CN114249835B - RXFP1 fusion protein, cell expressing RXFP1 fusion protein and application of RXFP1 fusion protein in activity determination of Relaxin-2 - Google Patents

Abstract

The invention discloses a RXFP1 fusion protein, a cell expressing the RXFP1 fusion protein and application of the RXFP1 fusion protein in the determination of the activity of Relaxin-2, wherein an EGFP fluorescent label is connected with RXFP1 receptor protein through flexible connecting peptide, and the amino acid sequence of the flexible connecting peptide is shown as SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3. The invention also relates to a cell expressing the RXFP1 fusion protein, which can detect the bioactivity of Relaxin-2 and has high sensitivity, good specificity, strong operability and good application prospect.

Description

RXFP1 fusion protein, cell expressing RXFP1 fusion protein and application of RXFP1 fusion protein in activity determination of Relaxin-2

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to RXFP1 fusion protein, a cell expressing the RXFP1 fusion protein and application of the RXFP1 fusion protein in determination of Relaxin-2 activity.

Background

As genetic engineering techniques mature and are applied, two or even a plurality of different genes can be easily connected in series or embedded into each other to form a chimeric gene, and once expressed into a product, a fusion protein is obtained. In structural biology, the construction of fusion proteins has become a very common and efficient method, and one of the methods for constructing fusion proteins is to link constituent proteins by means of a linker peptide. The constituent proteins are selected according to the function of the desired fusion protein product and are in most cases relatively simple. Selection of an appropriate linker peptide is difficult and can easily be overlooked during the design of the fusion protein. Direct fusion of functional domains without a linker peptide can lead to undesirable results such as misfolding of the fusion protein, low yields or impaired activity. The choice of linker peptide sequence is therefore particularly important in order to link the constituent functional proteins, and the folding of the entire linker peptide must be taken into account in addition to the necessary appropriate amino acid composition. Therefore, rational design and selection of the linker peptide is important for the construction of fusion proteins.

A variety of linker peptides have been found to be useful in the construction of fusion proteins, and are largely classified into three groups according to the function of the linker peptide: flexible linker peptides, rigid linker peptides and in vivo cleavable linker peptides, wherein flexible linker peptides are generally selected when certain interactions between functional domains in the linked constituent proteins are required. Flexible linker peptides are generally composed of small non-polar or polar amino acids, since small amino acids are more flexible and can allow the functional domains of the linker component proteins to function better without interfering with each other. However, improper selection of the length of the flexible linker peptide and its constituent amino acids and arrangement may affect the function of the constituent proteins, and may also affect the complete and stable expression of the entire fusion protein in the recombinant engineered cells.

Relaxin (Relaxin) is a reproduction-associated endocrine hormone first discovered by FederiskHisaw and is so named because it has the effect of relaxing the birth canal of female animals, and has a double-stranded structure similar to insulin. The human Relaxin family discovered to date has 7 polypeptides, among which Relaxin-2 is the major Relaxin family peptide in the blood circulation.

In recent years, the role of Relaxin-2 has been emphasized in many aspects: reducing blood vessel tension, increasing blood flow of heart and kidney, improving elasticity of artery and promoting wound healing; ② inhibiting cardiac muscle cell hypertrophy and inhibiting fibroblast activation and proliferation; thirdly, myocardial ischemia and reperfusion injury are resisted, and arrhythmia is reduced; softening reproductive tract tissues and maintaining normal pregnancy and delivery; regulating follicular development and stimulating mammary gland growth and differentiation; sixthly, the serum biomarker can be used as a novel serum biomarker and the like. Relaxin-2 plays a pharmacological role in acting on an endogenous receptor RXFP1(Relaxin familinepetide receptor1) and stimulating the generation of a second messenger cAMP (cyclic adenosine monophosphate), thereby performing a series of regulation and control in cells. Therefore, based on the potential value of Relaxin-2 in the aspect of cardiovascular disease treatment and the like, a method for sensitively and efficiently measuring the activity of Relaxin-2 in vitro is needed so as to evaluate the effectiveness of Relaxin-2 in disease treatment.

Currently, the drug detection of Relaxin-2 generally needs to be in the range of about 10-100 ng/mL, and the currently commonly used method for detecting the biological activity of Relaxin-2 mainly comprises an in vivo mode and an in vitro mode, wherein the in vivo detection method comprises an identification method for inhibiting uterine contraction, a pubic ligament relaxation method and the like, and an activity unit is calculated according to the dosage of Relaxin. The methods have large errors, are not suitable for large-scale measurement, and the sensitivity does not reach the measurement standard of the prior pharmacodynamic pharmacokinetics and quality control. In vitro determination methods are based on liquid chromatography-tandem mass spectrometry to indirectly determine the biological activity of relaxin, but the methods are complex to operate, time-consuming and costly. Therefore, it is highly desirable to develop a rapid, reliable and sensitive method for determining the biological activity of Relaxin-2.

Disclosure of Invention

The invention aims to provide the RXFP1 fusion protein, the designed flexible connecting peptide is used for connecting the RXFP1 protein and the EGFP label protein, and the recombinant engineering cell expressing the RXFP1 fusion protein is constructed to detect the biological activity of Relaxin-2, so that the fusion protein has the advantages of high sensitivity, good specificity, strong operability and good application prospect.

The invention adopts the following technical scheme:

in a first aspect, embodiments of the present invention provide a Flexible Connecting Peptide (FCP), an amino acid sequence of the FCP is shown in SEQ ID No.1, SEQ ID No.2, or SEQ ID No. 3.

Preferably, the amino acid sequence of the flexible connecting peptide is shown as SEQ ID No. 1.

In a second aspect, the embodiment of the present invention provides an RXFP1 fusion protein, wherein an EGFP fluorescent tag is connected to an RXFP1 receptor protein through a Flexible Connecting Peptide (FCP), and an amino acid sequence of the RXFP1 fusion protein is shown as SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.

Preferably, the amino acid sequence of the RXFP1 fusion protein is shown in SEQ ID No. 4.

In a third aspect, the embodiment of the invention provides a coding gene of RXFP1 fusion protein, and the nucleotide sequence is shown in SEQ ID No. 7.

In a fourth aspect, embodiments of the invention provide a recombinant expression vector comprising a gene encoding a RXFP1 fusion protein.

In a fifth aspect, embodiments of the invention provide a recombinantly engineered cell comprising a gene encoding a RXFP1 fusion protein.

In a sixth aspect, the embodiment of the invention provides an application of a recombinant engineering cell containing a coding gene of RXFP1 fusion protein in the determination of the activity of Relaxin-2.

The invention has the beneficial effects that:

the design of flexible connecting peptide solves the problem that the activity of receptor protein is influenced by the tag protein in the traditional construction.

Secondly, the addition of flexible connecting peptide can fully expose the ligand binding site and G protein binding site of RXFP1 protein, so that the effect of the action of Relaxin-2 and RXFP1 is fully displayed.

The addition of the fluorescent label is convenient for screening the recombinant engineering cells and detecting the activity of the recombinant engineering cells after the cells are frozen for a long time.

The recombinant engineering cell can be used for measuring the biological activity of the Relaxin-2 sample in a short time, does not need expensive instruments such as a mass spectrometer and the like, has the characteristics of high sensitivity, good specificity, strong operability, convenience in carrying out detection with large range and high flux, low cost and the like, has good application prospect, and is suitable for popularization and application.

Drawings

FIG. 1 is a standard curve fitted with the concentration of cAMP standard as the abscissa and the OD value corresponding to each concentration as the ordinate.

FIG. 2 is a curve fitted by taking the concentration of Relaxin-2 sample as abscissa and taking OD value of cAMP correspondingly generated by recombinant engineering cells expressing EGFP-FCP-RXFP1-1 protein as ordinate.

FIG. 3 is a curve fitted by taking the concentration of Relaxin-2 sample as abscissa and taking OD value of cAMP correspondingly generated by recombinant engineering cells expressing EGFP-FCP-RXFP1-2 protein as ordinate.

FIG. 4 is a curve fitted by taking the concentration of Relaxin-2 sample as abscissa and taking OD value of cAMP correspondingly generated by recombinant engineering cells expressing EGFP-FCP-RXFP1-3 protein as ordinate.

FIG. 5 is a sigmoidal dose-dependent curve of the biological activity of Relaxin-2 versus the concentration of cAMP.

FIG. 6 shows Western Blot to identify expression patterns of recombinant engineered cells expressing EGFP-FCP-RXFP1-3 protein and RXFP1 in HEK293 cells, 1, 2: a recombinant engineered cell protein sample group expressing the EGFP-FCP-RXFP1-3 protein; 3. 4HEK293 cell protein sample group.

FIG. 7 shows the expression of green fluorescent protein observed in HEK293 cell culture monoclone after virus infection for 8-9 days.

FIG. 8 shows the green fluorescent protein expression on cell membrane of the recombinant engineered cell strain HEK293/RXFP 1.

FIG. 9 shows the expression of green fluorescent protein on cell membrane of recombinant engineered cell line HEK293/RXFP 1.

For a person skilled in the art, without inventive effort, other relevant figures can be derived from the above figures.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

Examples

1. Design and selection of flexible connecting peptide

The invention provides a method for designing and selecting a flexible connecting peptide sequence. Specifically, 3 sequences are designed according to the design principle of the flexible connecting peptide, and the quality of the flexible connecting peptide is judged according to the pharmacodynamic standard, namely the activity detection of Relaxin-2: relaxin-2 stimulates the recombinant engineered cell RXFP1/HEK293 and the biological activity of Relaxin-2 is analyzed by the cAMP production amount. Finally, a suitable Flexible Connecting Peptide (FCP) for connecting the EGFP and RXFP1 proteins was identified.

1.1 design and Synthesis of EGFP-FCP-RXFP1

First, the polypeptide chains of both EGFP and RXFP1 should not interact and fold together to lose activity of each other; secondly, the active centers of EGFP and RXFP1 are far away from each other, so that steric hindrance is not formed to influence the expression of the activity, and the activity cannot be influenced or even inhibited; third, the flexible linker peptide sequence is too long, and the synergy of the two proteins may be reduced, or too short, which may affect the activity of the proteins. Only the suitable flexible connecting peptide can connect two proteins into a macromolecule, and the two proteins can not be influenced to keep respective functions, and the convenience of protein expression, purification, renaturation and the like can also be considered.

On the basis, 3 kinds of flexible connecting peptides are designed and synthesized in a whole gene way, and the sequence is SEQ ID No. 1: GGGTGRSGGG, SEQ ID No. 2: GSSSTGRQSSSG, SEQ ID No. 3: GGGGEQGRGAVGGG are provided.

Correspondingly, three RXFP1 fusion proteins are obtained, and the amino acid sequences of the RXFP1 fusion proteins are as follows:

(1)EGFP-FCP-RXFP1-1(SEQ ID No.4)：……GSSSTGRQSSSG……。

since the EGFP and RXFP1 sequences are known sequences, they are shown here in simplified form by the underlined sequence shown in SEQ ID No. 2.

Wherein, the underlined part is a sequence shown in SEQ ID No. 1.

(2)EGFP-FCP-RXFP1-2(SEQ ID No.5)：……GSSSTGRQSSSG……。

Wherein, the sequences of EGFP and RXFP1 are the same as those in SEQ ID No.4, and the representation is omitted here. The only difference is in the flexible linker peptide, the underlined part is the sequence shown in SEQ ID No. 2.

(3)EGFP-FCP-RXFP1-3(SEQ ID No.6)：……GGGGEQGRGAVGGG……。

Wherein, the sequences of EGFP and RXFP1 are the same as those in SEQ ID No.4, and the representation is omitted here. The only difference is in the flexible linker peptide, the underlined part is the sequence shown in SEQ ID No. 3.

Correspondingly, the nucleotide sequences of the corresponding coding genes of the three RXFP1 fusion proteins are as follows:

(a) the nucleotide sequence of the coding gene corresponding to EGFP-FCP-RXFP1-1 is shown in SEQ ID No.7, and the nucleotide sequences corresponding to EGFP and RXFP1 are known sequences and are briefly shown as follows: … …ggcggcggcaccgggcgcagcggcggcggc……。

(b) The nucleotide sequence of the coding gene corresponding to EGFP-FCP-RXFP1-2 is shown as SEQ ID No. 8: … …g gcagcagcagcaccgggcgccagagcagcagcggc … …. Nucleotide sequences corresponding to EGFP and RXFP1 are omitted here.

(c) EGFP-FCP-RXFP 1-3: the nucleotide sequence of the corresponding coding gene is shown as SEQ ID No. 9: … …ggcggcggcggggagcaggggcgcggggccgtgggcggcggc… … is added. The nucleotide sequences representing the correspondence of EGFP and RXFP1 are omitted here.

1.2 construction of pLJM1-EGFP-FCP-RXFP1 recombinant plasmid

According to the DNA sequences of EGFP-FCP-RXFP1-1, EGFP-FCP-RXFP1-2 and EGFP-FCP-RXFP1-3, a pair of gene specific primers are designed as follows, the 5' ends respectively have NheI and BstBI enzyme digestion sites, cDNA sequences synthesized by EGFP-FCP-RXFP1-1, EGFP-FCP-RXFP1-2 and EGFP-FCP-RXFP1-3 are taken as templates, respective gene fragments are obtained by PCR, and lentiviral plasmids pLJM1-Empty are taken as vectors, are subjected to enzyme digestion and connection to construct recombinant plasmids pLJM1-EGFP-FCP-RXFP1-1, pLJM1-EGFP-FCP-RXFP1-2, pLJM 1-EGFP-RXFP 1-3.

primer1：5’-CGGCTAGCTACCACTCGTTCCCGCTCCTCGAC-3’

NheI

primer2：5’-CCCTTCGAATGAATAGGAATTGAGTCTCGTTGA-3’

BstBI

1.3 transfection and infection of HEK293 cells

The linearized recombinant plasmids pLJM1-EGFP-FCP-RXFP1-1, pLJM1-EGFP-FCP-RXFP1-2 and pLJM1-EGFP-FCP-RXFP1-3 are co-transfected with lentivirus packaging plasmids pSPAX2 and pMD2.G to HEK293T cells respectively by using a liposome, the viruses are collected after 72 hours, polybrene is added to infect the HEK293 cells. And after 72 hours, using a DMEM culture medium containing 1000 mu G/mL G418 and added with 10% fetal calf serum to screen for 10-14 days, and gradually expanding and culturing 3 recombinant engineering cells stably expressing EGFP-FCP-RXFP1-1, EGFP-FCP-RXFP1-2 and EGFP-FCP-RXFP1-3 proteins by a limiting dilution method.

1.4 Relaxin-2 Activity assay

The concentration of cAMP standard is used as the abscissa, and the OD value corresponding to each concentration is used as the ordinate, and a standard curve is fitted and shown in FIG. 1.

3 kinds of recombinant engineering cells were inoculated in 24-well plates, and the number of cells was 3X 10⁵One/well in DMEM + 10% FBS + 1000. mu.g/mL G418 medium, 37 ℃, 5% CO₂When the cultured cells grow well and grow to 70% -80% (monolayer cells), the original culture solution is sucked out. Adding 50, 10, 2, 0.4, 0.08, 0.016 and 0.0032ng/mL of Relaxin-2 sample, and stimulating at 37 ℃ for 30 min. The cells were lysed by aspiration, centrifuged, and the cAMP content in the supernatant was measured by ELISA using 0.1M HCl. A curve (standard curve) is fitted by taking the concentration of the sample Relaxin-2 as the abscissa and the OD value of the corresponding cAMP as the ordinate, and is shown in FIGS. 2 to 4.

As can be seen from FIGS. 2 to 4, after Relaxin-2 with different concentrations stimulates the recombinant engineered cell RXFP1/HEK293 expressing the EGFP-FCP-RXFP1-1 protein, a complete S-shaped curve is fitted by the concentration of Relaxin-2 and the OD value of the generated cAMP. Therefore, the sequence GGGTGRSGGG of the first designed flexible connecting peptide is selected, and the subsequent experiment uses recombinant engineering cells RXFP1/HEK293 expressing EGFP-FCP-RXFP1-1 protein to measure the biological activity of Relaxin-2 and Western Blot experiment.

The concentration of cAMP was calculated from the OD values of cAMP produced after the cAMP standard curve and different concentrations of Relaxin-2 stimulated cells, and the curve fitted with the concentration of Relaxin-2 is shown in FIG. 5. It can be seen that the biological activity of Relaxin-2 and the concentration of cAMP present an S-shaped dose-dependent curve, which indicates that the established recombinant engineering cell RXPP 1/HEK293 can rapidly measure the biological activity of Relaxin-2 in vitro at high flux.

2. Western Blot method for detecting EGFP-FCP-RXFP1 protein expression

The expression of the EGFP-FCP-RXFP1 protein in the RXFP1/HEK293 cell is detected by using a Western Blot method. Specifically, RXFP1/HEK293 cells are cracked, total protein in the cells is extracted, and the expression of RXFP1 protein is determined by Western Blot.

2.1 extraction of Total protein from RXFP1/HEK293 cells

RXFP1/HEK293 cells were plated onto 6-well plates uniformly, and when the monolayer cells were grown to 80% confluency in DMEM + 10% FBS + 1000. mu.g/mL G418, the culture medium was aspirated. And (3) cracking the cells on ice by using a high-efficiency RIPA cracking solution for 30min, quickly scraping the cracked cells in the holes to one side by using a scraper, sucking out the cells, centrifuging the cells at the temperature of 4 ℃ and the rpm of 12000 for 10min, sucking out the floccule, and preparing a sample.

2.2 Western Blot method for detecting protein expression of EGFP-FCP-RXFP1

Electrophoresis: 1 piece of 1.5mm 8% SDS-PAGE gel was prepared, gel was concentrated at 80V for 20min and gel was separated at 130V for 70 min. Film transferring: placing from the anode to the cathode in sequence: sponge, 2 layers of filter paper, PVDF membrane, gel, 2 layers of filter paper, sponge, ice bath 90V 200mA70 min. Cleaning: wash 2 times 1 × TBST for 5 min. And (3) sealing: 200mL of 1 XTSSt +10g of skim milk powder, blocked overnight at room temperature. Incubating the primary antibody: 1mL of 1 XTSST + 6. mu.L primary antibody was incubated at room temperature for 2h by the reverse patch method. Cleaning: washing with 1 × TBST for 4 times, 1 time for 30 min. Incubation of secondary antibody: 1mL of 1 XTSSt + 2. mu.L secondary antibody was incubated for 1h at room temperature by the reverse patch method. Cleaning: wash 4 times 1 × TBST for 30 min. Color development: the hypersensitive ECL chemiluminescent substrate was dropped on PVDF membrane, exposed for 30s in dark clips, developed for about 1min until RXFP1 specific band appeared, and fixed, and the result is shown in FIG. 6.

3. Detection of RXFP1 fusion protein expression

After the recombinant engineering cell strain HEK293/RXFP1 is established, the expression of RXFP1 fusion protein in the recombinant engineering cell strain is detected so as to achieve the aim of identifying the recombinant engineering cell strain HEK293/RXFP 1. The detection of the expression of the fusion protein comprises two parts of detection of green fluorescent protein EGFP and detection of RXFP1 receptor protein. The detection of the RXFP1 receptor protein in the cell mainly uses a Western Blot experiment, comprises the extraction and quantification of the total protein in the recombinant engineering cell strain, and uses an antibody of RXFP1 to detect the RXFP1 receptor protein in the total protein of the recombinant engineering cell strain by using a Western Blot technology. In addition, cells were cultured continuously using medium containing G418 antibiotic to identify the resistance gene of the recombinant engineered cell strain HEK293/RXFP1 that grew stably under these conditions.

Identification of EGFP expression on cell membranes by fluorescence microscopy:

and (3) observing the recombinant engineering cell strain HEK293/RXFP1 with good growth state by using a fluorescence microscope, respectively observing the bright field and the fluorescence field of 4X, 10X, 20X and 40X times of objective lens under the same visual field, and determining the expression condition of the monoclonal recombinant engineering cell strain HEK293/RXFP1 and the green fluorescent protein on the cell membrane by comparing and superposing the bright field and the fluorescence field.

As a result:

the HEK293 cells were screened for virus infection in culture using limiting dilution. After culturing for 4-5 days, small cell clones growing in the holes can be observed under a microscope, under a 40 multiplied objective lens, obvious green fluorescence appears on cell membranes under a fluorescence microscope through comparison and superposition of a bright field and a fluorescence field, and green fluorescent protein is expressed as shown in figure 1; when the culture is carried out for 8-9 days, cell clusters can be observed to grow out of the holes under a microscope, under a 40X objective lens, obvious green fluorescence can still exist on cell membranes under a fluorescence microscope through comparison and superposition of a bright field and a fluorescence field, and the expression of green fluorescent protein is shown in figure 2. The screening culture medium is continuously used for gradually expanding culture, and observation under a fluorescent microscope objective shows that obvious green fluorescence appears on the cell membrane of the recombinant engineering cell HEK293/RXFP1 through comparison and superposition of a bright field and a fluorescent field, which indicates that green fluorescent protein is continuously expressed, and is shown in figure 3.

The above embodiments are merely preferred examples of the present invention and are not exhaustive of the possible implementations of the present invention. Any obvious modifications to the above would be obvious to those of ordinary skill in the art, but would not bring the invention so modified beyond the spirit and scope of the present invention.

SEQUENCE LISTING

<110> Hebei Jimega corporation of limited responsibility for science and technology development

<120> RXFP1 fusion protein, cell expressing RXFP1 fusion protein and application thereof in activity determination of Relaxin-2

Application in sex

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Thr Lys Cys Leu Pro Gln Leu Leu His Cys Asn Gly Val Asp Asp Cys

290 295 300

Gly Asn Gln Ala Asp Glu Asp Asn Cys Gly Asp Asn Asn Gly Trp Ser

305 310 315 320

Leu Gln Phe Asp Lys Tyr Phe Ala Ser Tyr Tyr Lys Met Thr Ser Gln

325 330 335

Tyr Pro Phe Glu Ala Glu Thr Pro Glu Cys Ser Phe His Phe Phe Leu

340 345 350

Phe Ile Thr Leu Leu Phe Leu Val Pro His Cys His His Ala Leu Pro

355 360 365

Leu Pro Leu Asp Ser Val Val Gly Ser Val Pro Val Gln Cys Leu Cys

370 375 380

Gln Gly Leu Glu Leu Asp Cys Asp Glu Thr Asn Leu Arg Ala Val Pro

385 390 395 400

Ser Val Ser Ser Asn Val Thr Ala Met Ser Leu Gln Trp Asn Leu Ile

405 410 415

Arg Lys Leu Pro Pro Asp Cys Phe Lys Asn Tyr His Asp Leu Gln Lys

420 425 430

Leu Tyr Leu Gln Asn Asn Lys Ile Thr Ser Ile Ser Ile Tyr Ala Phe

435 440 445

Arg Gly Leu Asn Ser Leu Thr Lys Leu Tyr Leu Ser His Asn Arg Ile

450 455 460

Thr Phe Leu Lys Pro Gly Val Phe Glu Asp Leu His Arg Leu Glu Trp

465 470 475 480

Leu Ile Ile Glu Asp Asn His Leu Ser Arg Ile Ser Pro Pro Thr Phe

485 490 495

Tyr Gly Leu Asn Ser Leu Ile Leu Leu Val Leu Met Asn Asn Val Leu

500 505 510

Thr Arg Leu Pro Asp Lys Pro Leu Cys Gln His Met Pro Arg Leu His

515 520 525

Trp Leu Asp Leu Glu Gly Asn His Ile His Asn Leu Arg Asn Leu Thr

530 535 540

Phe Ile Ser Cys Ser Asn Leu Thr Val Leu Val Met Arg Lys Asn Lys

545 550 555 560

Ile Asn His Leu Asn Glu Asn Thr Phe Ala Pro Leu Gln Lys Leu Asp

565 570 575

Glu Leu Asp Leu Gly Ser Asn Lys Ile Glu Asn Leu Pro Pro Leu Ile

580 585 590

Phe Lys Asp Leu Lys Glu Leu Ser Gln Leu Asn Leu Ser Tyr Asn Pro

595 600 605

Ile Gln Lys Ile Gln Ala Asn Gln Phe Asp Tyr Leu Val Lys Leu Lys

610 615 620

Ser Leu Ser Leu Glu Gly Ile Glu Ile Ser Asn Ile Gln Gln Arg Met

625 630 635 640

Phe Arg Pro Leu Met Asn Leu Ser His Ile Tyr Phe Lys Lys Phe Gln

645 650 655

Tyr Cys Gly Tyr Ala Pro His Val Arg Ser Cys Lys Pro Asn Thr Asp

660 665 670

Gly Ile Ser Ser Leu Glu Asn Leu Leu Ala Ser Ile Ile Gln Arg Val

675 680 685

Phe Val Trp Val Val Ser Ala Val Thr Cys Phe Gly Asn Ile Phe Val

690 695 700

Ile Cys Met Arg Pro Tyr Ile Arg Ser Glu Asn Lys Leu Tyr Ala Met

705 710 715 720

Ser Ile Ile Ser Leu Cys Cys Ala Asp Cys Leu Met Gly Ile Tyr Leu

725 730 735

Phe Val Ile Gly Gly Phe Asp Leu Lys Phe Arg Gly Glu Tyr Asn Lys

740 745 750

His Ala Gln Leu Trp Met Glu Ser Thr His Cys Gln Leu Val Gly Ser

755 760 765

Leu Ala Ile Leu Ser Thr Glu Val Ser Val Leu Leu Leu Thr Phe Leu

770 775 780

Thr Leu Glu Lys Tyr Ile Cys Ile Val Tyr Pro Phe Arg Cys Val Arg

785 790 795 800

Pro Gly Lys Cys Arg Thr Ile Thr Val Leu Ile Leu Ile Trp Ile Thr

805 810 815

Gly Phe Ile Val Ala Phe Ile Pro Leu Ser Asn Lys Glu Phe Phe Lys

820 825 830

Asn Tyr Tyr Gly Thr Asn Gly Val Cys Phe Pro Leu His Ser Glu Asp

835 840 845

Thr Glu Ser Ile Gly Ala Gln Ile Tyr Ser Val Ala Ile Phe Leu Gly

850 855 860

Ile Asn Leu Ala Ala Phe Ile Ile Ile Val Phe Ser Tyr Gly Ser Met

865 870 875 880

Phe Tyr Ser Val His Gln Ser Ala Ile Thr Ala Thr Glu Ile Arg Asn

885 890 895

Gln Val Lys Lys Glu Met Ile Leu Ala Lys Arg Phe Phe Phe Ile Val

900 905 910

Phe Thr Asp Ala Leu Cys Trp Ile Pro Ile Phe Val Val Lys Phe Leu

915 920 925

Ser Leu Leu Gln Val Glu Ile Pro Gly Thr Ile Thr Ser Trp Val Val

930 935 940

Ile Phe Ile Leu Pro Ile Asn Ser Ala Leu Asn Pro Ile Leu Tyr Thr

945 950 955 960

Leu Thr Thr Arg Pro Phe Lys Glu Met Ile His Arg Phe Trp Tyr Asn

965 970 975

Tyr Arg Gln Arg Lys Ser Met Asp Ser Lys Gly Gln Lys Thr Tyr Ala

980 985 990

Pro Ser Phe Ile Trp Val Glu Met Trp Pro Leu Gln Glu Met Pro Pro

995 1000 1005

Glu Leu Met Lys Pro Asp Leu Phe Thr Tyr Pro Cys Glu Met Ser

1010 1015 1020

Leu Ile Ser Gln Ser Thr Arg Leu Asn Ser Tyr Ser

1025 1030 1035

<210> 6

<211> 1037

<212> PRT

<213> Artificial Synthesis

<400> 6

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly

225 230 235 240

Gly Gly Gly Glu Gln Gly Arg Gly Ala Val Gly Gly Gly Met Thr Ser

245 250 255

Gly Ser Val Phe Phe Tyr Ile Leu Ile Phe Gly Lys Tyr Phe Ser His

260 265 270

Gly Gly Gly Gln Asp Val Lys Cys Ser Leu Gly Tyr Phe Pro Cys Gly

275 280 285

Asn Ile Thr Lys Cys Leu Pro Gln Leu Leu His Cys Asn Gly Val Asp

290 295 300

Asp Cys Gly Asn Gln Ala Asp Glu Asp Asn Cys Gly Asp Asn Asn Gly

305 310 315 320

Trp Ser Leu Gln Phe Asp Lys Tyr Phe Ala Ser Tyr Tyr Lys Met Thr

325 330 335

Ser Gln Tyr Pro Phe Glu Ala Glu Thr Pro Glu Cys Ser Phe His Phe

340 345 350

Phe Leu Phe Ile Thr Leu Leu Phe Leu Val Pro His Cys His His Ala

355 360 365

Leu Pro Leu Pro Leu Asp Ser Val Val Gly Ser Val Pro Val Gln Cys

370 375 380

Leu Cys Gln Gly Leu Glu Leu Asp Cys Asp Glu Thr Asn Leu Arg Ala

385 390 395 400

Val Pro Ser Val Ser Ser Asn Val Thr Ala Met Ser Leu Gln Trp Asn

405 410 415

Leu Ile Arg Lys Leu Pro Pro Asp Cys Phe Lys Asn Tyr His Asp Leu

420 425 430

Gln Lys Leu Tyr Leu Gln Asn Asn Lys Ile Thr Ser Ile Ser Ile Tyr

435 440 445

Ala Phe Arg Gly Leu Asn Ser Leu Thr Lys Leu Tyr Leu Ser His Asn

450 455 460

Arg Ile Thr Phe Leu Lys Pro Gly Val Phe Glu Asp Leu His Arg Leu

465 470 475 480

Glu Trp Leu Ile Ile Glu Asp Asn His Leu Ser Arg Ile Ser Pro Pro

485 490 495

Thr Phe Tyr Gly Leu Asn Ser Leu Ile Leu Leu Val Leu Met Asn Asn

500 505 510

Val Leu Thr Arg Leu Pro Asp Lys Pro Leu Cys Gln His Met Pro Arg

515 520 525

Leu His Trp Leu Asp Leu Glu Gly Asn His Ile His Asn Leu Arg Asn

530 535 540

Leu Thr Phe Ile Ser Cys Ser Asn Leu Thr Val Leu Val Met Arg Lys

545 550 555 560

Asn Lys Ile Asn His Leu Asn Glu Asn Thr Phe Ala Pro Leu Gln Lys

565 570 575

Leu Asp Glu Leu Asp Leu Gly Ser Asn Lys Ile Glu Asn Leu Pro Pro

580 585 590

Leu Ile Phe Lys Asp Leu Lys Glu Leu Ser Gln Leu Asn Leu Ser Tyr

595 600 605

Asn Pro Ile Gln Lys Ile Gln Ala Asn Gln Phe Asp Tyr Leu Val Lys

610 615 620

Leu Lys Ser Leu Ser Leu Glu Gly Ile Glu Ile Ser Asn Ile Gln Gln

625 630 635 640

Arg Met Phe Arg Pro Leu Met Asn Leu Ser His Ile Tyr Phe Lys Lys

645 650 655

Phe Gln Tyr Cys Gly Tyr Ala Pro His Val Arg Ser Cys Lys Pro Asn

660 665 670

Thr Asp Gly Ile Ser Ser Leu Glu Asn Leu Leu Ala Ser Ile Ile Gln

675 680 685

Arg Val Phe Val Trp Val Val Ser Ala Val Thr Cys Phe Gly Asn Ile

690 695 700

Phe Val Ile Cys Met Arg Pro Tyr Ile Arg Ser Glu Asn Lys Leu Tyr

705 710 715 720

Ala Met Ser Ile Ile Ser Leu Cys Cys Ala Asp Cys Leu Met Gly Ile

725 730 735

Tyr Leu Phe Val Ile Gly Gly Phe Asp Leu Lys Phe Arg Gly Glu Tyr

740 745 750

Asn Lys His Ala Gln Leu Trp Met Glu Ser Thr His Cys Gln Leu Val

755 760 765

Gly Ser Leu Ala Ile Leu Ser Thr Glu Val Ser Val Leu Leu Leu Thr

770 775 780

Phe Leu Thr Leu Glu Lys Tyr Ile Cys Ile Val Tyr Pro Phe Arg Cys

785 790 795 800

Val Arg Pro Gly Lys Cys Arg Thr Ile Thr Val Leu Ile Leu Ile Trp

805 810 815

Ile Thr Gly Phe Ile Val Ala Phe Ile Pro Leu Ser Asn Lys Glu Phe

820 825 830

Phe Lys Asn Tyr Tyr Gly Thr Asn Gly Val Cys Phe Pro Leu His Ser

835 840 845

Glu Asp Thr Glu Ser Ile Gly Ala Gln Ile Tyr Ser Val Ala Ile Phe

850 855 860

Leu Gly Ile Asn Leu Ala Ala Phe Ile Ile Ile Val Phe Ser Tyr Gly

865 870 875 880

Ser Met Phe Tyr Ser Val His Gln Ser Ala Ile Thr Ala Thr Glu Ile

885 890 895

Arg Asn Gln Val Lys Lys Glu Met Ile Leu Ala Lys Arg Phe Phe Phe

900 905 910

Ile Val Phe Thr Asp Ala Leu Cys Trp Ile Pro Ile Phe Val Val Lys

915 920 925

Phe Leu Ser Leu Leu Gln Val Glu Ile Pro Gly Thr Ile Thr Ser Trp

930 935 940

Val Val Ile Phe Ile Leu Pro Ile Asn Ser Ala Leu Asn Pro Ile Leu

945 950 955 960

Tyr Thr Leu Thr Thr Arg Pro Phe Lys Glu Met Ile His Arg Phe Trp

965 970 975

Tyr Asn Tyr Arg Gln Arg Lys Ser Met Asp Ser Lys Gly Gln Lys Thr

980 985 990

Tyr Ala Pro Ser Phe Ile Trp Val Glu Met Trp Pro Leu Gln Glu Met

995 1000 1005

Pro Pro Glu Leu Met Lys Pro Asp Leu Phe Thr Tyr Pro Cys Glu

1010 1015 1020

Met Ser Leu Ile Ser Gln Ser Thr Arg Leu Asn Ser Tyr Ser

1025 1030 1035

<210> 7

<211> 3102

<212> DNA

<213> Artificial Synthesis

<400> 7

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 720

ggcggcaccg ggcgcagcgg cggcggcatg acatctggtt ctgtcttctt ctacatctta 780

atttttggaa aatatttttc tcatgggggt ggacaggatg tcaagtgctc ccttggctat 840

ttcccctgtg ggaacatcac aaagtgcttg cctcagctcc tgcactgtaa cggtgtggac 900

gactgcggga atcaggccga tgaggacaac tgtggagaca acaatggatg gtctctgcaa 960

tttgacaaat attttgccag ttactacaaa atgacttccc aatatccttt tgaggcagaa 1020

acacctgaat gttcttttca tttctttctt ttcattaccc tcctcttcct ggtccctcac 1080

tgtcatcatg ccttaccact tccccttgat tcagtggtcg gttctgtgcc agtgcaatgt 1140

ctttgccaag gtctggagct tgactgtgat gaaaccaatt tacgagctgt tccatcggtt 1200

tcttcaaatg tgactgcaat gtcacttcag tggaacttaa taagaaagct tcctcctgat 1260

tgcttcaaga attatcatga tcttcagaag ctgtacctgc aaaacaataa gattacatcc 1320

atctccatct atgctttcag aggactgaat agccttacta aactgtatct cagtcataac 1380

agaataacct tcctgaagcc gggtgttttt gaagatcttc acagactaga atggctgata 1440

attgaagata atcacctcag tcgaatttcc ccaccaacat tttatggact aaattctctt 1500

attctcttag tcctgatgaa taacgtcctc acccgtttac ctgataaacc tctctgtcaa 1560

cacatgccaa gactacattg gctggacctt gaaggcaacc atatccataa tttaagaaat 1620

ttgactttta tttcctgcag taatttaact gttttagtga tgaggaaaaa caaaattaat 1680

cacttaaatg aaaatacttt tgcacctctc cagaaactgg atgaattgga tttaggaagt 1740

aataagattg aaaatcttcc accgcttata ttcaaggacc tgaaggagct gtcacaattg 1800

aatctttcct ataatccaat ccagaaaatt caagcaaacc aatttgatta tcttgtcaaa 1860

ctcaagtctc tcagcctaga agggattgaa atttcaaata tccaacaaag gatgtttaga 1920

cctcttatga atctctctca catatatttt aagaaattcc agtactgtgg gtatgcacca 1980

catgttcgca gctgtaaacc aaacactgat ggaatttcat ctctagagaa tctcttggca 2040

agcattattc agagagtatt tgtctgggtt gtatctgcag ttacctgctt tggaaacatt 2100

tttgtcattt gcatgcgacc ttatatcagg tctgagaaca agctgtatgc catgtcaatc 2160

atttctctct gctgtgccga ctgcttaatg ggaatatatt tattcgtgat cggaggcttt 2220

gacctaaagt ttcgtggaga atacaataag catgcgcagc tgtggatgga gagtactcat 2280

tgtcagcttg taggatcttt ggccattctg tccacagaag tatcagtttt actgttaaca 2340

tttctgacat tggaaaaata catctgcatt gtctatcctt ttagatgtgt gagacctgga 2400

aaatgcagaa caattacagt tctgattctc atttggatta ctggttttat agtggctttc 2460

attccattga gcaataagga atttttcaaa aactactatg gcaccaatgg agtatgcttc 2520

cctcttcatt cagaagatac agaaagtatt ggagcccaga tttattcagt ggcaattttt 2580

cttggtatta atttggccgc atttatcatc atagtttttt cctatggaag catgttttat 2640

agtgttcatc aaagtgccat aacagcaact gaaatacgga atcaagttaa aaaagagatg 2700

atccttgcca aacgtttttt ctttatagta tttactgatg cattatgctg gatacccatt 2760

tttgtagtga aatttctttc actgcttcag gtagaaatac caggtaccat aacctcttgg 2820

gtagtgattt ttattctgcc cattaacagt gctttgaacc caattctcta tactctgacc 2880

acaagaccat ttaaagaaat gattcatcgg ttttggtata actacagaca aagaaaatct 2940

atggacagca aaggtcagaa aacatatgct ccatcattca tctgggtgga aatgtggcca 3000

ctgcaggaga tgccacctga gttaatgaag ccggaccttt tcacataccc ctgtgaaatg 3060

tcactgattt ctcaatcaac gagactcaat tcctattcat ga 3102

<210> 8

<211> 3108

<212> DNA

<213> Artificial Synthesis

<400> 8

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 720

agcagcagca ccgggcgcca gagcagcagc ggcatgacat ctggttctgt cttcttctac 780

atcttaattt ttggaaaata tttttctcat gggggtggac aggatgtcaa gtgctccctt 840

ggctatttcc cctgtgggaa catcacaaag tgcttgcctc agctcctgca ctgtaacggt 900

gtggacgact gcgggaatca ggccgatgag gacaactgtg gagacaacaa tggatggtct 960

ctgcaatttg acaaatattt tgccagttac tacaaaatga cttcccaata tccttttgag 1020

gcagaaacac ctgaatgttc ttttcatttc tttcttttca ttaccctcct cttcctggtc 1080

cctcactgtc atcatgcctt accacttccc cttgattcag tggtcggttc tgtgccagtg 1140

caatgtcttt gccaaggtct ggagcttgac tgtgatgaaa ccaatttacg agctgttcca 1200

tcggtttctt caaatgtgac tgcaatgtca cttcagtgga acttaataag aaagcttcct 1260

cctgattgct tcaagaatta tcatgatctt cagaagctgt acctgcaaaa caataagatt 1320

acatccatct ccatctatgc tttcagagga ctgaatagcc ttactaaact gtatctcagt 1380

cataacagaa taaccttcct gaagccgggt gtttttgaag atcttcacag actagaatgg 1440

ctgataattg aagataatca cctcagtcga atttccccac caacatttta tggactaaat 1500

tctcttattc tcttagtcct gatgaataac gtcctcaccc gtttacctga taaacctctc 1560

tgtcaacaca tgccaagact acattggctg gaccttgaag gcaaccatat ccataattta 1620

agaaatttga cttttatttc ctgcagtaat ttaactgttt tagtgatgag gaaaaacaaa 1680

attaatcact taaatgaaaa tacttttgca cctctccaga aactggatga attggattta 1740

ggaagtaata agattgaaaa tcttccaccg cttatattca aggacctgaa ggagctgtca 1800

caattgaatc tttcctataa tccaatccag aaaattcaag caaaccaatt tgattatctt 1860

gtcaaactca agtctctcag cctagaaggg attgaaattt caaatatcca acaaaggatg 1920

tttagacctc ttatgaatct ctctcacata tattttaaga aattccagta ctgtgggtat 1980

gcaccacatg ttcgcagctg taaaccaaac actgatggaa tttcatctct agagaatctc 2040

ttggcaagca ttattcagag agtatttgtc tgggttgtat ctgcagttac ctgctttgga 2100

aacatttttg tcatttgcat gcgaccttat atcaggtctg agaacaagct gtatgccatg 2160

tcaatcattt ctctctgctg tgccgactgc ttaatgggaa tatatttatt cgtgatcgga 2220

ggctttgacc taaagtttcg tggagaatac aataagcatg cgcagctgtg gatggagagt 2280

actcattgtc agcttgtagg atctttggcc attctgtcca cagaagtatc agttttactg 2340

ttaacatttc tgacattgga aaaatacatc tgcattgtct atccttttag atgtgtgaga 2400

cctggaaaat gcagaacaat tacagttctg attctcattt ggattactgg ttttatagtg 2460

gctttcattc cattgagcaa taaggaattt ttcaaaaact actatggcac caatggagta 2520

tgcttccctc ttcattcaga agatacagaa agtattggag cccagattta ttcagtggca 2580

atttttcttg gtattaattt ggccgcattt atcatcatag ttttttccta tggaagcatg 2640

ttttatagtg ttcatcaaag tgccataaca gcaactgaaa tacggaatca agttaaaaaa 2700

gagatgatcc ttgccaaacg ttttttcttt atagtattta ctgatgcatt atgctggata 2760

cccatttttg tagtgaaatt tctttcactg cttcaggtag aaataccagg taccataacc 2820

tcttgggtag tgatttttat tctgcccatt aacagtgctt tgaacccaat tctctatact 2880

ctgaccacaa gaccatttaa agaaatgatt catcggtttt ggtataacta cagacaaaga 2940

aaatctatgg acagcaaagg tcagaaaaca tatgctccat cattcatctg ggtggaaatg 3000

tggccactgc aggagatgcc acctgagtta atgaagccgg accttttcac atacccctgt 3060

gaaatgtcac tgatttctca atcaacgaga ctcaattcct attcatga 3108

<210> 9

<211> 3114

<212> DNA

<213> Artificial Synthesis

<400> 9

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagggc 720

ggcggcgggg agcaggggcg cggggccgtg ggcggcggca tgacatctgg ttctgtcttc 780

ttctacatct taatttttgg aaaatatttt tctcatgggg gtggacagga tgtcaagtgc 840

tcccttggct atttcccctg tgggaacatc acaaagtgct tgcctcagct cctgcactgt 900

aacggtgtgg acgactgcgg gaatcaggcc gatgaggaca actgtggaga caacaatgga 960

tggtctctgc aatttgacaa atattttgcc agttactaca aaatgacttc ccaatatcct 1020

tttgaggcag aaacacctga atgttctttt catttctttc ttttcattac cctcctcttc 1080

ctggtccctc actgtcatca tgccttacca cttccccttg attcagtggt cggttctgtg 1140

ccagtgcaat gtctttgcca aggtctggag cttgactgtg atgaaaccaa tttacgagct 1200

gttccatcgg tttcttcaaa tgtgactgca atgtcacttc agtggaactt aataagaaag 1260

cttcctcctg attgcttcaa gaattatcat gatcttcaga agctgtacct gcaaaacaat 1320

aagattacat ccatctccat ctatgctttc agaggactga atagccttac taaactgtat 1380

ctcagtcata acagaataac cttcctgaag ccgggtgttt ttgaagatct tcacagacta 1440

gaatggctga taattgaaga taatcacctc agtcgaattt ccccaccaac attttatgga 1500

ctaaattctc ttattctctt agtcctgatg aataacgtcc tcacccgttt acctgataaa 1560

cctctctgtc aacacatgcc aagactacat tggctggacc ttgaaggcaa ccatatccat 1620

aatttaagaa atttgacttt tatttcctgc agtaatttaa ctgttttagt gatgaggaaa 1680

aacaaaatta atcacttaaa tgaaaatact tttgcacctc tccagaaact ggatgaattg 1740

gatttaggaa gtaataagat tgaaaatctt ccaccgctta tattcaagga cctgaaggag 1800

ctgtcacaat tgaatctttc ctataatcca atccagaaaa ttcaagcaaa ccaatttgat 1860

tatcttgtca aactcaagtc tctcagccta gaagggattg aaatttcaaa tatccaacaa 1920

aggatgttta gacctcttat gaatctctct cacatatatt ttaagaaatt ccagtactgt 1980

gggtatgcac cacatgttcg cagctgtaaa ccaaacactg atggaatttc atctctagag 2040

aatctcttgg caagcattat tcagagagta tttgtctggg ttgtatctgc agttacctgc 2100

tttggaaaca tttttgtcat ttgcatgcga ccttatatca ggtctgagaa caagctgtat 2160

gccatgtcaa tcatttctct ctgctgtgcc gactgcttaa tgggaatata tttattcgtg 2220

atcggaggct ttgacctaaa gtttcgtgga gaatacaata agcatgcgca gctgtggatg 2280

gagagtactc attgtcagct tgtaggatct ttggccattc tgtccacaga agtatcagtt 2340

ttactgttaa catttctgac attggaaaaa tacatctgca ttgtctatcc ttttagatgt 2400

gtgagacctg gaaaatgcag aacaattaca gttctgattc tcatttggat tactggtttt 2460

atagtggctt tcattccatt gagcaataag gaatttttca aaaactacta tggcaccaat 2520

ggagtatgct tccctcttca ttcagaagat acagaaagta ttggagccca gatttattca 2580

gtggcaattt ttcttggtat taatttggcc gcatttatca tcatagtttt ttcctatgga 2640

agcatgtttt atagtgttca tcaaagtgcc ataacagcaa ctgaaatacg gaatcaagtt 2700

aaaaaagaga tgatccttgc caaacgtttt ttctttatag tatttactga tgcattatgc 2760

tggataccca tttttgtagt gaaatttctt tcactgcttc aggtagaaat accaggtacc 2820

ataacctctt gggtagtgat ttttattctg cccattaaca gtgctttgaa cccaattctc 2880

tatactctga ccacaagacc atttaaagaa atgattcatc ggttttggta taactacaga 2940

caaagaaaat ctatggacag caaaggtcag aaaacatatg ctccatcatt catctgggtg 3000

gaaatgtggc cactgcagga gatgccacct gagttaatga agccggacct tttcacatac 3060

ccctgtgaaa tgtcactgat ttctcaatca acgagactca attcctattc atga 3114

Claims (8)

1. An RXFP1 fusion protein, characterized by: the EGFP fluorescent label is connected with the RXFP1 receptor protein through flexible connecting peptide, and the amino acid sequence of the EGFP fluorescent label is shown as SEQ ID No.4, SEQ ID No.5 or SEQ ID No. 6.

2. The RXFP1 fusion protein according to claim 1, wherein: the amino acid sequence of the flexible connecting peptide is shown in SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.

3. The RXFP1 fusion protein according to claim 2, wherein: the amino acid sequence of the flexible connecting peptide is shown as SEQ ID No. 1.

4. The RXFP1 fusion protein according to claim 1, wherein: the amino acid sequence is shown in SEQ ID No. 4.

5. The gene encoding the RXFP1 fusion protein according to claim 4, wherein: the nucleotide sequence is shown in SEQ ID No. 7.

6. A recombinant expression vector comprising the coding gene of claim 5.

7. A recombinantly engineered cell comprising the coding gene of claim 5.

8. Use of the recombinant engineered cell of claim 7 in the determination of Relaxin-2 activity.

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