CN1274830C - Salmon calcitonin analogues and expression method for the same in vegetable oils - Google Patents

Abstract

The invention clones a novel salmon calcitonin analog gene msCT (31aa) by using genetic engineering technology, and establishes a gene expression system in which oleosin and target protein are fused. These include: (1) Isolating and cloning the promoter of the rapeseed oleosin gene and the structural gene encoding the sesame oleosin; (2) Designing and synthesizing the coded salmon calcitonin analog msCT [Trp ¹ , Ser ⁶ , Phe ⁷ , del-Leu ¹⁹ ] gene msCT, it is inserted in the C terminal of sesame oleosin gene; (3) constructed the sesame oleosin-msCT fusion gene plant expression vector driven by rapeseed oleosin gene promoter, uses respectively Rapeseed rape and cotton were transformed by Agrobacterium method and pollen tube passage method, and transgenic plants and lines were obtained respectively. (4) Molecular biological detection of transgenic rapeseed and cotton proves the integration and expression of exogenous genes. The novel calcitonin analogue of the present invention has a biological activity of 6000 to 8000 U/mg, far exceeding natural salmon calcitonin 4,500 U/mg and human calcitonin 150 U/mg. The salmon calcitonin analogue has The effect of reducing blood calcium can be used to treat osteoporosis, Paget's disease, bone pain and various hypercalcemia.

Description

A novel salmon calcitonin analogue and method for its expression in vegetable oil bodies

field of invention

The present invention relates to a novel salmon calcitonin analogue msCT (31aa) and its expression method in vegetable oil bodies, in particular to the production of salmon calcitonin analogues using a fusion protein gene expression system composed of vegetable oil body protein-target protein Methods for msCT(31aa) and other target proteins.

Contents of the invention

Calcitonin is an important polypeptide hormone that regulates calcium and phosphorus metabolism and bone metabolism in the body. It can reduce blood calcium levels and also has antihistamine, anticholinergic, and inhibitory effects on gastric acid and pancreatic secretion. It is mainly used clinically for the treatment of hypercalcemia, osteoporosis, osteitis deformans (Pagete's disease), bone pain, spinal stenosis, bone metastasis of malignant tumors, renal osteoheterotrophy, fractures, hyperphosphatemia Hyperparathyroidism, peptic ulcer, acute pancreatitis, hyperparathyroidism and rheumatoid arthritis, etc., can also be used as a diagnostic drug for certain cancers, and is one of the important biochemical drugs. The new "calcium migration" theory believes that osteoporosis causes calcium in the bone to lose into the blood and deposit in the brain and blood vessels, thereby causing senile diseases such as senile dementia and arteriosclerosis. Therefore, calcitonin plays an important role in preventing It has a good application prospect in the treatment of senile osteoporosis. China is entering an aging society, and the elderly are prone to osteoporosis, which is also a worldwide problem. According to statistics, the United States spends about 10 billion U.S. dollars on the treatment of osteoporosis every year, and 1.3 million people suffer from fractures due to osteoporosis every year. Among them, hip fractures can cause serious complications, and about 5% to 20% of The patient died within 1 year after the fracture, and more than 50% of the survivors were disabled. Due to the extremely small content of calcitonin in biological materials (only 5-6mg calcitonin per kilogram of fresh pig thyroid gland), complicated extraction process and expensive chemical synthesis, etc., the large-scale application of calcitonin is limited, so the United States, Japan, Britain and other countries are actively developing genetically engineered calcitonin. How to obtain calcitonin with high biological activity, long half-life and non-antigenicity has always been concerned by people.

The use of transgenic plants to produce medical proteins or industrial enzymes has attracted widespread attention in recent years. The foreign protein is expressed in the plant oil body, and the target gene is inserted into the C-terminus or N-terminus of the oleosin gene to form a fusion protein gene. Since the Oleosin protein is embedded on the surface of the oil body, after the fusion protein gene is specifically expressed in the oil body of the recipient plant seed, the oil body is used to pulverize the seed → liquid extraction → centrifuge to recover the upper oil phase, that is The fusion protein or target protein can be separated from other components in the cell, thus significantly simplifying the separation and purification process of the target protein.

In order to complete the construction of the oil body expression system involved in the present invention, the inventors first isolated and cloned the promoter of the rapeseed oil body protein gene, the structural gene of the sesame oil body protein, and the mutant gene msCT[Trp ¹ of salmon calcitonin, Ser ⁶ , Phe ⁷ , del-Leu ¹⁹ ], then insert the msCT gene into the C-terminus of the sesame oleosin gene, construct a fusion protein gene plant expression vector driven by the promoter of the rape oleosin gene, and transform rapeseed and cotton , obtained transgenic plants and strains respectively.

The transgenic rapeseed was detected by PCR, which proved that the calcitonin gene had been integrated into the rapeseed genome. The transgenic cotton was tested by PCR-Southern and Western, which proved that the msCT gene was integrated in cotton and expressed in oil bodies. So far, 44 transgenic cotton lines of T2 generation have been obtained. Since the leaves of T1 and T2 generation plants are smeared with a high concentration (5000ppm) of Kanamycin (Kan) solution in the field, the Kanamycin-resistant (KanR) strains screened out should theoretically be individuals with high expression .

The amino acid sequence of natural salmon calcitonin is as follows:

Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr- Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂

After extensive and in-depth research, the inventor has now invented a novel salmon calcitonin analog msCT, whose amino acid sequence is: Trp-Ser-Asn-Leu-Ser-Ser-Phe-Val-Leu-Gly-Lys -Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro- _NH2 , ie natural salmon calcitonin The 1st amino acid Cys is mutated to Trp, the 6th Thr is mutated to Ser, the 7th Cys is mutated to Phe, and the 19th Leu is deleted (for its nucleotide sequence, see Sequence ID No. 3 in the Sequence Listing).

Invention effect

Compared with natural salmon calcitonin, the amino acids at the 1st, 6th, 7th, and 19th positions of msCT have been modified to significantly increase its biological activity (6,000-8,000U/mg). Natural salmon calcitonin and The biological activities of human calcitonin are 4,500 and 150 U/mg, respectively.

The use of transgenic plants to express exogenous target proteins such as salmon calcitonin analogues in vegetable oil bodies can greatly simplify the separation and purification process of target proteins and reduce production costs; the seeds of transgenic plants are easy to store and transport, which is conducive to industrial production and significantly improves The added value of agricultural by-products.

Brief introduction to the drawings

Fig. 1 is a schematic diagram of the structure of the plant expression vector pNOC121.

Fig. 2 is a construction diagram of pNOP vector.

Figure 3 is the enzyme digestion identification diagram of pNOP, in which 1: pNOP/HindIII+PstI double digestion; 2: λDNA/EcoRI+HindIII marker.

Fig. 4 is a construction diagram of pSO vector.

Figure 5 is the enzyme digestion identification diagram of pSO, in which 1: λDNA/EcoRI+HindIIImarker; 2: pSO/PstI+BamHI double digestion.

Fig. 6 is a construction diagram of pCT vector.

Figure 7 is the enzyme digestion identification diagram of pCT, in which 1: pBR322DNA/BstNI marker; 2: pCT/BamHI+XhoI double enzyme digestion.

Fig. 8 is a construction diagram of the intermediate vector pNOPM.

Figure 9 is the enzyme digestion identification diagram of pNOPM, in which 1: pNOPM/HindIII+PstI double digestion; 2: λDNA/EcoRI+HindIII marker.

Fig. 10 is a construction diagram of the intermediate vector pNsOM.

Figure 11 is the enzyme digestion identification diagram of pNsOM, in which 1: λDNA/EcoRI+HindIIImarker; 2: pNsOM/PstI+BamHI double digestion.

Figure 12 is a construction diagram of the intermediate vector pNOCM.

Figure 13 is the enzyme digestion identification diagram of pNOCM, in which 1: pNOCM/BamHI+XhoI double digestion; 2: λDNA/EcoRI+HindIII marker.

Figure 14 is a construction diagram of the plant expression vector pNOC121.

Figure 15 is the enzyme digestion identification diagram of pNOC121, in which 1: λDNA/EcoRI+HindIIImarker; 2: pNOC121/HindIII+EcoRI double digestion.

Figure 16 is the PCR detection of transgenic rapeseed nptII, wherein 1: λDNA/EcoRI+HindIII marker; 2-5: 750bp target fragment amplified by PCR.

Figure 17 is the PCR detection of transgenic rapeseed sOle/msCT, in which 1-4: 577bp PCR amplified target fragment; 5: λDNA/EcoRI+HindIII marker.

Figure 18 is the PCR-Southern hybridization (nptII) of T1 generation of transgenic cotton, wherein 1～11, 13～23, 25～34 are transgenic cotton; 12, 24, 36 are λDNA/EcoRI+HindIIImarker; 35 is non-transgenic cotton control .

Figure 19 is the Western detection of oleosin-msCT fusion protein in transgenic cottonseed oil bodies, wherein 1: protein molecular weight maker; 2: non-transgenic cotton control; 3: transgenic cotton line 351.

Figure 20 is a structural diagram of pEA. Figure 21 is a pMV structure diagram.

Below is the specific embodiment that realizes the present invention

In this study, we inserted the salmon calcitonin gene msCT into the 3' end of the sesame oil body protein gene (sOle), and constructed the plant expression vector pNOC121 of the sOle/msCT fusion protein gene driven by the rapeseed oil body protein promoter (NOP) . The schematic diagram of the structure of pNOC121 is shown in Fig. 1 .

Example 1 PCR amplification and cloning of rapeseed oil body protein gene promoter

Because rapeseed is an important bulk oil crop in my country, with high oil content (42-45%), and the amount of 20kD oil body protein in rapeseed oil body is 10 times that of 24kD oil body protein, so we start the gene according to the 20kD oil body protein Primers NP5 and NP3 were designed according to the sequence of the primer, and its sequence is as follows:

NP5: ATTCAAGCTTGTCGGATCATGACGTTTCCAG

NP3: GCTTCTGCAGAATTGAGAGAGATCGAAGAG

Using the total DNA of B. campestris green oil 14 varieties as a template, the promoter of the 20KD oleosin gene of 896 bp was amplified by PCR (PCR reaction conditions were: 94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 Minutes, 72°C extension for 10 minutes after the end of 35 cycles), it was ligated to pUC19 with HindIII+PstI double enzyme digestion to obtain pNOP. The construction process and enzyme digestion identification of the pNOP vector are shown in Figures 2 and 3, respectively. The result of pNOP sequencing showed that the cloned product was the promoter of the 896bp rapeseed oil body protein gene (see sequence number 1 in the sequence listing).

Example 2 Amplification and Cloning of Sesame Oleosin Gene

With the plasmid pEA (Fig. 20) DNA having the sesame oleosin gene as a template, primers Ole5 and Ole3 were designed, and their sequences were as follows:

Ole5: GTGCTGCAGACAACAATGGCGGACGAACCCCACG

Ole3: AAAGGATCCCGGTCTTATTACATCTTGGCTTC

A 440bp sesame oleosin gene without terminator sequence was obtained by PCR amplification. The PCR reaction conditions were: 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 1 minute, and 10 minutes at 72°C after 35 cycles. The PCR product was digested with PstI/BamHI, and connected to the vector pUC19 to obtain the cloning vector pSO. The vector construction and enzyme digestion identification of pSO are shown in Figures 4 and 5, respectively. For its sequence, refer to the sequence identification number 2 in the sequence listing.

Example 3 Artificial synthesis of salmon calcitonin analog gene

We designed the corresponding gene sequence according to the plant preferred codon, GC content and required enzyme cutting site, and carried out the artificial synthesis of the salmon calcitonin analogue msCT gene (138bp in total). The G/C content of this gene is 51.5%, and the cloning vector is pBluescriptSK. The cloning vector after inserting the msCT gene at the BamHI/XhoI site was named pCT. The construction and enzyme digestion identification of pCT vector are shown in Figures 6 and 7.

Example 4 Construction of intermediate vector pNOPM

Extract the plasmid DNA of pNOP, digest it with HindIII/PstI double enzymes, recover the 896bp target fragment (promoter of rapeseed oil body protein gene), connect it to the vector pMV (Figure 21), obtain the intermediate vector pNOPM, and pass the HindIII/PstI double enzyme Cut identification proves the connection is correct. The construction and enzyme digestion identification of the pNOPM intermediate vector are shown in Figures 8 and 9.

Example 5 Construction of intermediate vector pNsOM

Extract the pSO plasmid DNA, digest it with PstI/BamHI double enzymes, recover the 440bp target fragment (sesame oil body protein gene), connect it to pNOPM, and obtain the intermediate vector pNsOM, which is identified by PstI/BamHI double enzyme digestion, and the size of the digested fragment is 440bp. The construction and enzyme digestion identification of the pNsOM intermediate vector are shown in Figures 10 and 11.

Example 6 Construction of intermediate vector pNOCM

Extract the pCT plasmid DNA, and recover the 138bp target fragment (salmon calcitonin analogue msCT gene) after digestion with BamHI/XhoI, and connect it to the pNsOM vector to obtain the intermediate vector pNOCM (Figure 12). As identified by BamHI/XhoI double digestion, the size of the restriction fragment was 138bp (Figure 13).

Example 7 Construction of plant expression vector pNOC121

Extract pNOCM plasmid DNA, digest it with HindIII/EcoRI double enzymes, recover a DNA fragment of about 2.0 kb, and connect it to pBI121 (purchased from CLONTECH Company) (Figure 14), and identify the size of the digested fragment by HindIII/EcoRI double enzyme digestion is 2.0 kb (Fig. 15). So far, the construction of the NOP/sOle/msCT plant expression vector pNOC121 has been completed.

Example 8 Agrobacterium-mediated genetic transformation of rapeseed

1. Transformation of pNOC121 plasmid DNA into Agrobacterium

Add 2 micrograms of pNOC121 plasmid DNA to 100 microliters of LBA4404 competent cells, mix well, and ice-bath for 5 minutes. Freeze in liquid nitrogen for 5 minutes and then quickly transfer to a 37°C water bath for 5 minutes. Add 1 ml of YEB liquid medium, shake and culture at 250 rpm on a shaker at 28°C for 4-5 hours. Take an appropriate amount of bacterial liquid and smear it on the YEB solid medium containing 125 mg/L streptomycin and 100 mg/L kanamycin, culture it at 28°C for 24-48 hours, and screen to obtain a single colony of Agrobacterium with pNOC121 plasmid DNA .

2. Activation of Agrobacterium

Pick a single colony of Agrobacterium from the plate, inoculate it into 5 ml of YEB liquid medium (containing 100 mg/L of kanamycin and 100 mg/L of streptomycin), culture it with shaking overnight, and inoculate 1 ml of bacterial liquid into 50 ml In YEB liquid medium (containing 100 mg/L kanamycin and 100 mg/L streptomycin), shake vigorously until the OD ₆₀₀ is 0.4-0.5 (about 3-4 hours), centrifuge at 5000 rpm for 5 minutes, and use MS1 (MS+6BA 1.0 mg/L+NAA 0.05 mg/L) culture medium was washed once, and resuspended with MS ₀ (without any hormone and antibiotics), so that the OD ₆₀₀ was 0.1-0.2.

3. Genetic Transformation of Rapeseed

Cut the hypocotyls of rapeseed that had germinated for 7 days, soak them in the above-mentioned MS ₀ resuspended Agrobacterium solution for 1 minute, and place them on MS1 (MS+BA 1mg/L+NAA 0.05mg/L) solid medium for co-cultivation 4 After that, transfer to the MS1 solid medium containing kanamycin 10mg/L+ thiazoporomycin (Cef) 100mg/L to continue bud differentiation selection culture, and resistant buds will appear in about 4 weeks. When it stretches to 1-2 centimeters, transfer to the rooting medium MS2 (MS+NAA 0.1mg/L+Kan 100mg/L+Cef 100mg/L), and it will take about 10 days to take root.

Example 9 PCR detection of transgenic rapeseed

Genomic DNA of transgenic rapeseed was extracted, and two pairs of primers, npt5/npt3 and sOle/msCT, were used for PCR amplification detection. The sequences of each primer were:

npt5:GAACAAGATGGATTGCACGC

npt3: AGAAGGCGATAGAAGGCGATG

sOle: TGGCGGACGAACCCCACGACC

msCT: CTTACTCAGCCTCGAGTTACTATCCT

The PCR reaction conditions of npt5/npt3 are: 94°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute; the PCR reaction conditions of sOle/msCT are: 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1 minute. After 35 cycles each was extended at 72°C for 10 minutes. The expected target fragments of 750bpnptII and 578bp of sesame oleosin-msCT were amplified respectively. The PCR detection photos are shown in Figures 16 and 17, respectively.

Example 10 Transformation of Haidao Mianhai 7124 by pollen tube passage method

A large amount of pNOC 121 plasmid DNA was extracted, diluted to 0.02 μg/μl with sterile redistilled water, and when Beijing Danghai 7124 flowered for about 24 hours, the diluted DNA solution was diluted in an amount of 10 μl per flower (0.2 μg DNA) into the cotton ovary, and smear 1% gibberellin at the bud stalk to prevent the bud from coming off. A total of 5 kilograms of about 50,000 cotton seeds were harvested and sent to Hainan for propagation. After sowing, 5000 ppm of kanamycin solution was applied to the leaves at the seedling stage, and 101 kanamycin-resistant (Kan ^R ) plants were screened out.

Example 11 T2 generation plants of Hai 7124 transgenic cotton

Kanamycin resistance (Kan ^R ) test

The T2 generation seeds harvested from Hainan Nanfan Kan ^R strains were sown in Beijing, and 5000ppm of kanamycin was applied to each leaf of each row twice at the 4-leaf stage and before budding. The results of Kan ^R and Kan ^s (kanamycin sensitive) strains are listed in Table 1.

Table 1 Field investigation of Kan resistance of T2 generation plants in Beijing Beijing Zhuxing Hainan Number Kan ^R strain Kan ^S strain Total number of plants Beijing Zhuxing Hainan Number Kan ^R strain Kan ^S strain Total number of plants 1 1 5 1 6 48 91 0 25 25 2 2 12 9 twenty one 49 92 0 18 18 5 7 10 10 20 50 93 0 19 19 6 8 11 45 56 51 94 1 16 17 7 10 0 47 47 52 96 5 13 18 8 11 1 10 11 53 100 0 18 18 9 13 0 32 32 54 101 2 45 47 10 15 0 19 19 55 102 0 88 88 11 16 0 56 56 56 103 2 77 79 13 19 11 8 19 57 104 0 twenty two twenty two 14 20 11 17 28 58 105 1 58 59 15 twenty one 0 41 41 59 106 1 61 62 16 twenty three 3 44 47 60 107 3 41 44 17 twenty four 2 8 10 61 108 1 4 5 19 26 0 51 51 63 109 2 62 64 twenty one 28 1 25 26 64 110 6 86 92 twenty three 31 0 31 31 65 111 2 34 36 26 36 2 18 20 66 112 2 47 49 29 39 1 33 34 67 114 2 51 53 30 44 4 52 56 68 117 18 13 31 32 46 1 twenty one twenty two 69 120 5 29 34 34 48 0 33 33 71 351 4 twenty two 26 35 50 3 8 11 72 6 1 43 44 36 51 0 7 7 73 9 0 twenty one twenty one 37 76 1 46 47 74 12 1 2 3 38 77 1 36 37 75 18 0 3 3 39 78 1 67 68 76 41 3 10 13 40 80 3 70 73 77 79 0 30 30 41 82 0 20 20 78 90 0 3 3 42 84 1 41 42 79 95 0 2 2 44 86 0 15 15 80 97 2 3 5 45 87 0 47 47 81 98 1 1 2 46 88 2 twenty four 26 82 14 1 4 5 47 89 2 27 29

Example 12 PCR-Southern detection of T2 generation transgenic cotton

The genomic DNA of the Kan ^R strain was extracted, and the genomic DNA was amplified by PCR with two pairs of primers, npt5/npt3 and sOle/msCT. The reaction conditions of PCR are: npt5/npt3: 94°C for 30 seconds, 52°C for 30 seconds, 72°C for 1 minute; sOle/msCT: 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 1 minute. After 35 cycles all were extended at 72 °C for 10 min. The results showed that 63 strains were positive for nptII, and 44 strains were positive for sOle/msCT among these 63 strains.

Take 32 transgenic cotton plants of the T2 generation nptII and sOle/msCT PCR positive, and use the pNOC121 plasmid DNA/HindIII+EcoRI double-digested fragment as a probe to carry out PCR-Southern hybridization, and the result is 29 positive (Figure 18) .

Example 13 The expression of oleosin oil body protein-calcitonin target protein in cottonseed

Western detection

Extract oil body protein from cottonseed, SDS polyacrylamide gel electrophoresis. After transmembrane, goat anti-salmon calcitonin polyclonal antibody was used for Western analysis. The results showed that the salmon calcitonin analogue protein was expressed in the seeds of the transgenic cotton 351 line, and the size of the expression product was about 19kD, which was consistent with the expected size (sesame Oleosinn protein 15.5kD+msCT protein 3.45kD), as shown in the figure 19.

Example 14 Separation of Oleosin and Salmon Calcitonin Analogs

1. Extraction of oleosin-salmon calcitonin analog fusion protein from cottonseed

(1) Take 10 cotton seeds and remove the shell, add about 3 times the volume of buffer A (0.15MTricine-KOHpH7.5, 10mM KCl, 1mM MgCl ₂ , 1mM EDTA, 0.6M sucrose) to grind, 5000×g, 4°C Centrifuge for 10 minutes.

(2) The supernatant was resuspended with buffer A containing 0.6M sucrose.

(3) Cover the liquid surface with an equal volume of buffer A containing 0.1M sucrose.

(4) Centrifuge at 18000×g and 4°C for 20 minutes, repeat twice, and take the supernatant.

NOTE; Oil bodies are now separated from other components in the seeds. The components of oil bodies include: neutral lipids, phospholipids, and oil body proteins.

(5) Add 2 times the volume of diethyl ether in the supernatant, and centrifuge at 13600×g for 4 minutes. Most of the upper diethyl ether layer is neutral lipids, and the phospholipids are left in the lower water phase, and the middle protein layer is taken.

(6) Resuspend the protein in 250 microliters of buffer A containing 0.1M sucrose, add 750 microliters of chloroform/methanol (2:1) mixture, and extract 2 to 3 times.

(7) Take the intermediate protein layer, extract it once with ether, and freeze-dry it in a -70°C refrigerator for storage.

2. Isolation of Oleosins from Salmon Calcitonin Analogs

(1) The fusion protein of oleosin-salmon calcitonin analog was dissolved in as little sterile water as possible.

(2) Add 50 microliters of thrombin cleavage reaction buffer (0.1M triethanolamine pH8.4, 0.5% sarkosyl).

(3) Add 0.02U thrombin, digest overnight at 37°C.

(4) The salmon calcitonin analog was obtained by HPLC separation.

3. HPLC Purification Procedure of Salmon Calcitonin Analogs

Analytical reversed-phase column C18 (5μ, 8×250mm, Dalian Institute of Chemical Physics); ultraviolet wavelength 214nm; mobile phase A-0.1% trifluoroacetic acid, B-70% acetonitrile (0.1% trifluoroacetic acid). Gradient: B rises from 0% to 60% within 20 minutes, and then drops to 0% within 10 minutes, with a flow rate of 0.5 ml/min. Salmon calcitonin analogs are collected, freeze-dried, and stored at -70°C.

Sequence Listing

<110>Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> A Novel Salmon Calcitonin Analog and Its Expression Method in Vegetable Oil Body

<160>4

<170>PatentIn Version 2.1

<210>1

<211>896

<212>DNA

<213> Rapeseed (Brassica campestris)

<400>1

ATTCAACGTTGTCGGATCATGACGTTTCCAGAAAAACATCGAGCAAGCTCTC

AAAGCTGACCTCTTTCGGATCGTACTGAACCCGAACAATCTCGTTATGCCC

CGTCGTCTCCGAACAGACATCCTCGTAAGTAGGATTGTCGACGAATCCATG

GCTATACCCAACCTCCGTCTTCGTCACGCCTGGAACCCTCTGGTAAGCCAA

TTCCGCTCCCCAGAAGCAACCGGCGCCGAATTGCGCGAATTGCTGACCAG

GCGAAGGAACATCGTCGTCGGGTCCTTGTGCGATTGCGGAGGAAGCCGCC

GGGTCGGGTTGGGGACGAGACCCGAATCCGAGCCTGGTGAATAGGTTGTT

CATCGGAGATTTATAGACGGAGATGGATCGAGCGGTTTTGGGGAAAGGGG

AAGTGGGTTTGGCTCTTTTGGATAGAGAGAGTGCAGCTTTGGCGAGAGAC

TGGAGAGGTTTAGAGAGAGACACGGCGGAGATTACCGGAGGAGAGGCGA

CGATAGATAGCATTATCGAAGGGACGGGAGAAAGAGTGACGTGGAGAAAT

AAGAAACCGTTAAGAGTCGGATATTTATTATATTAAAAGCCCACTGGGCCTA

AACCCATTTAAACAAGACAAGATAAATGGGCCGTGTGTGAGTGTTAACGTT

CGGTTTCAAATGCCAACGCCATTGGAACAA AACAAACGTGTCCTCAAGTA

AACCCCTGTCGTTTACACCTCCAATGGCTGCATGGTGAAGCCATTAACACGT

GGCGTAGAATGCATGACGACGCCATTGACACCTGACTCTCTTTCCTTCTCT

TCATATATCTCTAATCAATTCAACACTCATTGTCATAGCTATTCGGAAAATAT

ATACACATCCTTTTCTCTTCGATCTCTCTCAATTC

<210>2

<211>440

<212>DNA

<213> Sesamum (Sesamum indicum L.)

<400>2

ATGGCGGacGAACCCCACGACCaGCGCCCCACCGACGTCATCaaGAGCTACC

TCCCCGAAAaGGGTCCCTCCACCTCCTCAAGTCCTCGCCGTCGTGACCCTCT

TCCCCCTCGGCGCCGTCCTCCTCTGCCTAGCCGGTCTCATTCTTACCGGGA

CCATCATCGGCCTCGCCGTCGCCACCCCGCTCTTCGTCATCTTCAGCCCCAT

CTTGGTCCCCGCCGCCCTAACCATCGCCCTAGCCGTCACCGGTTTCTTGAC

CTCCGGAGCTTTCGGCATCACCGCCCTGTCCTCGATTTCGTGGTTGCTGAA

CTACGTTAGGCGAATGCGGGGGAGCTTGCCAGAGCaGCTGGATCATGCACG

GCGGCGCGTGCaGGAGAcGGTGGGCCaGAaGACAAGGGAGGCGGGGCaGA

GAAGCCAAGATGTAATAAGACCGTGA

<210>3

<211>138

<212>DNA

<213> Artificial sequence

<400>3

GGATCCCATA TGCTTGTTCC AAGGGGATCT TGG AGC AAC CTC AGC AGC TTC GTG CTC GGA

AAG CTC AGC CAA GAG CTC CAC AAG CAA ACC TAC CCA AGG ACC AAC ACC GGA TCT

GGA ACC CCA GGA TAG TAA CTCGAG

<210>4

<211>31

<212>PRT

<213> Artificial sequence

<400>4

Trp-Ser-Asn-Leu-Ser-Ser-Phe-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-Thr-Asn- Thr-Gly-Ser-Gly-Thr-Pro-NH ₂

Claims (12)

1. One kind the coding salmon calcitonin analogues msCT (31aa) gene msCT[Trp ¹, Ser ⁶, Phe ⁷, del-Leu ¹⁹], the aminoacid sequence structure of its coded product is: Trp-Ser-Asn-Leu-Ser-Ser-Phe-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂, the 1st amino acids Cys that is about to natural salmon calcitonin see calcimar sports Trp, and the 6th Thr sports Ser, and the 7th Cys sports Phe, the 19th Leu disappearance.
2. 2. a recombinant vectors contains the described gene of claim 1.
3. 3. carrier according to claim 2, this carrier comprise the promotor of oil body protein (oleosin) gene, the gene of the structure gene of oil body protein and coding salmon calcitonin analogues.
4. 4. method for preparing calcitonin-like comprises:

   (1) C end or the N that the described gene of claim 1 is inserted in oil body protein gene holds;

   (2) place the oil body protein promotor to make up plant expression vector afterwards the fusion gene that obtains;

   (3) with the plant expression vector transformation receptor plant that makes up;

   (4) obtain transgenic plant;

   (5) separate and target protein that purifying gives expression in the transgenic plant oil body.
5. 5. preparation method according to claim 4, wherein said recipient plant and transgenic plant are oilseed plants.
6. 6. preparation method according to claim 5, wherein said oilseed plant is rape, Sunflower Receptacle, soybean, peanut, sesame, cotton, oil palm or Fructus oleae europaeae.
7. 7. the transgenic plant cells that is obtained by the described preparation method of claim 4-6 is organized or organ.
8. 8. salmon calcitonin analogues, its aminoacid sequence structure is: Trp-Ser-Asn-Leu-Ser-Ser-Phe-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH ₂
9. 9. contain the described gene of claim 1 or contain the fungi of the described recombinant vectors of claim 2.
10. 10. fungi according to claim 9, it is a primary yeast.
11. 11. contain the described gene of claim 1 or contain the bacterium of the described recombinant vectors of claim 2.
12. 12. bacterium according to claim 11, it is a kind of intestinal bacteria or Agrobacterium.

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