Kind Of Fusion Protein, Gene Encoding It, Expression Method And Use Thereof

Abstract

This invention provides a fusion protein (FP4) and the code gene, expression method, and clinical application; its goal is to provide a fusion protein, the code gene, and the expression method as well as to take this fusion protein as the active constituent anti-allergic medicine. In addition, the invention provides the sequence of fusion protein attached on SEQ ID 2 and any derivation protein of the fusion protein (FP4) which includes adding or deleting several amino acids of SEQ ID2 as well as increasing or deducing nuclear tides of SEQ ID 1. We designed to cross-link IgE active receptor and an IgG inhibitory receptor by endogens hinge without any extraneous chemical link. This invention fusion protein has function to block the IgE acceptor, moreover fragment of fusion protein has ability to specifically bind to a native IgG inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motif, thereby inhibiting the IgE-driven mediator release from mast cells and basophils. It will play important role in the allergic disease treatment.

Description

FIGURE EXPLANATION

FIG. 1: FP4 Molecular Structure

FIG. 2 FP4 Cloning Flow Chart

FIG. 3A: Electrophoresis Analysis of Fcε PCR products

FIG. 3B: Electrophoresis Analysis of Fcγ PCR products.

FIG. 4: Flow Analysis of FP4 binding ability to FcεRI receptor.

FIG. 5: Flow Analysis of FP4 binding ability to FcγRII receptor

FIG. 6: FP4 protein inhibited histamine release in human basophils

FIG. 7: FP4 fusion protein blocked allergic reaction in the transgenic mice.

BEST WAY TO CARRY OUT THE IMPLEMENTATION PROCESS

Implementation 1: fusion protein FP4's expression.

As FIG. 2 illustrates, fusion protein FP4's expression process includes the following steps the exact translation of the term), purify the B lymphocyte from human's circumference blood, specifically: Carries on the regular procedure with instruction below: separate (not sure extract genome DNA, direct things with the specificity:

P1 (Fcε):
5′-GTGGCCCAGCCGGCCTTCACCCCGCCCACCGTGAAG-3′;
P2 (Fcε):
5′-GTGGATCCTTTACCGGGATTTACAGACAC-3′;
P3 (Fcγ):
5′-GGGGATCCGAGCCCAAATCTTGTGAC-3′;
P4 (Fcγ):
5′-GTGCGGCCGCTCATTTACCCGGAGACAGGGAGAG-3′

We amplified Fcε and Fcγ genes by using Polymerase Chain Reaction, PCR products of Fcε and Fcγ were run on 1% agarose gel electrophoresis, the result was shown in FIG. 3, Fcε DNA PCR product is 1155 bp; Fcγ DNA PCR product is 928 bp. The PCR products were directly clone into pCR4-TOPO Vector (INVITROGEN,CA). After ligation, we obtained FPEFc-ε and FPGFc-γ and did the nucleotide sequence analysis. After confirming the nucleotide sequences correct, Fcε gene was cut by SfiI-BamHI (NEW ENGLAND BIOLABS); Fcγ gene was cut by BamHI-NotI. At mean time pSecTag expression vector (INVITROGEN,CA) was cut by Sfi I-Not I and then ligated with Fcε and Fcγ into pSecTagFP4. We confirmed the insertion sequence is the same as that in FIG. 1. The pSecTagFP4 vector was transfected by electroporation into mouse myeloma SP2/0 cell. After selection with ZEOCIN, the positive clones were screened by ELISA (coating the plate with the mouse anti-human IgE, adding the supernatants of clones, then adding goat anti-human IgE conjugated with AP). We obtained the positive clones highly expressing FP4 protein. We cultured the positive clones by using 10% FBS RPMI 1640 medium (Invitrogen, CA) in 37° C. 5% CO₂ for 15 days. We collected the supernatants by 2000 rpm centrifuge and purified the FP4 protein by using anti-human IgE affinity column (coupling mouse anti-human IgE to CNBr-activated Sepharose 4B(PHARMACIA).

Implementation 2:FP4 and FcεRI Receptor Binding Experiment

Incubated 1×10⁶ CHO3D10 cells with 5 μg of Purified FP4 at 4° C. 1 hour, then added 5 μl of FITC labeled anti-human IgE (CALTAG,CA), analyzed with flow cytometry. The result was shown in FIG. 4. FP4 protein binds FcεRI receptor, presenting FITC positive of CHO3D10; In FIG. 4, SSC-H is in the cytoplasmic granularity, FSC-H is the cell size, FL2-H is PE, FL 1-H is FITC.

Implementation 3: FP4 and FcγRII Receptor Binding Experiment

Incubated 1×10⁶ HMC-1 cell and 5 μg of Purifies FP4 protein at 4° C. 1 hour, added 5 μl of FITC labeled anti-human IgG (CALTAG, CA), analyzed with flow cytometry. The result shown that FP4 protein binds to FcγRII receptor, the HMC-1 cell presenting FITC positive(see FIG. 5), SSC-H is the cell granularity, FSC-H is the cell size, FL2-H is PE, FL1-H is FITC.

Implementation 4: FP4 Protein in vitro Function Experiment

We separated and purified human basophil from human peripheral blood. The purified basophils(1×10⁶) were incubated with 1-10 μg specific human anti-NP-IgE (SEROTECH) at 37° C. for 2 hours, and then added 2 μg NP-BSA, the histamine release was measured by ELISA. The results showed that FP4 protein significantly inhibited histamine release in human basophils in dose-dependent fashion (see FIG. 6). Hu IgE stands for human IgE, NS IgE stands non-specific human IgE, NP-BSA stands for allergen, +addition, −non addition.

Implementation 5:FP4 Protein in vivo Function Experiment

The human FcεRIα chain transgenic mouse (from University of California, San Diego) was injected intradermally with human anti-NP-IgE (SEROTECH), 4 hour later i.v. challenging 100 μg of NP-BSA plus 1% Evans (Sigma) blue dye. If allergic reaction occurs, the local skin color turns to blue because of the leakage of dye from blood vessel. When we added the same amount of fusion protein (FP4), then allergic reaction was completely blocked, the local skin color not presenting blue (see FIG. 6). This experimental result further proved the FP4 protein in vivo inhibited allergic reaction.

INDUSTRIAL APPLICATION

In this invention, fusion protein FP4's ingredients originate from human immunoglobulin. Therefore, this protein enters the human body as a medicine; it does not have any antigens (foreign body protein immunity source). Fusion protein FP4 area C (hinge) obtains characteristics such as being nimble, easily rotated, and able to connect FcεRI and FcγRII together, thus stimulating the cell to prohibit signal sending, which then prohibits cells from releasing various active biological particles and prevent allergic reactions from occurring. All these results from the body of the experiment prove that the fusion protein FP4 can effectively connect adipose cell or Fc_RI on the surface of basophil granular cell with Fc_RII, in order to prevent allergic reaction. Fusion protein FP4 from this invention not only obtains IgE monoclonal antibody to block the function of IgE acceptor, but more importantly it starts an allergic reaction inside the cell's signal transduction pathway by suppressing it. (starting allergic reaction more importantly in the cell the signal conduction system suppression system) Furthermore, it strengthens the suppression of the cell's allergic reaction, which will play a vital role in its allergic disease treatment.

Foreword tabulation (SEQ ID NO 1)
<160> 2
<210> 1
<211> 1665
<212> DNA
<213> Artificial sequence
<220>
<223>
<400> 1
ttcaccccgc ccaccgtgaa gatcttacag tcgtcctgcg acggcggcgg gcacttcccc 60
ccgaccatcc agctcctgtg cctcgtctct gggtacaccc cagggactat caacatcacc 120
tggctggagg acgggcaggt catggacgtg gacttgtcca ccgcctctac cacgcaggag 180
ggtgagctgg cctccacaca aagcgagctc accctcagcc agaagcactg gctgtcagac 240
cgcacctaca cctgccaggt cacctatcaa ggtcacacct ttgaggacag caccaagaag 300
tgtgcagatt ccaacccgag aggggtgagc gcctacctaa gccggcccag cccgttcgac 360
ctgttcatcc gcaagtcgcc cacgatcacc tgtctggtgg tggacctggc acccagcaag 420
gggaccgtga acctgacctg gtcccgggcc agtgggaagc ctgtgaacca ctccaccaga 480
aaggaggaga agcagcgcaa tggcacgtta accgtcacgt ccaccctgcc ggtgggcacc 540
cgagactgga tcgaggggga gacctaccag tgcagggtga cccaccccca cctgcccagg 600
gccctcatgc ggtccacgac caagaccagc ggcccgcgtg ctgccccgga agtctatgcg 660
tttgcgacgc cggagtggcc ggggagccgg gacaagcgca ccctcgcctg cctgatccag 720
aacttcatgc ctgaggacat ctcggtgcag tggctgcaca acgaggtgca gctcccggac 780
gcccggcaca gcacgacgca gccccgcaag accaagggct ccggcttctt cgtcttcagc 840
cgtctagagg tgaccagggc cgaatgggag cagaaagatg agttcatctg ccgtgcagtc 900
catgaggcag ctagcccctc acagaccgtc cagcgagcgg tgtctgtaaa tcccggtaaa 960
ggatccgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1020
ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1080
tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1140
aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1200
gagcagtaca acagcacgta ccgggtggtc agcgtcctca ccgtcctgca ccaggactgg 1260
ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1320
aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1380
tcccgggatg agctgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1440
cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1500
acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1560
aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1620
aaccactaca cgcagaagag cctctccctg tctccgggta aatga 1665
SEQ ID NO 2
<210> 2
<211> 554
<212> PRT
<213> Artificial sequence
<220>
<223>
<400> 2
Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly
1               5                  10                    15
Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser
20                  25                    30
Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly
35                  40                    45
Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu
50                  55                    60
Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys
65                  70                    75
His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln
80                  85                    90
Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn
95                 100                   105
Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp
110                115                   120
Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp
125                130                   135
Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala
140                145                   150
Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln
155                160                   165
Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr
170                175                   180
Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His
185                190                   195
Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser
200                205                   210
Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu
215                220                   225
Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln
230                235                   240
Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu
245                250                   255
Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys
260                265                   270
Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr
275                280                   285
Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val
290                295                   300
His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser
305                310                   315
Val Asn Pro Gly Lys Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr
320                325                   330
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
335                340                   345
Ser Val Pro Leu Pro Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
350                355                   360
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
365                370                   375
Glu Asp Pro Glu Val Lys Pro Asn Trp Tyr Val Asp Gly Val Glu
380                385                   390
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
395                400                   405
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
410                415                   420
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
425                430                   435
Pro Ala Pro Ile Glu Lys Thr Ile Set Lys Ala Lys Gly Gln Pro
440                445                   450
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
455                460                   465
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Pro Tyr
470                475                   480
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
485                490                   495
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
500                505                   510
Pro Pro Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
515                520                   525
Gln Gly Asn Val Pro Ser Cys Ser Val Met His Glu Ala Leu His
530                535                   540
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
545                550                   554

Claims

1. A fusion protein, comprising SEQ ID 2 amino acid sequence protein, or is SEQ ID 2 amino acid sequence with a several amino acid remnant base substitution, the flaw or increases also has with SEQ ID 2 amino acid sequence same active by SEQ ID 2 derivation protein.

2. The fusion protein of claim 1 wherein the fusion protein comprising sequecing of SEQ ID 2

3. Code gene of a fusion protein, is one of following nucleotide sequences: DNA sequence of SEQ ID 1;DNA sequence of SEQ ID 2 of protein;With foreword tabulation in SEQ ID1 definition DNA sequence has 95% above homology, also code of DNA sequence same fusion protein, a function protein of SEQ ID 2.

4. The fusion protein of claim 3 wherein its characteristic in: States the fusion protein code gene which is in foreword tabulation SEQ ID 1.

5. The fusion protein of claim 3 wherein its express vectors, carriers, and the cell lines.

6. The fusion protein of claim 3 wherein expands to the right of any fragment derive from SEQ ID 1 or SEQ ID 2.

7. The method of fusion protein includes the code gene of fusion protein (SEQ ID2) clone into SP2/0, obtains the masculine clone, raises states the masculine clone, expression fusion protein FP4; States the fusion protein code gene is one of following nucleotide sequences: foreword tabulation SEQ ID 1 DNA sequence;foreword tabulation SEQ ID 2 multi-nucleotides protein sequence;With foreword tabulation in SEQ ID 1 definition DNA sequence has 95% above homology, also code same DNA sequence of function protein.

8. The fusion protein of claim 7 wherein its characteristic in: claim includes express carrier, vector of the code gene of fusion protein to express function proteinn protein; expression of a fusion protein (function protein) in carrier pSecTag.

9. The fusion protein of claim 1 wherein its fusion protein as the active constituent anti-allergic medicine.

10. The fusion protein of claim 1 wherein its fusion protein to treat in the anti-allergic medicine in the preparation the application.