CN101003575A - Fusion protein in soluble receptor II - antibody Fc section of human tumor necrosis factor - Google Patents

Abstract

This invention discloses fusion protein of soluble receptor II of human tumor necrosis factor and Fc fragment of antibody. The amino acid sequence of the fusion protein is shown in SEQ ID No.2. This invention utilizes IRES sequence of double-cistronic vector pTandem-1 to ligate the gene sequence of the fusion protein with DHFR gene, which is used as a screening marker. This invention can reduce the screening intensity and raise the expression level. This invention can effectively and stably produce fusion protein of soluble receptor II of human tumor necrosis factor and Fc fragment of antibody.

Description

The human tumor necrosis factor fusion protein in soluble receptor II-antibody Fc section

Technical field

The invention belongs to gene engineering technology field, relate in particular to a kind of human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein.

Background technology

The research and development of recombinant soluble acceptor class medicine successfully are important breakthroughs of biotech drug development in recent years, and the disease of treatment comprises cancer, cardiovascular disorder and autoimmune disorder etc.Last century, early eighties in the rheumatoid arthritis patient, found that (tumor necrosis factor-α, TNF) content in serum far surpasses the normal people to tumour necrosis factor.In mouse rheumatoid arthritis animal model afterwards, show that also blood serum tumor necrosin level obviously increases.Utilize among the tumour necrosis factor soluble receptors-II (TNFR-II) that extracts and the interior too high TNF of body, treatment suffers from the mouse of rheumatoid arthritis, and curative effect is obvious.Also obtained gratifying effect afterwards clinically.Rheumatoid arthritis pharmacological agent at present comprises hormone and immunosuppressor etc., DeGrain, and also drug side effect is bigger.The major advantage of the soluble receptors of tumour necrosis factor is the composition that belongs to human body self, be difficult for causing the generation of immunne response in treatment, thereby security is better.Can be effectively, stably manufacturer's tumour necrosis factor soluble receptors II-antibody Fc section fusion rotein can satisfy clinical needs.

Summary of the invention

The objective of the invention is to utilize engineered principle, a kind of human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein is provided;

Technical scheme of the present invention is summarized as follows:

A kind of human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein, it has the described aminoacid sequence of SEQ ID No.2 in the sequence table.

The described aminoacid sequence of described a kind of human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein is obtained or is obtained by the protein synthesis technology is synthetic by the prepared carrier transfectional cell of the described nucleotide sequence of SEQ ID No.1 in the sequence table.

Advantage of the present invention is the IRES sequence of utilizing among the two-cistron expression vector pTandem-1, TNFR-Fc gene and selection markers DHFR gene are linked together, can alleviate proof strength, improve expression level, the present invention can effectively, stably make TNFRII-Fc.

Description of drawings

Fig. 1 .PCR amplification DHFR gene fragment;

Fig. 2 .PCR amplification TNFR-Fc gene fragment;

Fig. 3. the expression vector structural map;

Fig. 4 .rTNFR-Fc purifying SDS-PAGE figure;

The western blot figure of Fig. 5 .rTNFR-II.

Embodiment

A kind of preparation method of recombinant human tumor necrosis factor's soluble receptors II-antibody Fc section fusion rotein: make up cells of mamma animals dual-expression vector pT-dhfr-TRFc → transfection Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary epithelial cell → usefulness and select substratum screening positive clone → enlarged culturing → collection substratum → low ternperature separation process to keep supernatant → Protein A affinity chromatography column purification → product evaluation.

Below in conjunction with the drawings and specific embodiments the present invention is described in further detail, but the present invention is not limited to following embodiment:

Embodiment 1PCR amplification gene: with SEQ ID No.4 (5 '-AAAAAA GTTTAAACATGGTTCGACCATAGAACT-3 ') for forward primer is, with SEQ ID No.5 (5 ' AAAAAA GCTAGCTTAGTCTTTTCCCTCGTAGACT-3 ') is reverse primer, (Clontech company) is template with Chinese hamster ovary celI cDNA library, PCR is reflected in the 50 μ L cumulative volumes and carries out, reaction conditions is for beginning circulation after 5 minutes 94 ℃ of sex change, 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 1.5 minutes, 72 ℃ were extended 1 minute, after totally 30 circulations, extended 10 minutes in 72 ℃ again, amplify one with the dna fragmentation SEQ ID No.3 that estimates that big or small (564bp) conforms to.

The structure of embodiment 2 pT-dhfr expression vectors: PCR product and carrier pTandem-1 (Novagen company) that gel reclaims use PmeI and BlpI double digestion respectively, and from 1% sepharose, reclaim test kit and reclaim by gel, connect (16 ℃ then, 16h), transformed into escherichia coli DH-5 α competent cell, select positive colony, carry out the double digestion analysis verification after extracting plasmid, and carry out dna sequencing and identify, sequencing result and the described sequence of SEQ ID No.3 are identical, the expression plasmid that makes up is called as pT-dhfr, and construction strategy is seen Fig. 3.

Embodiment 3 is according to the synthetic SEQ ID No.8 of SEQ ID No.6 and two primers of SEQ ID No.9, with people's lung cDNA library (Clontech company) is that template is carried out the PCR reaction, amplify human tumor necrosis factor soluble receptors II gene order (TNFRII), the PCR product is TNFRII-pcrp.

Embodiment 4 is according to the synthetic SEQ ID No.10 of SEQ ID No.6 and two primers of SEQ ID No.11, with human B lymphocyte library (Clontech company) is that template is carried out the PCR reaction, amplify people's IgG antibody 1 Fc end group because of, PCR product called after: Fc-pcrp;

Embodiment 5 utilizes overlapping extension PCR method to synthesize TNFR-Fc fusion molecule gene fragment: with SEQ ID No.6 is that forward primer, SEQ ID No.7 are reverse primer, the TNFRII-pcrp and the Fc-pcrp that are obtained with embodiment 3 and embodiment 4 are masterplate, PCR is reflected in the 50 μ L cumulative volumes and carries out, reaction conditions is for beginning circulation after 5 minutes 94 ℃ of sex change, 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 2.5 minutes, and 72 ℃ were extended 3 minutes, after totally 30 circulations, extended 10 minutes in 72 ℃ again.Amplify one with estimate size (1857bp) the described gene order of SEQ ID No.1 (Fig. 2) that conforms to.

The structure of embodiment 6 pT-dhfr-TRFc expression vectors: PCR product and carrier pT-dhfr that gel reclaims example 5 use NcoI and XhoI double digestion respectively, and from 1% sepharose, reclaim test kit and reclaim by gel, connect (16 ℃ then, 16h), transformed into escherichia coli DH-5 α competent cell, select positive colony, carry out the double digestion analysis verification behind the extraction plasmid, and carry out dna sequencing and identify.Sequencing result shows: it is identical with the described gene order of SEQ ID No.1 to insert sequence dna fragment, and the expression plasmid of structure is called as pT-dhfr-TRFc.

Embodiment 7 expression plasmid pT-dhfr-TRFc transfection CHO/DHFR-cells (host China hamster ovarian epithelial cell Chinese hamster ovary cell, CHO): the pT-dhfr-TRFc expression plasmid transfection CHO/DHFR-cell with sequence verification (contains 10% foetal calf serum, 2mM glutamine, 10ug/ml glycine, 15ug/ml xanthoglobulin 5ug/m, thymus pyrimidine at F12/DEME; 37 ℃, 5%CO ₂) cultivate, the cell in vegetative period of taking the logarithm, cell cover and obtain about 50-70%, adopt the above-mentioned cell of liposome transfection.Behind the cell transfecting, earlier with above-mentioned culture medium culturing 2 days, every day observation of cell form.After the cell cultures 2 days, use phosphoric acid buffer thorough washing cell 5 times, change and select substratum (the F12/DEME substratum that does not contain glycine, xanthoglobulin, thymus pyrimidine; 7% dialysis serum) cultivate, cultivated 7-10 days, filter out positive colony.After the enlarged culturing, collect and select the substratum supernatant then, and low-temperature centrifugation separates preservation.

The purifying of embodiment 8 expression products: wash out the protective material solution of the Protein A affinity column of 2ml with the phosphoric acid buffer of 20ml, and then with 3 times of column volume phosphoric acid buffer balance chromatography columns.With syringe embodiment 6 is separated the sample of preserving and inject chromatography column.With the phosphoric acid buffer wash-out foreign protein of at least 8 times of volumes, then with the composition on the citrate buffer solution wash-out adsorption column of the pH3.0 of 6 times of volumes and collect.Measure the protein content of wash-out, and the gained sample is analyzed (Fig. 4) with SDS-PAGE.

The immunoblotting of embodiment 9 rTNFR-Fc detects: conventional SDS-PAGE and western blotting method, the monoclonal antibody of the anti-soluble tumor necrosis factor receptor of specificity-II extracellular region is as first antibody, and second antibody is the anti-mouse antibody of horseradish peroxidase-labeled.The result proves that rTNFR-Fc can be discerned (Fig. 5) by monoclonal antibody specific.

Sequence table

<160>11

<210>1

<211>1857

<212>DNA

<213>Homo?sapiens

<221>gene

<222>(1)…(1857)

<400>1

atggcgcccg?tcgccgtctg?ggccgcgctg?gccgtcggac?tggagctctg?ggctgcggcg

cacgccttgc?ccgcccaggt?ggcatttaca?ccctacgccc?cggagcccgg?gagcacatgc

cggctcagag?aatactatga?ccagacagct?cagatgtgct?gcagcaagtg?ctcgccgggc

caacatgcaa?aagtcttctg?taccaagacc?tcggacaccg?tgtgtgactc?ctgtgaggac

agcacataca?cccagctctg?gaactgggtt?cccgagtgct?tgagctgtgg?ctcccgctgt

agctctgacc?aggtggaaac?tcaagcctgc?actcgggaac?agaaccgcat?ctgcacctgc

aggcccggct?ggtactgcgc?gctgagcaag?caggaggggt?gccggctgtg?cgcgccgctg

cgcaagtgcc?gcccgggctt?cggcgtggcc?agaccaggaa?ctgaaacatc?agacgtggtg

tgcaagccct?gtgccccggg?gacgttctcc?aacacgactt?catccacgga?tatttgcagg

ccccaccaga?tctgtaacgt?ggtggccatc?cctgggaatg?caagcaggga?tgcagtctgc

acgtccacgt?cccccacccg?gagtatggcc?ccaggggcag?tacacttacc?ccagccagtg

tccacacgat?cccaacacac?gcagccaact?ccagaaccca?gcactgctcc?aagcacctcc

ttcctgctcc?caatgggccc?cagcccccca?gctgaaggga?gcactggcga?cggaggtggc

ggatctggag?gtggcgggag?tatcctctgc?gcctgggccc?agctctgtcc?cacaccgcgg

tcacatggca?ccacctctct?tgcagcctcc?accaagggcc?catcggtctt?ccccctggca

ccctcctcca?agagcacctc?tgggggcaca?gcggccctgg?gctgcctggt?caaggactac

ttccccgaac?cggtgacggt?gtcgtggaac?tcaggcgccc?tgaccagcgg?cgtgcacacc

ttcccggctg?tcctacagtc?ctcaggactc?tactccctca?gcagcgtggt?gaccgtgccc

tccagcagct?tgggcaccca?gacctacatc?tgcaacgtga?atcacaagcc?cagcaacacc

aaggtggaca?agaaagttga?gcccaaatct?tgtgacaaaa?ctcacacatg?cccaccgtgc

ccagcacctg?aactcctggg?gggaccgtca?gtcttcctct?tccccccaaa?acccaaggac

accctcatga?tctcccggac?ccctgaggtc?acatgcgtgg?tggtggacgt?gagccacgaa

gaccctgagg?tcaagttcaa?ctggtacgtg?gacggcgtgg?aggtgcataa?tgccaagaca

aagccgcggg?aggagcagta?caacagcacg?taccgggtgg?tcagcgtcct?caccgtcctg

caccaggact?ggctgaatgg?caaggagtac?aagtgcaagg?tctccaacaa?agccctccca

gcccccatcg?agaaaaccat?ctccaaagcc?aaagggcagc?cccgagaacc?acaggtgtac

accctgcccc?catcccggga?ggagatgacc?aagaaccagg?tcagcctgac?ctgcctggtc

aaaggcttct?atcccagcga?catcgccgtg?gagtgggaga?gcaatgggca?gccggagaac

aactacaaga?ccacgcctcc?cgtgctggac?tccgacggct?ccttcttcct?ctacagcaag

ctcaccgtgg?acaagagcag?gtggcagcag?gggaacgtct?tctcatgctc?cgtgatgcat

gaggctctgc?acaaccacta?cacgcagaag?agcctctccc?tgtctccggg?taaatga 1857

<210>2

<211>618

<212>PRT

<213>Homo?sapiens

<221>

<222>(1)…(618)

<400>2

MetAlaProValAlaValTrpAlaAlaLeuAlaValGlyLeuGluLeuTrpAlaAlaAlaHisAlaLeuProAlaGlnValAlaPheT

hrProTyrAlaProGluProGlySerThrCysArgLeuArgGluTyrTyrAspGlnThrAlaGlnMetCysCysSerLysCysSerPr

oGlyGlnHisAlaLysValPheCysThrLysThrSerAspThrValCysAspSerCysGluAspSerThrTyrThrGlnLeuTrpAsn

TrpValProGluCysLeuSerCysGlySerArgCysSerSerAspGlnValGluThrGlnAlaCysThrArgGluGlnAsnArgIleC

ysThrCysArgProGlyTrpTyrCysAlaLeuSerLysGlnGluGlyCysArgLeuCysAlaProLeuArgLysCysArgProGlyPh

eGlyValAlaArgProGlyThrGluThrSerAspValValCysLysProCysAlaProGlyThrPheSerAsnThrThrSerSerThr

AspIleCysArgProHisGlnIleCysAsnValValAlaIleProGlyAsnAlaSerArgAspAlaValCysThrSerThrSerProT

hrArgSerMetAlaProGlyAlaValHisLeuProGlnProValSerThrArgSerGlnHisThrGlnProThrProGluProSerTh

rAlaProSerThrSerPheLeuLeuProMetGlyProSerProProAlaGluGlySerThrGlyAspGlyGlyGlyGlySerGlyGly

GlyGlySerIleLeuCysAlaTrpAlaGlnLeuCysProThrProArgSerHisGlyThrThrSerLeuAlaAlaSerThrLysGlyP

roSerValPheProLeuAlaProSerSerLysSerThrSerGlyGlyThrAlaAlaLeuGlyCysLeuValLysAspTyrPheProGl

uProValThrValSerTrpAsnSerGlyAlaLeuThrSerGlyValHisThrPheProAlaValLeuGlnSerSerGlyLeuTyrSer

LeuSerSerValValThrValProSerSerSerLeuGlyThrGlnThrTyrIleCysAsnValAsnHisLysProSerAsnThrLysV

alAspLysLysValGluProLysSerCysAspLysThrHisThrCysProProCysProAlaProGluLeuLeuGlyGlyProSerVa

lPheLeuPheProProLysProLysAspThrLeuMetIleSerArgThrProGluValThrCysValValValAspValSerHisGlu

AspProGluValLysPheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerT

hrTyrArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAlaLe

uProAAlaProIleGluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSerArgGluGlu

MetThrLysAsnGlnValSerLeuThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGlnP

roGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrValAspLysSerAr

gTrpGlnGlnGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSerPro

GlyLysUmb 618

<210>3

<211>583

<212>DNA

<213>Homo?sapiens

<221>gene

<222>(1)…(583)

<400>3

atggttcgac?cattgaactg?catcgtcgcc?gtgtcccaaa?atatggggat?tggcaagaac

ggagacctac?cctggcctcc?gctcaggtac?cctggcctcc?gctcaggaac?gagttcaagt

acttccaaag?aatgaccaca?acctcttcag?tggaaggtaa?acagaatctg?gtgattatgg

gtaggaaaac?ctggttctcc?attcctgaga?agaatcgacc?tttaaaggac?agaattaata

tagttctcag?tagagaactc?aaagaaccac?cacgaggagc?tcattttctt?gccaaaagtt

tggatgatgc?cttaagctta?ttgaacaacc?ggaattggca?agtaaagtag?acatggtttg

gatagtcgga?ggcagttctg?tttaccagga?agccatgaat?caaccaggcc?acctcagact

ctttgtgaca?aggatcatgc?aggaatttga?aagtgacacg?tttttcccag?aaattgattt

ggggaaatat?aaacttctcc?cagaataccc?aggcgtcctc?tctgaggtcc?aggaggaaaa

aggcatcaag?tataagtttg?aagtctacga?gaagaaagac?taa 583

<210>4

<211>33

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(33)

<400>4

aaaaaa gttt?aaacatggtt?cgaccataga?act 33

<210>5

<211>34

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(34)

<400>5

aaaaaagcta?gcttagtctt?ttccctcgta?gact 34

<210>6

<211>27

<212>DNA

＜213〉synthetic

<221>

<222>(1)…?(27)

aaaaaaccat?ggcgcccgtc?tgggccg 27

<400>7

<210>5

<211>32

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(32)

<400>

aaaaaactcg?agtcatttac?ccggagacag?gg 32

<210>8

<211>19

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(19)

<400>8

atggcgcccg?tctgggccg 19

<210>9

<211>20

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(20)

<400>9

gtcgccagtg?ctcccttcag 20

<210>10

<211>37

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(37)

<400>10

agtggaggga?ggaggatcta?tcctctgcgc?ctgggcc 37

<210>11

<211>32

<212>DNA

＜213〉synthetic

<221>

<222>(1)…(32)

<400>11

aaaaaactcg?agtcatttac?ccggagacag?gg 32

Claims (2)

1. a human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein is characterized in that it has the described aminoacid sequence of SEQ ID No.2 in the sequence table.

2. a kind of human tumor necrosis factor soluble receptors II-antibody Fc section fusion rotein according to claim 1 is characterized in that described aminoacid sequence is obtained or obtained by the protein synthesis technology is synthetic by the prepared carrier transfectional cell of the described nucleotide sequence of SEQ ID No.1 in the sequence table.

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