCT?GCAGGACAGG?GGACAGATGA?CCAGGTGTGT?CCACCTGGGC

* * * * *ATACTTAAGG?ACGTCGGGCC?CCCTAGGTGA?TCAAGATCTA?GAGGGCCCTA

1600

* * * * *TTCTATAGTG?TCACCTAAAT?GCTAGAGCTC?GCTGATCAGC?CTCGACTGTG

* * * * *CCTTCTAGTT?GCCAGCCATC?TGTTGTTTGC?CCCTCCCCCG?TGCCTTCCTT

1700

* * * * *GACCCTGGAA?GGTGCCACTC?CCACTGTCCT?TTCCTAATAA?AATGAGGAAA

* * * * *TTGCATCGCA?TTGTCTGAGT?AGGTGTCATT?CTATTCTGGG?GGGTGGGGTG

1800

* * * * *GGGCAGGACA?GCAAGGGGGA?GGATTGGGAA?GACAATAGCA?GGCATGCTGG

* * * * *GGATGCGGTG?GGCTCTATGG?AACCAGCTGG?GGCTCGAGGG?GGGATCCCCA

1900

* * * * *CGCGCCCTGT?AGCGGCGCAT?TAAGCGCGGC?GGGTGTGGTG?GTTACGCGCA

* * * * *GCGTGACCGC?TACACTTGCC?AGCGCCCTAG?CGCCCGCTCC?TTTCGCTTTC

2000

* * * * *TTCCCTTCCT?TTCTCGCCAC?GTTCGCCGGC?TTTCCCCGTC?AAGCTCTAAA

* * * * *TCGGGGCATC?CCTTTAGGGT?TCCGATTTAG?TGCTTTACGG?CACCTCGACC

2100

* * * * *CCAAAAAACT?TGATTAGGGT?GATGGTTCAC?GTAGTGGGCC?ATCGCCCTGA

* * * * *TAGACGGTTT?TTCGCCCTTT?GACGTTGGAG?TCCACGTTCT?TTAATAGTGG

2200

* * * * *ACTCTTGTTC?CAAACTGGAA?CAACACTCAA?CCCTATCTCG?GTCTATTCTT

* * * * *TTGATTTATA?AGGGATTTTG?GGGATTTCGG?CCTATTGGTT?AAAAAATGAG

2300

* * * * *CTGATTTAAC?AAAAATTTAA?CGCGAATTTT?AACAAAATAT?TAACGTTTAC

* * * * *AATTTAAATA?TTTGCTTATA?CAATCTTCCT?GTTTTTGGGG?CTTTTCTGAT

2400

* * * * *TATCAACCGG?GGTGGGTACC?GAGCTCGAAT?TCTGTGGAAT?GTGTGTCAGT

* * * * *TAGGGTGTGG?AAAGTCCCCA?GGCTCCCCAG?GCAGGCAGAA?GTATGCAAAG

2500

* * * * *CATGCATCTC?AATTAGTCAG?CAACCAGGTG?TGGAAAGTCC?CCAGGCTCCC

* * * * *CAGCAGGCAG?AAGTATGCAA?AGCATGCATC?TCAATTAGTC?AGCAACCATA

2600

* * * * *GTCCCGCCCC?TAACTCCGCC?CATCCCGCCC?CTAACTCCGC?CCAGTTCCGC

* * * * *CCATTCTCCG?CCCCATGGCT?GACTAATTTT?TTTTATTTAT?GCAGAGGCCG

2700

* * * * *AGGCCGCCTC?GGCCTCTGAG?CTATTCCAGA?AGTAGTGAGG?AGGCTTTTTT

* * * * *GGAGGCCTAG?GCTTTTGCAA?AAAGCTCCCG?GGAGCTTGGA?TATCCATTTT

2800

* * * * *CGGATCTGAT?CAAGAGACAG?GATGAGGATC?GTTTCGCATG?ATTGAACAAG

* * * * *ATGGATTGCA?CGCAGGTTCT?CCGGCCGCTT?GGGTGGAGAG?GCTATTCGGC

2900

* * * * *TATGACTGGG?CACAACAGAC?AATCGGCTGC?TCTGATGCCG?CCGTGTTCCG

* * * * *GCTGTCAGCG?CAGGGGCGCC?CGGTTCTTTT?TGTCAAGACC?GACCTGTCCG

3000

* * * * *GTGCCCTGAA?TGAACTGCAG?GACGAGGCAG?CGCGGCTATC?GTGGCTGGCC

* * * * *ACGACGGGCG?TTCCTTGCGC?AGCTGTGCTC?GACGTTGTCA?CTGAAGCGGG

3100

* * * * *AAGGGACTGG?CTGCTATTGG?GCGAAGTGCC?GGGGCAGGAT?CTCCTGTCAT

* * * * *CTCACCTTGC?TCCTGCCGAG?AAAGTATCCA?TCATGGCTGA?TGCAATGCGG

3200

* * * * *CGGCTGCATA?CGCTTGATCC?GGCTACCTGC?CCATTCGACC?ACCAAGCGAA

* * * * *ACATCGCATC?GAGCGAGCAC?GTACTCGGAT?GGAAGCCGGT?CTTGTCGATC

3300

* * * * *AGGATGATCT?GGACGAAGAG?CATCAGGGGC?TCGCGCCAGC?CGAACTGTTC

* * * * *GCCAGGCTCA?AGGCGCGCAT?GCCCGACGGC?GAGGATCTCG?TCGTGACCCA

3400

* * * * *TGGCGATGCC?TGCTTGCCGA?ATATCATGGT?GGAAAATGGC?CGCTTTTCTG

* * * * *GATTCATCGA?CTGTGGCCGG?CTGGGTGTGG?CGGACCGCTA?TCAGGACATA

3500

* * * * *GCGTTGGCTA?CCCGTGATAT?TGCTGAAGAG?CTTGGCGGCG?AATGGGCTGA

* * * * *CCGCTTCCTC?GTGCTTTACG?GTATCGCCGC?TCCCGATTCG?CAGCGCATCG

3600

* * * * *CCTTCTATCG?CCTTCTTGAC?GAGTTCTTCT?GAGCGGGACT?CTGGGGTTCG

* * * * *AAATGACCGA?CCAAGCGACG?CCCAACCTGC?CATCACGAGA?TTTCGATTCC

3700

* * * * *ACCGCCGCCT?TCTATGAAAG?GTTGGGCTTC?GGAATCGTTT?TCCGGGACGC

* * * * *CGGCTGGATG?ATCCTCCAGC?GCGGGGATCT?CATGCTGGAG?TTCTTCGCCC

3800

* * * * *ACCCCAACTT?GTTTATTGCA?GCTTATAATG?GTTACAAATA?AAGCAATAGC

* * * * *ATCACAAATT?TCACAAATAA?AGCATTTTTT?TCACTGCATT?CTAGTTGTGG

3900

* * * * *TTTGTCCAAA?CTCATCAATG?TATCTTATCA?TGTCTGGATC?CCGTCGACCT

* * * * *CGAGAGCTTG?GCGTAATCAT?GGTCATAGCT?GTTTCCTGTG?TGAAATTGTT

4000

* * * * *ATCCGCTCAC?AATTCCACAC?AACATACGAG?CCGGAAGCAT?AAAGTGTAAA

* * * * *GCCTGGGGTG?CCTAATGAGT?GAGCTAACTC?ACATTAATTG?CGTTGCGCTC

4100

* * * * *ACTGCCCGCT?TTCCAGTCGG?GAAACCTGTC?GTGCCAGCTG?CATTAATGAA

* * * * *TCGGCCAACG?CGCGGGGAGA?GGCGGTTTGC?GTATTGGGCG?CTCTTCCGCT

4200

* * * * *TCCTCGCTCA?CTGACTCGCT?GCGCTCGGTC?GTTCGGCTGC?GGCGAGCGGT

* * * * *ATCAGCTCAC?TCAAAGGCGG?TAATACGGTT?ATCCACAGAA?TCAGGGGATA

4300

* * * * *ACGCAGGAAA?GAACATGTGA?GCAAAAGGCC?AGCAAAAGGC?CAGGAACCGT

* * * * *AAAAAGGCCG?CGTTGCTGGC?GTTTTTCCAT?AGGCTCCGCC?CCCCTGACGA

4400

* * * * * GCATCACAAA?AATCGACGCT?CAAGTCAGAG?GTGGCGAAAC?CCGACAGGAC

* * * * *TATAAAGATA?CCAGGCGTTT?CCCCCTGGAA?GCTCCCTCGT?GCGCTCTCCT

4500

* * * * *GTTCCGACCC?TGCCGCTTAC?CGGATACCTG?TCCGCCTTTC?TCCCTTCGGG

* * * * *AAGCGTGGCG?CTTTCTCAAT?GCTCACGCTG?TAGGTATCTC?AGTTCGGTGT

4600

* * * * *AGGTCGTTCG?CTCCAAGCTG?GGCTGTGTGC?ACGAACCCCC?CGTTCAGCCC

* * * * *GACCGCTGCG?CCTTATCCGG?TAACTATCGT?CTTGAGTCCA?ACCCGGTAAG

4700

* * * * *ACACGACTTA?TCGCCACTGG?CAGCAGCCAC?TGGTAACAGG?ATTAGCAGAG

* * * * *CGAGGTATGT?AGGCGGTGCT?ACAGAGTTGT?TGAAGTGGTG?GCCTAACTAC

4800

* * * * *GGCTACACTA?GAAGGACAGT?ATTTGGTATC?TGCGCTCTGC?TGAAGCCAGT

* * * * *TACCTTCGGA?AAAAGAGTTG?GTAGCTCTTG?ATCCGGCAAA?CAAACCACCG

4900

* * * * *CTGGTAGCGG?TGGTTTTTTT?GTTTGCAAGC?AGCAGATTAC?GCGCAGAAAA

* * * * *AAAGGATCTC?AAGAAGATCC?TTTGATCTTT?TCTACGGGGT?CTGACGCTCA

5000

* * * * *GTGGAACGAA?AACTCACGTT?AAGGGATTTT?GGTCATGAGA?TTATCAAAAA

* * * * *GGATCTTCAC?CTAGATCCTT?TTAAATTAAA?AATGAAGTTT?TAAATCAATC

5100

* * * * *TAAAGTATAT?ATGAGTAAAC?TTGGTCTGAC?AGTTACCAAT?GCTTAATCAG

* * * * *TGAGGCACCT?ATCTCAGCGA?TCTGTCTATT?TCGTTCATCC?ATAGTTGCCT

5200

* * * * *GACTCCCCGT?CGTGTAGATA?ACTACGATAC?GGGAGGGCTT?ACCATCTGGC

* * * * *CCCAGTGCTG?CAATGATACC?GCGAGACCCA?CGCTCACCGG?CTCCAGATTT

5300

* * * * *ATCAGCAATA?AACCAGCCAG?CCGGAAGGGC?CGAGCGCAGA?AGTGGTCCTG

* * * * *CAACTTTATC?CGCCTCCATC?CAGTCTATTA?ATTGTTGCCG?GGAAGCTAGA

5400

* * * * *GTAAGTAGTT?CGCCAGTTAA?TAGTTTGCGC?AACGTTGTTG?CCATTGCTAC

* * * * *AGGCATCGTG?GTGTCACGCT?CGTCGTTTGG?TATGGCTTCA?TTCAGCTCCG

5500

* * * * *GTTCCCAACG?ATCAAGGCGA?GTTACATGAT?CCCCCATGTT?GTGCAAAAAA

* * * * *GCGGTTAGCT?CCTTCGGTCC?TCCGATCGTT?GTCAGAAGTA?AGTTGGCCGC

5600

* * * * *AGTGTTATCA?CTCATGGTTA?TGGCAGCACT?GCATAATTCT?CTTACTGTCA

* * * * *TGCCATCCGT?AAGATGCTTT?TCTGTGACTG?GTGAGTACTC?AACCAAGTCA

5700

* * * * *TTCTGAGAAT?AGTGTATGCG?GCGACCGAGT?TGCTCTTGCC?CGGCGTCAAT

* * * * *ACGGGATAAT?ACCGCGCCAC?ATAGCAGAAC?TTTAAAAGTG?CTCATCATTG

5800

                                                                            

* * * * *GAAAACGTTC?TTCGGGGCGA?AAACTCTCAA?GGATCTTACC?GCTGTTGAGA

                                                                 

* * * * *TCCAGTTCGA?TGTAACCCAC?TCGTGCACCC?AACTGATCTT?CAGCATCTTT

5900

* * * * *TACTTTCACC?AGCGTTTCTG?GGTGAGCAAA?AACAGGAAGG?CAAAATGCCG

* * * * *CAAAAAAGGG?AATAAGGGCG?ACACGGAAAT?GTTGAATACT?CATACTCTTC

6000

* * * * *CTTTTTCAAT?ATTATTGAAG?CATTTATCAG?GGTTATTGTC?TCATGAGCGG

* * * * *ATACATATTT?GAATGTATTT?AGAAAAATAA?ACAAATAGGG?GTTCCGCGCA

* *CATTTCCCCG?AAAAGTGCCA?CCTGACGTC

Among the plasmid pE/C sequence dna fragment of coding Chinese erythropoietin measure utilize the Sequenase test kit (United States Biochemical Inc.USA), is a template with the denatured double stranded dna, with α- ³⁵S-dATP is a mark, adopts the two deoxidation cessation method of Sanger to measure recombinant plasmid pE/C Chinese erythropoietin coding region dna sequence dna, and measurement result sees Table 2.

The sequence of the dna fragmentation of coding Chinese erythropoietin among the table 2. plasmid pE/C

ATGGGGGTG?CACGAATGTCCTGCCTGGCT?GTGGCTTCTC?CTGTCCCTGC?TGTCGCTCCC?TCTGGGCCTCCCAGTCCTGG?GCGCCCCACC?ACGCCTCATC?TGTGACAGCC?GAGTCCTGGAGAGGTACCTC?TTGGAGGCCA?AGGAGGCCGA?GAATATCACG?ACGGGCTGTGCTGAACACTG?CAGCTTGAAT?GAGAATATCA?CTGTCCCAGA?CACCAAAGTTAATITCTATG?CCTGGAAGAG?GATCGAGGTC?GGGCAGCAGG?CCGTAGAAGTCTGGCAGGGC?CTGGCCCTGC?TGTCGGAAGC?TGTCCTGCGG?CCCCAGGCCCTGTTGGTCAA?CTCTICCCAG?CCGTGGGAGC?CCCTGCAGCT?GCATGTGGATAAAGCCGTCA?GTGGCCTTCG?CAGCCTCACC?ACTCTGCTTC?GGGCTCTGCGAGCCCAGAAG?GAAGCCATCT?CCCCTCCAGA?TGCGGCCTCA?GCTGCTCCACTCCGAACAAT?CACTGCTGAC?ACTTTCCGCA?AACTCTTCCG?AGTCTACTCCAATTTCCTCC?GGGGAAAGCT?GAAGCTGTAC?ACAGGGGAGG?CCTGCAGGACAGGGGACAGA

According to the dna sequence dna of measuring, the maturation protein amino acid composition sequence of deriving Chinese's erythropoietin sees Table 3..

The amino acid composition sequence of table 3. Chinese erythropoietin maturation protein:

APP?RLICDSRVLE?RYLLEAKEAENITTGCAEHC?SLNENITVPD?TKVNFYAWKR?MEVGQQAVEV?WQGLALLSEAVLRGQALLVN?SSQPWEPLQL?HVDKAVSGLR?SLTTLLRALR?AQKEAISPPDAASAAPLRTI?TADFFRKLFR?VYSNFLRGKL?KLYTGEACRT?GDR

3. the structure of expression cell line

Host cell is Chinese hamster ovary cell (the CHO dhfr of Tetrahydrofolate dehydrogenase defective type available from American Type Culture Collecti (ATCC) ^-).With this cell cultures in the 100mm culture dish, treat that cell covered with to 50～60% o'clock,, add by 5ml serum free medium, 10ug pE/C, 10ug pDHFR (see figure 2) with serum free medium drip washing cell, the transfection mixed solution that 60ug lipofectin forms was cultivated 4 hours for 37 ℃.The sucking-off substratum adds the F12 substratum that contains 10% foetal calf serum, 37 ℃ of overnight incubation.In the DMEM that contains penicillin, Streptomycin sulphate and 10% foetal calf serum, cultivate subsequently, with trypsin digestion cell, by dilution in 1: 5, in substratum, add MTX to final concentration be 1nM, continue to cultivate and occurred to resistance clone in 10～14 days.With the cell that the trysinization resistance clone is turned out, dilution in 1: 5 raises MTX concentration by 1nM → 5nM → 25nM → 100nM → 200nM → 1000nM subsequently gradually, the screening resistance clone.The resistance clone that will occur in containing 200nM MTX substratum is incubated in the 100mm culture dish, by dilution in 1: 500, continues to cultivate 3-5 days, reaches 2～4mm until the cell clone diameter.Utilize enzyme-linked immunosorbent assay to confirm that resulting cell can the expressing human erythropoietin.

Caption:

Fig. 1. the structural representation of plasmid pE/C

Fig. 2. the sub-1-656 tripartite leader[of the structural representation pE/C plasmid structure C MV promotor 209-864T7 promotor 865-883SP6 promotor 138-1645 people EPO gene 890-1520SV40 promotor 2674-2799 neomycin resistance gene 2805-3599SV40 polyadenylic acid tailing 3603-3812PDHFR plasmid structure gland virus stage starting 657-945 multiple clone site 956-1056 dihydrofolate reductase gene 1080-1678SV40 polyadenylic acid tailing 1856-2012 of plasmid pDHFR

Claims (6)

1. erythropoietin albumen, it is characterized in that: (1) SDS-PAGE apparent molecular weight is that 36～45KD (2) peptide chain portion is APP RLICDSRVLE RYLLEAKEAENITTGCAEHC SLNFNITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEAVLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALR AQKEAISPPDAASAAPLRTI TADFFRKLFR VYSNFLRGKL KLYTGEACRT GDR

(3) the 113rd amino acids residues are Arg

(4), be a glycoprotein by expressing cho cell.

2. the derivative of erythropoietin protein is characterized in that: be made up of all or part of aminoacid sequence in the claim 1, have the erythropoietin activity, comprise glycosylation form and non-glycosylated form.

3. the dna molecular of the erythropoietin of encoding is characterized in that:

( 1 ) ： ATGGGGGTG CACGAATGTCCTGCCTGGCT GTGGCTTCTC CTGTCCCTGC TGTCGCTCCC TCTGGGCCTCCCAGTCCTGG GCGCCCCACC ACGCCTCATC TGTGACAGCC GAGTCCTGGAGAGGTACCTC TTGGAGGCCA AGGAGGCCGA GAATATCACG ACGGGCTGTGCTGAACACTG CAGCTTGAAT GAGAATATCA CTGTCCCAGA CACCAAAGTTAATTTCTATG CCTGGAAGAG GATGGAGGTC GGGCAGCAGG CCGTAGAAGTCTGGCAGGCC CTGGCCCTGC TGTCGGAACC TGTCCTGCGG GGCCAGGCCCTGTTGGTCAA CTCTTCCCAG CCGTGGGAGC CCCTGCAGCT GCATGTGGATAAAGCCGTCA GTGGCCTTCG CAGCCTCACC ACTCTGCTTC GGGCTCTGCGAGCCCAGAAG GAAGCCATCT CCCCTCCAGA TGCGGCCTCA GCTGCTCCACTCCGAACAAT CACTGCTGAC ACTTTCCGCA AACTCTTCCG AGTCTACTCCAATTTCCTCC GGGGAAAGCT GAAGCTGTAC ACAGGGGAGG CCTGCAGGACAGGGGACAGA

(2) the 418th bit bases are C

4. new recombinant plasmid pE/C is characterized in that:

(1) this plasmid is by 6079 based compositions in the table 1.

(2) 899-1477 base is dna molecular in the claim 3 in this plasmid

(3) Chinese hamster ovary cell of this plasmid and plasmid pDHFR cotransfection Tetrahydrofolate dehydrogenase defective type can be used for expressing the erythropoietin in the claim 1.The plasmid pDHFR here is available from Invitrogen.

5. the genetically engineered cell strain of expressing human erythropoietin is characterized in that:

(1) obtains by plasmid pE/C in the claim 4 and plasmid pDHFR cotransfection Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell.The plasmid pDHFR here is available from Invitrogen, and Chinese hamster ovary cell is available from American Type Culture Collecti.

(2) through cultivating the erythropoietin that to express in the claim 1.

(3) this cell strain reaches 10000U/ml.10 to the expression amount of the erythropoietin in the claim 1 ⁶Cell.

6. one kind has the active medicine of erythropoietin, it is characterized in that: the albumen in the claim 1 or 2 is as all or part of active ingredient of this medicine.

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