CN102399283B - Mink Growth Hormone Releasing Hormone cDNA and Its Application - Google Patents

Abstract

The invention discloses a full-length nucleotide sequence and a mature peptide nucleotide sequence of a mink growth hormone releasing hormone cDNA gene, and encoded protein amino acid sequences thereof. The mink growth hormone releasing hormone cDNA sequence is a complete open reading frame with 321 nucleotide sequences, and 106 amino acids are encoded. The mature peptide is encoded by 132 nucleotide sequences, and 44 amino acid residues are encoded. The mink growth hormone releasing hormone cDNA can be used for promoting the growths of economic animals such as minks.

Description

Mink Growth Hormone Releasing Hormone cDNA and Its Application

technical field

The invention relates to mink growth hormone releasing hormone cDNA obtained by cloning and application thereof.

Background technique

Mink is a fur animal with high economic value. Its growth is mainly controlled by the growth hormone produced and released by the pituitary gland. After reaching body maturity, the production and release of growth hormone decreases. Growth hormone is under the dual control of growth hormone releasing hormone and growth hormone releasing inhibitory hormone produced by the hypothalamus of the animal itself. Growth hormone-releasing hormone stimulates the production and release of growth hormone by the pituitary gland, while growth hormone-releasing inhibitory hormone inhibits the production of growth hormone. The two work together to maintain the growth rate of mink. Growth hormone-releasing hormone is produced in the arcuate nucleus and ventromedial nucleus of the hypothalamus. After synthesis, it is transported through the axon and secreted to the median eminence, thereby reaching the adenohypophysis through the pituitary portal system. The pituitary gland is then stimulated to produce growth hormone. Take human growth hormone-releasing hormone as an example: the precursor of human growth hormone-releasing hormone is a polypeptide chain composed of 108 amino acid residues, of which the first 20 amino acid residues at the N-terminal are the signal peptide sequence. After being excised and reprocessed, the mature human growth hormone releasing hormone contains 44 amino acid residues, and the sequence of 44 peptides is: NH ₂ -Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr- Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn- Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-COOH. The mature protein reaches the adenohypophysis through the pituitary portal system, and binds to the GHRH receptor (GHRH-RP) on the membrane of eosinophilic cells that secretes GH. After GHRH binds to the receptor, it first activates a Gs protein that is sensitive to cholera toxin , and then Gs activates the adenylate cyclase (AC) on the membrane to convert ATP into cAMP, resulting in an increase in the level of cAMP. The increase of cAMP level will promote the conformational change and activation of the regulatory subunit of cAMP-dependent proteinase kinase A, promote the phosphorylation of related proteins, and then affect the activity of cells. GH secretion caused by cAMP is Ca ²⁺ dependent. When GHRH acts on growth hormone-secreting cells, the concentration of Ca ²⁺ will increase within 60 seconds, thereby promoting GH secretion. There is evidence that Ca ²⁺ blockade can terminate the release of cAMP on GH. It can be seen that the secretion of GH by GHRH is mediated by cAMP and Ca ²⁺ , through which the synthesis and release of GH are regulated ^[1] . Since GHRH is released in pulses, the corresponding pituitary GH also fluctuates in pulses.

Growth hormone releasing hormone (GHRH) is generally a polypeptide of 44 amino acids, its complete biological activity depends on the 1st-29th amino acid, and its half-life is 7-50 minutes, which shows that its action time is short. The molecular size of growth hormone releasing hormone in different animals is slightly different. The active form of GHRH protein in several animals such as pigs, cattle, dogs, humans and sheep is a small peptide with 44 amino acid residues. Their homology is high. As follows:

Dog: YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNREQGAKVRL

Pig: YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQGARVRL

Cattle: YADAIFTNSYRKVLGQLSARKLLQDIMNRQQGERNQEQGAKVRL

Person: YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGESNQERGARARL

Sheep: YADAIFTNSYRKILGQLSARKLLQDIMNRQQGERNQEQGAKVRL

The size of the cDNA nucleotide sequence of the corresponding growth hormone releasing hormone in different animals is also different. Human growth hormone releasing hormone was first isolated, purified and identified in 1974 ^[2] . At present, many animals including human growth hormone releasing hormone cDNA genes have been cloned and made public. The human cDNA sequence is 327 nucleotides, 312 for mice, 315 for rats, and 321 for dogs, cattle, and pigs. Most sequences are patented. like:

Chinese patent 02100065.4 protects the cDNA gene of sheep growth hormone releasing hormone and the application of its expression product.

00813372.7 (superactive porcine growth hormone releasing hormone analogue) preserves the optimized nucleotide and amino acid sequence of porcine GHRH.

03822291.4 (GHRH analogs) protects the optimized nucleotide sequence and amino acid of human GHRH.

200610017214.5 protects the GHRH medicine for promoting the growth of piglets and improving the immunity of piglets and its preparation method.

200510061609.0 protects a GHRH drug and its preparation that can promote the growth of human body.

200710068659.0 protects the extraction of velvet antler growth-promoting releasing factor (DEER GHRF) and its preparation method.

CN1615151 (Plasmid-Mediated Supplementation for Treatment of Individuals with Chronic Diseases) and CN1649496 (Compositions and Methods for Altering Lean Body Mass and Bone Properties of Individuals) protect artificial sequences of GHRH optimized according to different animal gene sequences.

CN1635898 protects porcine growth hormone releasing hormone analogs with super activity.

US Patents 7,351,815 and 7,361,642 both protect the nucleotide sequence and amino acid sequence of canine growth hormone releasing hormone.

As early as 1997, Draghia-Akli et al achieved the effect of promoting pig weight gain by intramuscular injection of pig GHRH expression plasmid ^[3] Subsequent experiments further confirmed the same result ^[4,5,6] . In dogs ^[7] , sheep ^[8] also obtained more successful experimental results.

Contents of the invention

The purpose of the present invention is to provide a mink GH-releasing hormone cDNA obtained by cloning and its application.

In order to achieve the above purpose, we designed a pair of primers according to the conserved sequence of GHRH in other animals: the upstream primer is (as shown in SEQ ID No.5): 5'ATGCCACTCTGGGTGTTC 3', and the downstream primer is (as shown in SEQ ID No.6 Shown): 5'TCATCCTTGGGAGTTCC 3'. Then the total RNA of the mink hypothalamus was prepared, then reverse transcription was performed, and then PCR was performed, and finally the cDNA of mink GHRH was cloned. Its full sequence is shown in SEQ ID No.4, the nucleotide sequence of GHRH mature peptide is shown in SEQ ID No.3, and the amino acid sequences encoded by them are shown in SEQ ID No.2 and SEQ ID No.1 respectively .

According to the cDNA of the mink GHRH mature peptide provided by the present invention, it can be used as the target gene, and a eukaryotic expression vector such as commonly used pVAX1 is used to insert the target gene into the vector to construct a eukaryotic expression vector, which is injected intramuscularly into the mink leg muscles , Mink growth speed can be obtained, and the results of weight gain are obvious. The full sequence of mink GHRH provided by the present invention can also be used to express it in a large amount in a conventional expression system to obtain mink GHRH, which is used to prepare active substances that can promote the growth of mink, an economic animal.

Description of drawings

Fig. 1 is the PCR identification figure of the recombinant plasmid that the embodiment of the present invention constructs, wherein 1: DL-2000plus Marker (5kb, 3kb, 2kb, 1kb, 750bp, 500bp, 250bp, 100bp); 2,3: Negative clone; 4, 5: Positive clone.

Fig. 2 is the recombinant plasmid pMD19-GHRHS digestion identification map constructed in the embodiment of the present invention, wherein 1: DL-2000 Marker (2kb, 1kb, 750bp, 500bp, 250bp, 100bp); 2: plasmid pMD19-GHRHS; 3: PCR Identification; 4: negative control; 5: Sph I single cut; 6: BamH I single cut; 7: Sph I+BamH I double cut.

Fig. 3 is the recombinant plasmid pVAX1-GHRHS enzyme digestion identification diagram constructed in the embodiment of the present invention, wherein 1: DL-2000 Marker (2kb, 1kb, 750bp, 500bp, 250bp, 100bp); 2: plasmid pVAX1-GHRHS; 3: Not I single cut; 4: BamH I single cut; 5: PCR identification; 6: negative control; 7: Not I+BamH I double cut.

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example:

Taking the nucleotide sequence of mink GHRH mature peptide as the target gene and pVAX1 as the eukaryotic expression vector, the target gene is inserted into the vector to construct a eukaryotic expression vector, and injected intramuscularly into the leg muscles of the mink to obtain the growth rate of the mink Accelerated, noticeable results in weight gain.

The vector construction process is as follows: the signal peptide gene sequence of human growth hormone and the mature peptide gene sequence of mink growth hormone releasing hormone were connected in series by PCR method, connected to the T vector (pMD19-GHRHS), and then double-linked with BamH I and EcoR I respectively. Cut the target gene on the pMD19-GHRHS vector and pVAX1 empty vector. The target gene was cloned into the pVAX1 vector, and after identification by PCR and sequencing (see Figure 1), the pMD19-GHRHS and pVAX1-GHRHS vectors were identified by enzyme digestion (see Figures 2 and 3). It was confirmed that the pVAX1-GHRHS expression vector was obtained. The purified expression vector was injected into the leg muscles of 30 2-4 month-old young minks at 20 μg-30 μg/mint, and weighed when the skin was taken. The average weight gain of the experimental group was as high as 300 g compared with the control group.

Among them, the upstream primer containing the human growth hormone signal peptide: ATGGCTACAGGCTCCCGGAC (as shown in SEQ ID No. 7); the downstream primer: CTATCAGAGTCGTACCTTTGCT (as shown in SEQ ID No. 8); PCR reaction conditions: 95 ° C for 10 min, 94 ° C for 30 sec , 53°C for 30sec, 72°C for 30sec for 30 cycles; 72°C for 10min; enzyme digestion conditions: 37°C.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

references

1. Gracia-Navarro F, JP, Malagon MM, Sánchez-Hormigo A, Luque RM, Hickey GJ, Peinado JR, Delgado E, Martínez-Fuentes AJ. Research progress in the stimulating inputs regulating growth hormone (GH) secretion. Comp BiochemPhysiol B Biochem Mol Biol. 2002132(1 ): 141-50.

2. Currie B.L, Johansson K.N., Greibrokk T., Folkers K., Bowers C.Y. Identification and purification of factor A-GHRH from hypothalami which releases growth hormone. Biochem. Biophys. Res. Commun. 1974, 60(2): 605-9 .

3. Draghia-Akli R, Fiorotto ML. A new plasma-mediated approach to supplement somatotropin production in pigs. J Anim Sci. 200482(13_suppl): E264-9.

4. Chen Liyin, Guo Rongfu, Xiang Zhentian, Feng Yu. Application research of pGRF gene plasmid in pig production, Porcine Industry Science, 2006 8: 14-6.

5. Zhang Yong, Zhou Anguo, Wu De, Zhu Yujing, Wang Kangning, Yang Feng. Study on growth-promoting and metabolic regulation effects of pGRF gene plasmid injected into piglet skeletal muscle, Journal of Animal Husbandry and Veterinary Medicine, 2003 34(2): 147-51.

6. Zhang Liyu, Sun Baozhong, Wang Min, Chong Jinghua. Research progress of growth hormone releasing factor (GRF) gene plasmid, Heilongjiang Animal Husbandry and Veterinary Medicine, 2006 9: 19-21.

7. Draghia-Akli R, Cummings KK, Khan AS, Brown PA, Carpenter RH. Effects of plasma-mediated growth hormone releasing hormone supplementation in young, healthy Beagle dogs. J Anim Sci. 200381(9): 2301-10.

8.Meng QY, Chen ZQ, Yu ZQ, Xie QF, Li N.Increased body weight viainjecting myogenic Expression gowth hormone-releasing hormone(GHRH)plasmidDNA into sheep.Anim Biotech.2004 15(2):175-92.

Claims (9)

1. a mink growth hormone releasing hormone, characterized in that its amino acid sequence is as shown in SEQ ID No.1. 2. A mink growth hormone releasing hormone precursor peptide, characterized in that its amino acid sequence is as shown in SEQ ID No.2. 3. the cDNA of the mink growth hormone releasing hormone described in coding claim 1 is characterized in that, its nucleotide sequence is as shown in SEQ ID No.3. 4. the cDNA of the mink growth hormone releasing hormone precursor peptide of coding claim 2, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.4. 5. An expression vector comprising the cDNA according to claim 3 or 4. 6. A host containing the expression vector of claim 5. 7. the application of mink growth hormone releasing hormone described in claim 1 in promoting the growth of mink. 8. the application of the cDNA described in claim 3 or 4 in the preparation of the active substance that promotes mink growth. 9. An active substance for promoting the growth of mink, characterized in that it contains the mink growth hormone releasing hormone as claimed in claim 1.

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