阅读说明：本技术 一种新型fgf19类似物及其应用 (Novel FGF19 analogue and application thereof ) 是由 朱升龙 陈永泉 王振 于 2021-03-12 设计创作，主要内容包括：本发明公开了一种新型FGF19类似物及其应用,属于医药技术领域。本发明在FGF19突变体NGM282基础上进行改造,得到的新型FGF19类似物的与NGM282相比具有更长效、更稳定的效果,能更好的改善肝脏损伤及纠正代谢紊乱、胖症、超重、代谢综合征、糖尿病、血脂异常等病症,且治疗过程中均未出现原始FGF19突变体NGM282治疗过程引起胆固醇升高及饮食下降的副作用。(The invention discloses a novel FGF19 analogue and application thereof, belonging to the technical field of medicines. The novel FGF19 analogue obtained by modifying the FGF19 mutant NGM282 has longer acting and more stable effects compared with the NGM282, can better improve liver injury and correct metabolic disorders, obesity, overweight, metabolic syndrome, diabetes, dyslipidemia and other diseases, and has no side effect of cholesterol increase and diet decrease caused by the treatment process of the original FGF19 mutant NGM282 in the treatment process.)

The FGF19 protein analog is characterized in that the amino acid sequence of the FGF19 protein analog is shown in any one of SEQ ID NO. 1-4.

2. A gene encoding an analog of the FGF19 protein of claim 1.

3. A vector and/or a microbial cell carrying the gene of claim 2.

4. A medicament or pharmaceutical composition for the treatment of diabetes or obesity, comprising an FGF19 protein analog of claim 1.

5. The medicament or pharmaceutical composition of claim 4, wherein the treatment of diabetes or obesity comprises inhibiting weight gain, reducing blood lipid and blood glucose, increasing insulin sensitivity.

6. A medicament or pharmaceutical composition for treating hepatitis or related diseases, comprising an analog of the FGF19 protein of claim 1.

7. The drug or pharmaceutical composition of claim 6, wherein the treatment of hepatitis or related diseases comprises reducing liver weight and triglyceride levels, repairing liver damage, inhibiting the expression of inflammatory factors, ameliorating non-alcoholic steatohepatitis, atherosclerosis, liver damage, cirrhosis and liver cancer, primary biliary cholangitis and/or primary sclerosing cholangitis.

8. Use of an analog of the FGF19 protein of claim 1, or a vector or microbial cell of claim 3, in the manufacture of a medicament for the treatment of one or more of diabetes, obesity, hepatitis, or a hepatitis-related disorder.

9. The use of claim 8, wherein the FGF19 protein analog is administered in a dose of 1-100 mg/kg.

10. The use of claim 9, wherein the route of administration of the medicament comprises oral, intraperitoneal, subcutaneous, intravenous, or intramuscular.

Technical Field

The invention relates to a novel FGF19 analogue and application thereof, belonging to the technical field of medicines.

Background

Fibroblast growth factor 19(FGF19) is a newly discovered metabolic regulator that stimulates intestinal secretion and expression after bile acids are secreted into the intestine. FGF19 can enter liver along with circulation after being secreted by intestinal tract and has the function of combining with FGFR4 in liver, has hormone-like effect, and plays an important role in metabolic regulation, such as regulating bile acid metabolism, regulating gallbladder filling, improving energy metabolism, reducing body mass, improving blood sugar and the like. Since a plurality of previous studies show that FGF19 has mitogenic effect, FGFR4 can promote the proliferation of FGF19 in liver and has carcinotropic effect. 2014, it was found that the terminal domain of FGF19N is a region essential for interaction with FGFR, therefore, selective knockout of the region recognizing FGFR4 receptor abolishes the mitogenic activity of FGF19, and thus, many articles focus on the N-terminal mutation of FGF 19.

NGM282 is a non-tumorigenic engineered variant of human FGF19, and belongs to a mutant modified from the N-terminus of FGF 19. NGM282 has just completed a phase II clinical study in the united states and the results show that 79% of patients reach the primary treatment endpoint and 34% reach normal liver fat content at 12 weeks. The mutant improves serum biomarkers of liver function, lipid metabolism and fibrosis of a patient and shows curative effect on metabolic diseases. However, in addition to the observation of some common digestive tract symptoms, nausea and erythema at the injection site in the above clinical studies, FGF19 mutant NGM282 was found to significantly increase cholesterol levels after injection, and many studies have shown that elevated cholesterol levels are one of the significantly higher risk factors for metabolic diseases, which is a great risk for the treatment of metabolic diseases. In addition, FGF19 can cause symptoms such as anorexia and appetite reduction, and has certain hidden troubles for future treatment processes, so how to optimize FGF19 to reduce side effects is a key problem for promoting the application of FGF19 to the treatment of various diseases at present.

Disclosure of Invention

Therefore, the invention constructs 4 mutant proteins by prediction and test and modification on the basis of the original non-carcinogenic sequence, and prepares 4 FGF19 mutant proteins with biological activity by optimizing production and purification processes. The results show that 4 mutants can play the efficacy of treating obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), atherosclerosis, liver injury, liver cirrhosis, liver cancer, Primary Biliary Cholangitis (PBC) and Primary Sclerosing Cholangitis (PSC), and the treatment effect of the 4 mutants is obviously better than that of NGM282 protein.

The invention provides an FGF19 protein analogue, wherein an amino acid sequence of the FGF19 protein analogue is shown in any one of SEQ ID NO. 1-4.

In one embodiment, the FGF19 protein analog is encoded by a gene.

In one embodiment, the nucleotide sequences of the coding genes corresponding to the amino acids shown in SEQ ID NO. 1-4 are shown in SEQ ID NO. 5-8, respectively.

The invention provides vectors and/or host cells carrying said genes.

The invention provides a medicine or a pharmaceutical composition for treating diabetes or obesity, which contains the FGF19 protein analogue.

In one embodiment, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

In one embodiment, the treatment of diabetes or obesity comprises inhibiting weight gain, reducing blood lipids and blood glucose, increasing insulin sensitivity.

The invention provides a medicament or a pharmaceutical composition for treating hepatitis or related diseases, which contains the FGF19 protein analogue.

In one embodiment, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

In one embodiment, the treatment of hepatitis or related diseases includes reducing liver weight and triglyceride levels, repairing liver damage, inhibiting the expression of inflammatory factors, ameliorating non-alcoholic steatohepatitis, atherosclerosis, liver damage, cirrhosis and liver cancer primary biliary cholangitis and/or primary sclerosing cholangitis.

The invention provides application of the FGF19 protein analogue in preparing a medicament for treating one or more diseases of diabetes, obesity, hepatitis or hepatitis related diseases.

In one embodiment, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable carrier or adjuvant.

In one embodiment, the FGF19 protein analog is administered at a dose of 1-100 mg/kg.

In one embodiment, the FGF19 protein analog is present in a dose of 2 to 30 mg/kg.

In one embodiment, the route of administration of the drug comprises oral, intraperitoneal, subcutaneous, intravenous, or intramuscular injection.

The invention has the beneficial effects that:

(1) compared with the original FGF19 mutant NGM282, the 4 novel FGF19 analogues have the effects of being long-acting, stable and better in treating obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis (NASH), atherosclerosis, liver injury, cirrhosis, liver cancer, Primary Biliary Cholangitis (PBC) and Primary Sclerosing Cholangitis (PSC).

(2) In the treatment process of the 4 novel FGF19 analogues, the side effects of cholesterol increase and diet decrease caused by the treatment process of the original FGF19 mutant NGM282 do not occur, and the influence on the normal life activities of the organism is small.

Drawings

FIG. 1 is an SDS-PAGE electrophoretic analysis of the expression level of purified proteins in E.coli, including FGF19-1, FGF19-2, FGF19-3, FGF19-4 and NGM282 proteins;

FIG. 2 is a graph comparing the in vivo half-lives of 5 proteins;

FIG. 3 is a graph of the effect of 5 proteins on body weight and diet in db/db mice;

FIG. 4 is a graph of the effect of 5 proteins on blood lipids in db/db mice;

FIG. 5 is a graph of the effect of 5 proteins on a diabetes related indicator in db/db mice;

FIG. 6 is a graph showing the effect of 5 proteins on the relative indices of NASH model mouse steatohepatitis and hepatic fibrosis;

FIG. 7 is a graph showing the effect of 5 proteins on tumor proliferation in mice with liver cancer transplantable tumors.

Detailed Description

Experimental animals and breeding: nude mice and db/db mice were purchased from shanghai srek. Raising in animal center of Wuxi medical college in south of the Yangtze river, and alternately illuminating at 20 +/-2 ℃ every 12 hours.

Cell culture: the hepatoma cell line HepG2 was provided by the institute of biochemistry and cell biology of the chinese academy of sciences; DMEM, 0.05% Trypsin, available from bosd; fetal bovine serum was purchased from Sijiqing.

Other medicines are made in China and analyzed to be pure.

The hepatoma cell line HepG2 was grown adherently in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing in a humidified incubator, and carrying out subculture once every other day.

Example 1: construction, expression and purification of recombinant protein

(1) Construction of expression vectors for FGF19-1, FGF19-2, FGF19-3 and FGF19-4

According to computer simulation replacement and codon preference of escherichia coli, 4 novel FGF19 genes are designed, and the nucleotide sequences of the genes are respectively shown as FGF19-1 (the nucleotide sequence is shown as SEQ ID NO. 5), FGF19-2 (the nucleotide sequence is shown as SEQ ID NO. 6), FGF19-3 (the nucleotide sequence is shown as SEQ ID NO. 7) and FGF19-4 (the nucleotide sequence is shown as SEQ ID NO. 8) in the sequence table. The 4 genes are synthesized by Shanghai Czeri biological company, and NdeI and BamHI enzyme cutting sites are designed at two ends of each gene. The 4 synthesized vectors containing the respective target gene fragments and pET30a (+) are subjected to double enzyme digestion by NdeI and BamHI respectively, and after the enzyme digestion is finished, the respective required target fragments are recovered by glue. The 4 target fragments were ligated to the prokaryotic expression vector pET30a (+) using T4DNA ligase in a ligation system of 10. mu.L, mixed well, ligated overnight at 4 ℃ and then transformed into E.coli DH 5. alpha. each. The positive clone is selected and is subjected to enzyme digestion identification, and then 4 recombinant plasmids pET30a-FGF19-1, pET30a-FGF19-2, pET30a-FGF19-3 and pET30a-FGF19-4 are respectively constructed.

(2) Expression and purification of proteins

Recombinant plasmids pET30a-FGF19-1, pET30a-FGF19-2, pET30a-FGF19-3 and pET30a-FGF19-4 containing the correct sequence were transformed into competent cells expressing the strain Rosseta (DE 3). The transformed single colonies were inoculated into 20mL LB medium containing Kan (50. mu.g/mL), cultured at 37 ℃ for 8 hours, inoculated into another 20mL LB medium containing Kan (50. mu.g/mL) at a volume ratio of 1:100, cultured at 37 ℃, induced at about 0.35 with IPTG to a final concentration of 0.25mmol/L, harvested at 30 ℃ for 5 hours, resuspended in Lysisbuffer (20mmol/LTris, 150mmol/LNaCl, pH8.0), disrupted, centrifuged, and the supernatant and precipitate were analyzed by 12 wt% SDS-PAGE electrophoresis, respectively. The results show that the expression levels of FGF19-1, FGF19-2, FGF19-3 and FGF19-4 proteins in Escherichia coli are remarkably increased, and the target protein mostly exists in the form of inclusion bodies.

Collecting a large amount of induced thallus, adding lysozyme (1mg/mL) into the thallus, standing on ice for 30min, and ultrasonically disrupting thallus cells (working for 1s, interval of 1s, 4 min/time, total 3 cycles). After the thalli are completely crushed, a QuixStand pretreatment system (750kD ultrafiltration hollow fiber column) is used for treating cell crushing liquid, enriching inclusion bodies, and removing membrane permeation end liquid. When the total volume was about 60mL, the inclusion bodies were washed by adding 100mL of washbuffer (20mmol/LTris, 2mol/LUrea, 150mmol/LNaCl, pH 8.0). When the volume of the solution was 50mL, 100mL of the washing solution was added thereto, and the above experiment was repeated 4 times. After washing, when the volume of the solution was 50mL, the permeation end was closed, and 150mL of denaturation solution (20mmol/L Tris, 10mol/LUrea, 150mmol/L NaCl, pH8.0) was added to the washed inclusion bodies, followed by cyclic denaturation for 2 hours. And opening the permeation end, and collecting liquid at the membrane permeation end to obtain the mFGF21 denatured liquid. The denatured mFGF21 was concentrated to a volume of 80mL using a 5KD hollow fiber column, and then renatured, and a vessel containing a renaturation solution (20mmol/LTris, 50mmol/LNaCl, pH8.0) was connected to the reservoir of the hollow fiber column via a rubber tube. After the liquid storage device is sealed, liquid flows out from the penetrating end, and the renaturation liquid is dripped into the denaturation liquid at a certain speed due to negative pressure generated in the storage device, and renaturation is slowly carried out at a constant speed. When the volume of the renaturation solution is 6 times of that of the denaturation solution, after renaturation is finished, the mixture is centrifuged at 8000rpm/min and 4 ℃ for 20min, and supernatant is collected. After the renaturation supernatant fluid is completely combined with a CaptoQ column (filled in an XK16/20 empty column, the height of the column is 10cm, and the flow rate is 300cm/h) with 5 times of column volume IEXbafferA (20mmol/LTris, 10mmol/LNaCl, pH8.0) through an AKTApurifier100 system, the renaturation supernatant fluid is washed by 3-4 times of column volume IEXbafferA; when the UV curve reached a stable baseline, the mixture of IEXbafferA and IEXbafferB (20mmol/LTris, 1mol/LNaCl, pH8.0) was used for elution, the mixture of 15 wt% and 100 wt% IEXbafferB was used for washing the hetero-proteins, the mixture of 18.5 wt% to 19 wt% IEXbafferB was used for eluting the target protein, and each elution peak was collected and subjected to 15 wt% SDSPAGE electrophoresis analysis. The result shows that the purity of the purified protein is more than 95%, as shown in FIG. 1, lane 1 is a protein standard molecular weight Marker; lanes 2-6 are purified FGF19-1, FGF19-2, FGF19-3, and FGF19-4, respectively.

Example 2: detection of in vivo half-life of recombinant protein

5 proteins NGM282, FGF19-1, FGF19-2, FGF19-3 and FGF 19-4.

25 rabbits weighing about 2kg were selected and randomly divided into 5 groups. 5 proteins NGM282, FGF19-1, FGF19-2, FGF19-3 and FGF19-4 are injected into each group subcutaneously respectively, the dose is 30mg/kg, and blood is collected in the marginal veins of the ear for about 800 mu L at 0h, 1h, 3h, 5h, 7h and 24h after administration. Centrifuging at 12000r/m for 10min, collecting supernatant, and storing at-20 deg.C. ELISA indirect method the in vivo half-lives of 5 proteins were determined: the standard curves of protein concentration content are respectively established by using NGM282, FGF19-1, FGF19-2, FGF19-3 and FGF19-4 proteins (2 mu g/mL, 0.2 mu g/mL, 200ng/mL, 20ng/mL and 2ng/mL) with different diluted concentrations, the diluted standard proteins and serum are coated on an enzyme label plate, the content of target proteins in each serum is determined by using an ELISA indirect method, and the in vivo half-life of 6 proteins is statistically analyzed and calculated.

Half life t in vivo_1/20.301 (t2-t1)/log (OD1/OD2), wherein OD1 and OD2 represent t1 and t2 respectively, and the blood serum is taken out of the microplate corresponding to the blood serumAverage light absorption value of (a).

The results are shown in FIG. 2, the in vivo half-lives of the NGM282 protein and the mutated proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 are calculated by a formula to be about 36min, 79min, 66min, 67min and 69min respectively, and the in vivo half-lives of the 4 novel FGF19-1, FGF19-2, FGF19-3 and FGF19-4 are remarkably increased.

Example 3: effect of recombinant proteins on body weight, diet, blood lipid and diabetes related indices

4 proteins, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, were prepared according to the method of example 1.

50 SPF-grade 8-week-old male db/db mice are taken, pre-fed for 1 week and weighed, fasted for 6 hours next day without water prohibition, tail vein blood is taken to measure fasting blood glucose of the mice, abnormal body weight is eliminated, 42 molded mice with blood glucose and body weight values close to mean values are screened, and the mice are randomly divided into a normal Saline injection group (Saline), an NGM282 group, an FGF19-1 group, an FGF19-2 group, an FGF19-3 group and an FGF19-4 group, wherein each group comprises 6 mice. The test substance corresponding to the experimental group is administered once at about 8 o' clock in the morning and half a day, the intraperitoneal injection is carried out, the dose is 2mg/kg, the physiological saline group is injected with the physiological saline with the same volume, and the administration is continuously carried out for 8 weeks. During the experiment, the patient can eat and drink water freely. During which time the mice were monitored for diet and body weight. After 8 weeks of administration, the mice of each experimental group were sacrificed (fasting overnight), and blood was collected from the eyeballs to measure blood sugar, Triglyceride (TG), Total Cholesterol (TC), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) levels of the experimental mice. The experimental data obtained were statistically analyzed.

The experimental data are shown in fig. 3-5, and the results in fig. 3 show that, compared with the normal saline control group, the NGM282 protein and the 4 novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 after mutation modification can both significantly reduce the weight of the mice, but the NGM282 protein can significantly reduce the diet of the mice and inhibit the appetite of the mice after injection, and compared with the NGM282, the 4 novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 after administration can not only inhibit the weight of the mice more powerfully and significantly, but do not affect the feeding of the mice, which indicates that the side effect of the reduction of the original FGF19 is successfully improved by mutation modification.

After 8 weeks of administration, the results of the blood lipid levels of the blood serum of mice in each experimental group are shown in fig. 4, compared with the normal saline group, the NGM282 significantly increases the contents of TG, TC and LDL-c in the blood serum of the mice, but the contents of various HDL-c have no obvious difference, which is consistent with a plurality of previous clinical reports, and a plurality of studies show that the increase of cholesterol and blood lipid contents is one of significant high-risk factors of metabolic diseases, which is a huge risk for the treatment of the metabolic diseases. After multiple replacement and transformation, after injection of 4 novel proteins including FGF19-1, FGF19-2, FGF19-3 and FGF19-4, the side effect of increasing TG, TC and LDL-c by original FGF19 protein is avoided, the content of TG in serum can be remarkably reduced, and the results further show that the side effect of increasing blood fat by original FGF19 is improved by successful mutation and transformation, and the safety and the effectiveness of clinical application of FGF19 are greatly improved.

During the administration period, fasting blood glucose was measured at 0 week, 2 weeks, 4 weeks and 8 weeks, respectively, and the fasting blood glucose level results of mice in each experimental group are shown in fig. 5A, wherein after 2 weeks of treatment, NGM282 did not have significant effect of improving blood glucose, but FGF19-1, FGF19-3 had significantly reduced fasting blood glucose in mice; after 4 weeks of treatment, the NGM282 starts to play a role in reducing blood sugar, but the treatment effect is obviously lower than that of FGF19-3 and FGF19-4, and no obvious difference exists between groups after 8 weeks, and the result shows that the mutant recombinant FGF19 protein has a quick effect of reducing blood sugar and is better than that of the original NGM 282. After 8 weeks of administration, glucose tolerance test and insulin tolerance test are carried out, and the results are shown in fig. 5B and 5C, compared with NGM282 protein, the mutant modified 4 novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 can more remarkably improve the glucose sensitivity and insulin sensitivity of diabetic mice.

Example 4: effect of recombinant proteins on non-alcoholic steatohepatitis (NASH) -related indices

4 proteins, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, were prepared according to the method of example 1.

60 SPF-grade 8-week-old male C57BL/6 mice are taken, fed with methionine choline deficiency MCD feed after being pre-fed for 1 week, and fed with 8 weeks, then the abnormal body weight is removed, and 42 adult mice with approximate mean blood sugar and body weight values are screened, and the adult mice are randomly divided into a normal Saline injection group (Saline), an NGM282 group, an FGF19-1 group, an FGF19-2 group, an FGF19-3 group and an FGF19-4 group, and 6 mice in each group. The test substance corresponding to the experimental group is administered once at about 8 o' clock in the morning and half a day, the intraperitoneal injection is carried out, the dose is 2mg/kg, the physiological saline group is injected with the physiological saline with the same volume, and the administration is continuously carried out for 8 weeks. During the experiment, the patient can eat and drink water freely. After 8 weeks of administration, mice of each experimental group were sacrificed (fasting overnight), and hepatic Triglyceride (TG), alkaline phosphatase (ALP), glutamic-pyruvic transaminase (ALT) levels of the experimental mice were measured and subjected to tissue section staining and inflammation index detection. The experimental data obtained were statistically analyzed.

The experimental data are shown in FIG. 6, and the results in FIG. 6A show that, compared with a Saline control group (Saline), the NGM282 protein and the 4 mutant and modified novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 can both significantly reduce the weight of the liver and the content of Triglyceride (TG) in the liver of a mouse, but the therapeutic effects of the 4 novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 are significantly better than those of the NGM 282. FIG. 6B transaminase results further show that 4 novel proteins, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, have significantly better protective functions against liver injury than NGM 282. Furthermore, HE staining directly showed that 4 novel proteins FGF19-1, FGF19-2, FGF19-3, and FGF19-4 significantly reduced liver fat vacuoles upon injection, with few microscopic vacuoles observed, while a portion of fat vacuoles were present after treatment with NGM282 (fig. 6C). FIG. 6D is a result of sirius red staining for observing deposition of collagen fibers in liver, which reflects liver fibrosis, and shows that 4 novel proteins after mutation modification, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, can reverse liver fibrosis, while NGM282 treatment can still have a partial fibrosis state, which indicates that the reversing effect of the modified recombinant protein on liver fibrosis is significantly better than that of NGM 282. The main pathological state of NASH is inflammation of liver, the expression condition of a marker inflammatory factor is detected by qPCR, and the result shows that 4 novel proteins of FGF19-1, FGF19-2, FGF19-3 and FGF19-4 after mutation modification can obviously inhibit the expression of the inflammatory factor, and the inhibition effect is obviously superior to that of NGM282 (figure 6E). Through the detection of the multiple indexes, the treatment effect of the 4 mutant modified novel proteins FGF19-1, FGF19-2, FGF19-3 and FGF19-4 on NASH and liver injury is obviously better than that of the original sequence.

Example 5: effect of recombinant proteins on liver cancer

4 proteins, FGF19-1, FGF19-2, FGF19-3 and FGF19-4, were prepared according to the method of example 1.

Human liver cancer cell HepG2 cell according to 1 × 10⁶The cells/mouse are inoculated subcutaneously to 6-week-old male nude mice, and the tumor grows to 200mm³The groups were randomly divided into a Saline injection group (Saline), an NGM282 group, an FGF19-1 group, an FGF19-2 group, an FGF19-3 group and an FGF19-4 group, and 6 were each group. The test substance corresponding to the experimental group is administered once at about 8 o' clock in the morning and half a day, the intraperitoneal injection is carried out, the dose is 2mg/kg, the physiological saline group is injected with the same volume of physiological saline, and the administration is continuously carried out for 21 days. Tumor volume was monitored daily, mice were sacrificed three weeks later, and tumor weights were weighed. The results show that: all 5 were able to suppress the volume and final tumor weight of the transplanted tumors, but the NGM282 inhibitory effect was significantly lower than the mutated recombinant protein (as shown in fig. 7).

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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agcgcaaaac agcgtcagct gtataaaaat cgtggttttc tgccgctgtg ccattttctg 420

ccgatgctgc cgatggttcc ggaagaaccg gaagatctgc gtggtcatct ggaaagcgat 480

atgtttagca gcccgcccga tgtgggctcc tcggaccctc tgagcatggt gggaccttcc 540

cagggccgaa gccccagcta cgcttcc 567

<210> 6

<211> 537

<212> DNA

<213> Artificial sequence

<400> 6

atgcgtgata gcagcccgct ggttcattat ggttggggtg atccgattcg tctgcgtcat 60

ctgtatacca gcggtccgca tggtctgagc agctgttttc tgcgtattcg tgcagatggt 120

gttgttgatt gtgcacgtgg tcagagcgca catagcctgc tggaaattaa agcagttgca 180

ctgcgtaccg ttgcaattaa aggtgttcat agcgttcgtt atctgtgtat gggtgcagat 240

ggtaaaatgc agggtctgct gcagtatagc gaagaagatt gtgcatttga agaagaaatt 300

cgtccggatg gttataatgt ttatcgtagc gaaaaacatc gtctgccggt ttgcctgagc 360

agcgcaaaac agcgtcagct gtataaaaat cgtggttttc tgccgctgcc aggcctgccc 420

cccgcactcc cggagccacc cggaatcctg gccccccagc cccccgatgt gggctcctcg 480

gaccctctga gcatggtggg accttcccag ggccgaagcc ccagctacgc ttcctga 537

<210> 7

<211> 576

<212> DNA

<213> Artificial sequence

<400> 7

atggcagact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 60

acagatgatg cccagcgtac agaagcccac ctggagatca gggaggatgg gacggtgggg 120

ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 180

attcaaatct tgggagtccg tacaccgagg ttcctgtgcc agcggccaga tggggccctg 240

tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 300

ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 360

tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact atgccatttt 420

ctgccgatgc tgccgatggt tccggaagaa ccggaagatc tgcgtggtca tctggaaagc 480

gatatgttta gcagcccgct ggaaaccgat agcatggacc cgtttggtct ggttaccggt 540

ctggaagcag ttcgtagccc gagctttgaa aaataa 576

<210> 8

<211> 543

<212> DNA

<213> Artificial sequence

<400> 8

atggcagact ccagtcctct cctgcaattc gggggccaag tccggcagcg gtacctctac 60

acagatgatg cccagcgtac agaagcccac ctggagatca gggaggatgg gacggtgggg 120

ggcgctgctg accagagccc cgaaagtctc ctgcagctga aagccttgaa gccgggagtt 180

attcaaatct tgggagtccg tacaccgagg ttcctgtgcc agcggccaga tggggccctg 240

tatggatcgc tccactttga ccctgaggcc tgcagcttcc gggagctgct tcttgaggac 300

ggatacaatg tttaccagtc cgaagcccac ggcctcccgc tgcacctgcc agggaacaag 360

tccccacacc gggaccctgc accccgagga ccagctcgct tcctgccact accaggcctg 420

ccccccgcac tcccggagcc acccggaatc ctggcccccc agcccctgga aaccgatagc 480

atggacccgt ttggtctggt taccggtctg gaagcagttc gtagcccgag ctttgaaaaa 540

taa 543