CN1054399C - Human gene P53BP3 interacting with anti-cancer gene P53 - Google Patents

Abstract

The present invention relates to a human protein gene P53BP3 interacting with an anti-cancer gene coding product P53. The CNX of the human protein gene P53BP3 is composed of 5055 nucleic acids, and a coding sequence codes protein of 815 amino acids. The human protein gene P53BP3 is widely expressed in cell strains of normal tissues and tumours of a human body. The P53BP3 gene is positioned in a twelfth chromosome p11.2-12.1 section.

Description

A human gene P53BP3 who acts on mutually with antioncogene P53

The present invention relates to the human gene P53BP3 that acts on mutually with antioncogene coded product P53 and it in each tissue of human body expression and the location on karyomit(e), to explore the new way of diagnosing tumor and treatment.

P53 is a kind of coded product of antioncogene, it can the wide participation cell cycle, the regulation and control of genetic expression, dna replication dna and reparation, cytodifferentiation and growth, suppress the formation of tumour and keep genomic every physiological process (Levine such as stable, A.J., Cell, 1997,88:323-331).Its core link is exactly DNA and protein, and protein-protein interacts, and wherein the interaction between P53 and other rho factor is more important, has become the focus that people note.In the past few years, people have cloned gene (Goga, A.and Liu, X., Oncogene, 1995, the 11:791-799 that many coded products can act on mutually with P53; Walker, K.K.and Levine, A.J.Proc.Natl.Acad.Sci.USA, 1996,93:15335-15340; Wang, X.W.and Yeh, H.Nature genet, 1995,10:188-193; Maheswaran, S.and Englert, A.J.Genes Der.1995,9:2143-2156; Horikoshi, N.and Vsheva, A., Mol.Cell.Biol.1995 15:227-234.), studies on the one hand the constitutional features of these factors, seeks the action rule of they and P53, is devoted to study their function on the other hand.The research of this two aspect is that the regulatory mechanism of illustrating the every physiological process of P53 participation cell is laid a good foundation.

Normal P53 has antitumous effect, wherein the apoptosis process of P53 mediation is that P53 controls tumorigenic a kind of effective way (Symonds, H., et al.Cell, 1994,78:703-711), but the mutant of some P53 causes cell carcinogenesis owing to having lost normal function, and itself promptly has oncogenic function the mutant of other P53.In the human cancer of 50-55%, can detect the P53 gene sudden change (Hollsteim, M., Rice, K.et al.Nucleic Acid Res.1994,22:355l-3555).The interaction of research P53 and other rho factors helps to inquire into the pathogenesis of tumour, for the diagnosis and the treatment of tumour provides new imagination and scheme.

Yeast two-hybrid system is that development in recent years is got up a kind of study in vivo interactional system between the protein (Chien, C.T.et al.Proc.Acad.Sci.USA, 1991,88:9578-9582).The principle of this system is based on the reconstruction of a kind of transcription factor GAL4 function in the yeast.The GAL4 molecule contains two relatively independent functional domains: DNA land and transcriptional activation domain.Exist the desire checking gene of interactional two protein (X and Y) to merge with DNA land and the transcriptional activation domain of GAL4 respectively, again these two fusion expression vectors are changed in the special yeast cell, on this saccharomycetic karyomit(e), integrated and can have been discerned and bonded site and downstream reporter gene (HIS3 or LacZ) by GAL4 DNA land, if interact between two protein, reporter gene just can be expressed.Protein Y is gene library also, just can therefrom screen the interactional new protein gene with protein X.

For this reason, the purpose of this invention is to provide a kind of human gene P53BP3 full length cDNA sequence that can act on mutually with P53, and analyze it in the expression level of 16 kinds of health adult tissues and 7 kinds of tumor cell lines and the location on karyomit(e).

The present invention is a kind of human gene P53BP3 that acts on mutually with antioncogene P53, it is according to above situation, utilize P53 to merge the DNA land of GAL4, as protein X, the transcriptional activation domain that merges GAL4 with the HeLa cell cdna library is protein Y, has screened a human gene P53BP3.Angled the full-length cDNA fragment through in-situ hybridization method, measured complete sequence, and carried out chromosomal localization and the Northern hybridization analysis in 16 kinds of health adult tissues and 7 kinds of tumor cell lines, for the action principle of research P53 has been opened up new approach.

The present invention is target molecule with P53, utilize the two-way crossing system of yeast from the HeLa cell cdna library, to screen a plurality of positive colonies, measure the cDNA sequence of each positive colony, one of them length is that the cDNA sequence of 1312bp is not found homologous sequence through the gene data library searching, 437 amino acid whose peptide sections of this cDNA sequence encoding (Fig. 1).The result of Northern hybridization shows that this full length gene mRNA is about 5.0kb.CDNA fragment with above-mentioned 1312bp is a probe, obtains 4.0kb and 1.8kb cloned sequence (Fig. 2) from HeLa cDNA library again.After measuring nucleotide sequence, they can be spliced into the full-length cDNA (Fig. 3) of 5055bp.Wherein 5 ' non-translational region is 2046bp, and the coding region is 2445bp, the protein that 815 amino acid of encoding are formed, and 3 ' non-translational region is 564bp.

There is a leucine zipper structure (63-84) in the coded product N petiolarea of this gene, there is a nuclear localization sequence (386-402) at the middle part, have 4 PEST sequence Fu Ji districts at the nearly N of molecule end and nearly C end in addition, show this albumen comparatively fast in the endocellular metabolism turnover, stability a little less than.It may be a kind of DNA conjugated protein (Fig. 4).Through protein database search, do not find and other proteic homologys that this gene is " orphan " gene, and is still undiscovered with other genes of family.

Carry out the Northern hybridization analysis of 16 kinds of mRNA of health adult tissue, the result shows this gene wide expression (Fig. 5) in various tissues.Wherein in testis tissue, express the highest, placenta, heart and thymus gland take second place, certain expression is all arranged in brain, liver, kidney, spleen, skeletal muscle, ovary, small intestine, colon, prostate gland, pancreas and peripheral blood leucocyte, in lung tissue, then express very weak.Carry out Northern hybridization analysis (Fig. 6) with 7 kinds of total RNA of human tumor cell line, the result shows that the expression of this gene in teratoma and lymphoma cell strain is high at the cell strain of uterus carcinoma, mammary cancer, liver cancer and neurogliocytoma, then do not detect expression in lung cancer cell line.

The present invention has also carried out the chromosomal localization analysis of P53BP3 gene, is probe with P53BP3, with the human lymphocyte karyomit(e) hybridization of exhibition layer on slide glass.Write down FISH signal and DADI signal in the same visual field simultaneously, the two contrast can determine that the P53BP3 assignment of genes gene mapping is in No. 12 chromosomal p11.2-12.1 section (Fig. 7).

Advantage of the present invention:

1, a kind of cDNA sequence of brand-new P53BP3 gene is provided, and its coded product can act on mutually with the product P 53 of antioncogene, and is significant in the diagnosis of tumour and treatment.

2, P53BP3 encoded protein product contains leucine zipper structure and nuclear localization sequence, may be a kind of transcriptional regulator, might become engineered product.

3, P53BP3 wide expression in 16 kinds of health adult tissues shows the tool critical function effect in cell of this gene, can be used as diagnosis index.

4, the present invention with the P53BP3 assignment of genes gene mapping at No. 12 chromosomal p11.2-12.1 sections, should carry out the linkage analysis of gene on the karyomit(e), be applied to the diagnosis of disease.

The present invention is further elaborated by the following drawings and embodiment, but does not limit the scope of the invention.

Description of drawings:

Fig. 1. reach the encoding amino acid sequence of releasing thus with the P53BP3 Partial cDNA nucleotide sequence that P53 acts on mutually

The point of contact of segmental clone of Fig. 2 .P53BP3 cDNA and restriction enzyme

Nucleotide sequence among the figure in the 2723-4007 representative graph 1

1-3943 is 5 ' end parts cDNA sequence

3247-5055 is 3 ' end parts cDNA sequence

Fig. 3 .P53BP3 cDNA complete nucleotide sequence and amino acid sequence coded

The aminoacid sequence of Fig. 4 .P53BP3 gene encoding production

The L that slightly writes among the figure represents the leucic position in the leucine zipper structure

Two-wire partly is a nuclear localization sequence

Single line partly is the PEST sequence

Fig. 5. with P53BP3 is probe, the Northern hybridization of people's different tissues mRNA

Among the figure: 1. heart 2. brains 3. placentas 4. lungs

5. liver 6. skeletal muscle 7. kidneys 8. pancreases

9. spleen 10. thymus gland 11. prostate glands 12. testis

13. ovary 14. small intestines 15. colons 16. peripheral blood leucocyte

Fig. 6. with P53BP3 is probe, the Northern hybridization analysis of the total RNA of different tumour cells

Among the figure: 1. lung cancer (A549) 2.HeLa 3. liver cancer (HepG2)

4. 5. neurospongioma (U251MG) of mammary cancer (MCF-7)

6. 7. teratoma (PA-1) of lymphoma (JurKatt)

The location of Fig. 7 .P53BP3 gene on karyomit(e)

Among the figure: the arrow indication is the fluorescence location of P53BP3 on karyomit(e) among the A, and B is No. 12 karyomit(e)s under the same visual field, and C is No. 12 chromosomal mode charts, and stain is represented the position of P53BP3 on No. 12 karyomit(e).

The two-way crossing system screening of embodiment 1 yeast P5 3BP3 cDNA fragment

P53 is combined with GAL4 DNA among territory fusion expression plasmid and HeLa cell cdna library and GAL4 transcriptional activation domain fusion expression plasmid (all available from Clontech company) the cotransformation yeast HF7c.Is the nutrition selection markers of albumen plasmid the TRP1 gene, the nutrition selection markers of library plasmid is LEU2, if mutually combine between target protein and the detected albumen, just activate the expression of reporter gene HIS3 among the HF7c, so cell just can be grown on the minimum medium that lacks Leu, Trp and His.1.5 * 10 ⁶Individual transformant is (at L ^-And W ^-On the flat board) in obtain 35 energy at SD (W ^-L ^-H ^-) transformant of growth on the flat board.In order to get rid of the false positive clone, measure these clones' galactoside enzyme activity, find only to have 6 to be cloned on the X-gal flat board apparent blue.Positive transformant is inoculated in the nutrient solution that lacks Leu cultivates, extract plasmid DNA, transformed into escherichia coli, refabrication plasmid DNA.In this plasmid and target protein plasmid transformed yeast SFY526, on the X-gal flat board, all show blue, showing has the galactoside enzyme activity, proves true positive clone.CDNA library plasmid in 6 positive colonies is carried out analysis of Restriction Endonuclease Profile and partial dna sequence respectively to be measured, one of them cDNA fragment length is 1.3kb (Fig. 1), in gene library, do not inquire homologous sequence, its commissarial new gene, and called after P53BP3.

Embodiment 2 P53BP3 Full Length cDNA Cloning and order-checkings

Above-mentioned 1.3kb cDNA fragment is cut to 850bp and 450bp fragment with EcoRI/XbaI and XbaI/XhoI enzyme respectively, the method of recommending by random primer labelling test kit (Boehringer Mannheim company) is carried out isotopic labeling respectively, screening hybridization clone from HeLa cell cdna library (Clontech company), the benton-Davis hybridization method is undertaken by cDNA library specification sheets recommend method.Through the two-wheeled screening, obtain 13 positive colonies.Each clone is carried out restriction enzyme digestion to be identified, two clones wherein, length is respectively 4.0kb and 1.8kb, can be spliced into the P53BP3 full-length cDNA (Fig. 2) of 5.0kb, according to the point of contact of the restriction enzyme of Fig. 2, make the endonuclease bamhi of different lengths, be cloned into respectively among M13mp18 and the M13mp19 (available from BRL company), measure dna sequence dna with two deoxidation cessation method.P53BP3 cDNA total length 5055bp, 5 ' non-translational region is 2046bp, and the coding region is 2445bp, and 3 ' non-translational region is 564bp (Fig. 3).

Embodiment 3 P53BP3 are at the expression analysis of people's healthy tissues and tumor cell line

The nylon membrane (Clontech company) of 16 kinds of total mRNA of healthy tissues of people will be fixed with, put in people's hybrid pipe, the quick hybridization liquid (Clontech company) that adds 68 ℃ of preheatings of 5ml, 68 ℃ of prehybridizations 30 minutes, the 1.3kb fragment that adds the isotope-labeled P53BP3 of sex change was hybridized 2 hours for 68 ℃.Take out hybrid pipe and wash film, 2 * SSC, room temperature is 10 minutes among the 0.05%SDS, 0.1 * SSC, 0.1%SDS washes twice for 50 ℃, totally 30 minutes.Film placed on the filter paper dry, compressing tablet promptly gets Fig. 5.

The preparation of the total RNA of tumor cell line, electrophoresis and commentaries on classics film, all the method for introducing by " molecular cloning experiment guide " is carried out (Sambrook, J., Fritsch, E.F., and Maniatis, T.MolecularCloning, Cold Spring Harbor Laboratory Press, 1989), the same with probe hybridization, can get Fig. 6.

The location of embodiment 4 P53BP3 on karyomit(e)

Isolated lymphocytes from human blood is incubated in the minimum medium (a-MEM) that contains 10% foetal calf serum and PHA, handles with BrdU after 68-72 hour, makes cell synchronization.With the nutrient solution washed cell of serum-free 3 times, cultivated 6 hours with the a-MEM nutrient solution that contains thymus pyrimidine more then.Collecting cell is also coated on the slide glass, makes cell rupture through hypotonic processing, and dry in air fixing back.Handle the slide glass that is loaded with cell lysate with Rnase, use 70% formaldehyde/2 * SSC again, make the chromosomal DNA sex change, use ethanol dehydration then in 70 ℃ of processing.With biotinylated dATP mark P53BP3 cDNA fragment, the probe of mark is joined in the hybridization mixed solution 75 ℃ of sex change 5 minutes with the BioNickLabeling Kit of BRL company.This hybridization mixed solution also comprises 50% formaldehyde, 10% T 500 and people Cotl DNA.The hybridization mixed solution is added on the slide glass that is loaded with the sex change chromosomal DNA 37 ℃ of incubated overnight.After the hybridization, mixed solution on the flush away slide glass, detect hybridization signal with enzymatic reaction, remember FISH signal (A among Fig. 7) and DAP1 signal (B among Fig. 7) in the same visual field simultaneously, can determine that P53BP3 is positioned in the section of No. 12 chromosomal p11.2-12.1, shown in the C among Fig. 7 after the two contrast.

Claims (1)

1, a kind of human body protein P53gene BP3 that acts on mutually with antioncogene coded product P53; ,P53BP3DNA：-2046 GAATTCTACTCGGTGGTTTCCCTCTGTAGGGTAGTTGGGAGCTTTGGAAGGATGTATGGG-1986 CCTGGAGGTACAAGCTCCAAGCCCTTTAGCCAACCAGTTGTTGACGCCCTGGGGCGCAGC-1926 ATCATAGGCTGTATGAGGAGAGTAGATACCGACTCTGTTCTCCAGAGCCCGGATCACCAG-1866 GCGCTCCTTCCATGTGTTCCAGGTTATGCGCTTCATGGGTCGGAAGATAGGCGGATGGTA-1806 GGAGAGAATGAGGTCTGCCTTCTTTTGCAGCACCTCCTCCATCACTTCCTCAGTCAGGTC-1746 ATTGGTCAGGAAGAGTGTATTTACAGTATGTGGTGGGCTTGGTTCCACCAGTAATCCAAC-1686 ATTGTCCCAACTCTCAGCAAACGAGAGGGATGCAAAGTCATTCAAGGAAGAAAGGAGAGC-1626 CTTCAAATCCATGAAGGAACGGGAAGAATTGCAGATCAGGGAATCTACAAACCGGACTGT-1566 CGTGGGGACTGGGCGTACGCAaGATGACAACATACAGAAATCAGGTAAAGTTCAAGTTTC-1506 TGATGTCAAAACAAAGAAAATGGTACACAGGTATACTTTAGGCCTGGATCTTTGAGGCAT-1446 CAAAACATATGGGCAAAAGAGCTGTCGTCATAGAGATCTGGGTTCAAATAGTGGCTCTGC-1386 CACTTACCAGCACACTGTAACTTAGGTAAGCCATTTAACCTCTCCAATCTTGTTTTCTCA-1326 CCTACGAAACAGAGATACTATAACCCACTTCACAGGCCATCCAGAGGAGCTAATGAATTA-1266 TGTAACCTAGCAAAGCCTGGTACATAATACACAAACGTCCATCCCATCTCCTGCCTTGCA-1206 CACCCTTGTTCTTATTCTCCCTACCCTTGCTTCAACCAACCTCCTCCCTAATCCATTTCT-1146 CTAAATATTACCTTTCCTTTTCCCAAGCCCCTCCAACTTCTTAAATAAAACTTTTCTTGA-1086 CCTCCCTTTTCAGCATCCTTCCCAAGGCTCCCCACCCCTCAGTATTCTCATAATTCTGTG-1026 TGTGCCCATTATGCTAAAGCTACTATGTAATAGGAACTAACTTCAATAAATCTCAGTATC-966 ACTAATATAAGCCAGCACTAAATGCTTACTGCATAGAATGACACTGCCCGCCTCCCCTGA-906 TTGCAACAGAATTTGGAGTTGGGGTGGTATATACTGGCATTACAATGTGCTTTACCAAAA-846 AACAGAATAATGTGGCGGGGCGCCTGTAATCCCGCGGCCGCGTCGACATAGGGCCTTTCC-786 GCCTGCGGGCCCAGTGAGTCGACACGGTGGGGGCCCGCGATCCCCGGGACTTACCACTGA-726 ATAAATCCAGTCCAGCGTGCGACGACTTACTGGCTTCATGGGGTCGCAGCCGCCGCTGGG-666 GTCTCCGCTGTCTCGCGAGGAGGGTGAAGCGCCCCGCCTGCTCCCGCTTCGGAGGGTAGG-606 CGGAGAAGTCGCCGGGTACGCCTTCGCGGATCCTGCCGACACCGACCTAGCTTTCTGGGC-546 TGCCGGGAGCTCGCGGCGAGCGCCCCAGCCAGGCCTGCGCCGGCATCCTCCGAGATAATG-486 GCATCAGCTGCTAAGGAATTTAAAATGGACAACTTTTCACCTAAAGCTGGCACTAGCAAA-426 TTGCAACAGACAGTACCAGCTGATGCATCTCCTGATTCTAAGTGTCCTATATGCTTGGAT-366 AGATTTGATAATGTGTCTTACTTAGATCGCTGCTTACATAAGTTCTGAAAACGCTGTGTA-306 CAGGAGTGGTCAAAAAACAAAGCTGAATGCCCACTATGTAAACAGCCCTTTGATTCTATT-246 TTCCATTCTGTGAGGGCAGAAGATGACTTCAAGGAGTATGTCCTAAGGCCTTCGTATAAT

(follow-up)-186 GGTTCTTTTGTCACCCCTGATCGACGATTTCGCTACCGTACAACTCTGACAAGGGA ACGA-126 AATGCTTCTGTGTATTCACCTAGTGGTCCTGTGAACAGAAGAACAACAACTCCACC GGAT-66 AGTGGAGTACTGTTTGAAGGGTTAGGCATTTCAACAAGACCTAGAGATGTTGAAAT TCCT-6 CAGTTTATGAGACAGATTGCAGTAAGGAGGCCAACTACGGCAGATGAAAGATCTTT GCGG

M R Q I A V R R P T T A D E R S L R 55 AAAATTCAAGAACAAGATATTATTAATTTTAGACGAACTCTTTATCGTGCTGGTGCTCGA

K I Q E Q D I I N F R R T L Y R A G A R 115 GTTAGAAATATTGAAGATGGTGGCCGCTACAGGGATATTTCAGCTGAATTTTTCCGTAGA

V R N I E D G G R Y R D I S A E F F R R 175 AATCCAGCTTGCCTTCACAGATTAGTCCCCTGGTTAAAACGTGAACTTACAGTTCTTTTT

N P A C L H R L V P W L K R E L T V L F 235 GGAGCTCATGGATCTTTAGTGAATATTGTCCAGCATATTATCATGAGTAATGTTACTCGC

G A H G S L V N I V Q H I I M S N V T R 295 TATGACTTGGAGAGTCAGGCATTTGTGTCTGATTTAAGACCATTTTTACTTAATCGAACT

Y D L E S Q A F V S D L R P F L L N R T 355 GAGCATTTTATACATGAATTTATCAGTTTTGCCCGATCTCCTTTTAACATGGCAGCCTTT

E H F I H E F I S F A R S P F N M A A F 415 GACCAGCATGCCAATTATGATTGCCCTGCTCCTTCATACGAAGAAGGCAGCCATTCTGAT

D Q H A N Y D C P A P S Y E E G S H S D 475 TCTTCAGTCATAACAATATCTCCAGATGAGGCTGAGACCCAAGAGCTGGATATTAATGTA

S S V I T I S P D E A E T Q E L D I N V 535 GCCACTGTTAGTCAGGCACCATGGGATGATGAAACTCCAGGACCATCTTACTCAAGCTCA

A T V S Q A P W D D E T P G P S Y S S S 595 GAGCAGGTACACGTTACTATGTCTTCTCTTTTAAATACTTCTGACAGTTCAGATGAAGAA

E Q V H V T M S S L L N T S D S S D E E 655 CTTGTCACAGGAGGAGCCACGTCTCAGATACAAGGAGTACAAACCAATGACGACCTAAAT

L V T G G A T S Q I Q G V Q T N D D L N 715 AATGACAGTGATGATTCTTCAGATAATTGTGTCATTGTTGGGTTTGTTAAACCACTAGCT

N D S D D S S D N C V I V G F V K P L A 775 GAGAGGACCCCAGAACTTGTTGAACTGTCCTCTGATTCTGAGGACTTAGGTTCTTATGAG

E R T P E L V E L S S D S E D L G S Y E 835 AAAATGGAGACAGTGAAGACACAAGAACAGGAGCAATCTTACAGTTCTGGTGATAGCGAT

K M E T V K T Q E Q E Q S Y S S G D S D 895 GTTAGTAGATGCTCATCTCCACACTCTGTCCTTGGAAAGGATGAACAAATAAATAAAGGT

V S R C S S P H S V L G K D E Q I N K G 955 CATTGTGATTCTAGTACAAGAATCAAATCAAAGAAGGAAGAGAAACGATCTACATCATTG

H C D S S T R I K S K K E E K R S T S L?1015 TCATCTCCCAGAAACCTGAACTCATCTGTAAGAGGAGACAGAGTATATTCTCCATATAAC

S S P R N L N S S V R G D R V Y S P Y N?1075 CATAGACACAGAAAGAGGGGAAGATCAAGAAGTTCAGATTCACGTTCTCAGAGTAGAAGT

H R H R K R G R S R S S D S R S Q S R S?1135 GGGCATGATCAGAAGAATCATAGAAAGCATCATGGGAAGAAAAGAATGAAAAGTAAACGA

G H D Q K N H R K H H G K K R M K S K R?1195 TCCAGAAGCAGGGAAAGTAGCAGACCTAGAGGGAGAAGAGACAAAAAGAGATCAAGAACT

S R S R E S S R P R G R R D K K R S R T?1255 AGAGATAGCAGTTGGTCCAGAAGAAGCCAAACTCTGTCTCTAAGTAGTGAAAGCACAAGC

R D S S W S R R S Q T L S L S S E S T S?1315 AGATCAAGGTCTCGTAGCAGTGATCATGGTAAAAGAAGATCACGGAGCAGAAATAGAGAT

R S R S R S S D H G K R R S R S R N R D?1375 CGTTATTATTTAAGAAATAATTATGGAAGCAGATACAAATGGGAGTATACTTATTACAGT

R Y Y L R N N Y G S R Y K W E Y T Y Y S

(follow-up) 1435 AGAAACAAGGACAGGGATGGGTACGAATCATCTTACAGGAGGAGGACTCTGTCCAG AGCT

R N K D R D G Y E S S Y R R R T L S R A1495 CATTATTCTAGACAGTCTTCAAGTCCAGAATTTAGAGTTCAGTCCTTTTCTGAAAGAACA

H Y S R Q S S S P E F R V Q S F S E R T1555 AATGCTAGGAAAAAAAATAATCACAGTGAGAGGAAGTATTACTACTATGAAAGGCACAGA

N A R K K N N H S E R K Y Y Y Y E R H R1615 TCAAGGAGCCTGTCTAGTAACAGATCAAGGACTGCATCTACCGGGACTGACCGGGTGAGA

S R S L S S N R S R T A S T G T D R V R1675 AATGAAAAGCCTGGAGGGAAACGAAAATACAAAACACGGCATTTGGAGGGTACTAACGAA

N E K P G G K R K Y K T R H L E G T N E1735 GTGGCTCAGCCATCTCGTGAATTTGCTTCTAAAGCAAAGGACAGTCATTACCAAAAATCT

V A Q P S R E F A S K A K D S H Y Q K S1795 TCATCAAAATTGGATGGAAACTACAAAAATGAGAGTGATACCTTTTCAGACAGCCGATCA

S S K L D G N Y K N E S D T F S D S R S1855 TCAGACAGAGAGACAAAACACAAAAGGAGAAAAAGGAAGACCCGGAGCCTAAGTGTAGAG

S D R E T K H K R R K R K T R S L S V E1915 ATAGTTTATGAAGGAAAAGCTACTGATACAACTAAACACCATAAAAAGAAAAAGAAGAAA

I V Y E G K A T D T T K H H K K K K K K1975 CATAAGAAGAAGCATAAGAAACACCATGGAGATAATGCTTCACGTTCCCCAGTTGTAATT

H K K K H K K H H G D N A S R S P V V I2035 ACCATTGACAGTGACAGTGATAAGGATTCTGAAGTAAAGGAGGATACAGAATGTGACAAT

T I D S D S D K D S E V K E D T E C D N2095 AGTGGTCCTCAAGACCCTCTACAAAATGAGTTTTTGGCTCCTTCCTTGGAACCATTTGAA

S G P Q D P L Q N E F L A P S L E P F E2155 ACTAAAGATGTAGTTACAATAGAAGCTGAATTTGGTGTGCTGGACAAGGAATGTGATATT

T K D V V T I E A E F G V L D K E C D I2215 GCCACACTTAGTAACAACTTGAATAATGCCAACAAAACTGTAGATAATATTCCACCTCTG

A T L S N N L N N A N K T V D N I P P L2275 GCAGCTTCAGTTGAACAAACTCTCGATGTAAGAGAAGAGAGCACCTTTGTTTCTGATTTG

A A S V E Q T L D V R E E S T F V S D L2335 GAGAACCAGCCCAGTAACATTGTGTCTCTTCAAACTGAGCCATCAAGGCAATTGCCATCG

E N Q P S N I V S L Q T E P S R Q L P S2395 CCACGGACATCATTAATGTCAGTATGTCTTGGTAGAGACTGTGATATGTCTTAAAACTGC

P R T S L M S V C L G R D C D M S *2455 CAAAGCATTTCATTGAGAATTATGATGTTATAAAAAGGAAAAAGGAAGAATGTCGTCTAC2515 TGCAGTCTATTTAAAGATGACATTTGGTGAAAACTCTCTTCCTCCTTACAATATTTTAAA2575 TGATTTTTTTTTTGGTGTTAATTTGTAAAAATCATTATTTGTTCAAAATGTATGTCCCAC2635 CCTCAAAGATATGCACTTTTAAGTGAAGAAAATGATACTGCTAGCAGCTTATTTAAAACT2695 TGGGGTCCTTTTTAAATAAGAAAAATTATATAAATTTTAGAAGTTATTTCATAAAGCCAT2755 ACGGTATTGACATATTTTTAAGGTAGTCAATGAGTATTTTTGAATTTTTTTTTTTTGAGA2815 GTTATTCTGGAAATGTGTTATAAGCTAGGAGAATCCCTTTGGACAGTCTTTATTTTTCTT2875 CTTAAAAATTTATATGATTCAAAACCATTTCTTCAGGTTAAATTGAGGCATTTTAATCTG2935 CACAGTTTATCTTCTGCCAAAATAAAAATTTACTATTTCCTTTATATACTTGTTTATCTG2995 GACTGCCAGTAAAAC

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