Human growth hormone fusion protein and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically relates to a human growth hormone fusion protein and its application. The fusion protein hGH-linker-HSA and the hGH mutant fusion protein hGHmutF31Q / V90E-linker-HSA are constructed by connecting hGH and HSA through a connecting peptide. Mouse experiments show that hGHmutF31Q / V90E-linker-HSA can improve the metabolic stability of hGH, prolong the half-life of hGH, and enhance the administration effect of hGH, and has a broad application prospect in clinical practice.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a human growth hormone fusion protein and an application thereof. Background Art

[0002] Growth hormone is a protein secreted by the anterior pituitary gland of the human body. It is composed of 191 amino acids and has the functions of promoting growth, regulating substance metabolism and degrading fat. It can promote bone growth and plays a very important role in human growth and development. If growth hormone is lacking, it can cause slow growth and development of children and cause short stature. Now recombinant human growth hormone has the same effect as human growth hormone, and can also promote the growth of bones, internal organs and the whole body. It is often used to treat children's dwarfism and dwarfism.

[0003] However, since its half-life in the body is only about 0.5 to 2 hours, it needs daily, long-term injections (several years, 5-10 years) to exert its therapeutic effect. The long treatment cycle requires longer-acting drugs. For this reason, the market is in urgent need of long-acting drugs, especially those suitable for children. Genetically modified growth hormone and fusion proteins are currently the most promising research directions.

[0004] The main factors that affect the application of hGH are the following three aspects: (1) Natural human growth hormone is very rare, so large-scale production using genetic engineering methods is of great significance to meet the large clinical demand. (2) Pharmaceutical-grade recombinant human growth hormone products have high purity requirements. How to purify them efficiently and with high purity? (3) Natural growth hormone has a short half-life in the body. It is difficult to extend the half-life of growth hormone and increase the residence time of the drug in the tissue to achieve long-term effect and reduce the frequency of administration. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a recombinant human growth hormone, a construction method thereof, and an application thereof.

[0006] In a first aspect, the present invention provides a fusion protein of human growth hormone, denoted as hGH-linker-HSA and hGHmutF31Q / V90E-linker-HSA.

[0007] Wherein linker is a commonly used connecting peptide in the art;

[0008] Preferred linkers can be (EAAAK)3, (AP)5, (GGGGS)3, S3N 10 Any of .

[0009] A more preferred linker is (AP)5.

[0010] The amino acid sequence of hGH is shown in SEQ ID NO:1.

[0011] SEQ ID NO: 1:

[0012] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.

[0013] The amino acid sequence of hGHmutF31Q / V90E is shown in SEQ ID NO:2.

[0014] SEQ ID NO:2:

[0015] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEQEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPEQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF.

[0016] The nucleotide sequence of hGH after codon optimization is shown in SEQ ID NO:3.

[0017] SEQ ID NO:3:

[0018] TTCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCGGGCCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGTTTGAAGAGGCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAACACAACAGAAATCCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCCAGTCGTGGCTCGAGCCCGTGCAGTTCCTGAGGAGTGTCTTCGCCAACAGCCTGGTCTACGGCGCCTCTGATTCGAACGTGTACGACCTGCTGAAGGACCTAGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCGCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCT TCTAG。

[0019] The nucleotide sequence of hGHmutF31Q / V90E is shown in SEQ ID NO:4.

[0020] SEQ ID NO:4:

[0021] TTCCCAACTATACCACTAAGTCGACTATTCGATAACGCTATGCTTCGGG

[0022] CCCATCGTCTTCATCAGCTAGCCTTTGACACCTACCAGGAGCAGGAAGAG

[0023] GCCTATATCCCCAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGAC

[0024] CTCCCTCTGTTTCTCAGAATCGATTCCGACACCCTCCAATCGCGAGGAAAC

[0025] ACAACAGAAATCCAACCTAGAGCTCCTCCGCATAAGCTTGCTGCTCATCC

[0026] AGTCGTGGCTCGAGCCCGAGCAGTTCCTGAGGAGTGTCTTCGCCAACAGC

[0027] CTGGTCTACGGCGCCTCTGATTCGAACGTGTACGACCTGCTGAAGGACCT

[0028] AGAGGAAGGGATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCG

[0029] CGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTC

[0030] ACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCA

[0031] GGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCT TCTAG。

[0032] The amino acid sequence of the HSA is shown in SEQ ID NO:5.

[0033] SEQ ID NO:5:

[0034] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL。

[0035] The nucleotide sequence of the HSA is shown in SEQ ID NO: 6.

[0036] SEQ ID NO: 6:

[0037]

[0038] The nucleotide sequence of (AP)5 is shown in SEQ ID NO:7.

[0039] SEQ ID NO:7: GCTCCAGCTCCAGCTCCAGCTCCAGCTCCA.

[0040] The second aspect of the present invention provides a method for constructing a fusion protein of human growth hormone, wherein human growth hormone or its mutant is sequentially connected with a rigid linker peptide (AP) 5 and HSA to form hGH-(AP)5-HSA and hGHmutF31Q / V90E-(AP)5-HSA fusion plasmids.

[0041] The fusion protein of the present invention is based on the amino acid sequence, utilizing the degeneracy of the amino acid codons, and based on the codon preferences of different expression hosts, including Escherichia coli, yeast, CHO cells, and other expression hosts. Those skilled in the art can encode different nucleotide sequences so that they can be efficiently expressed in different hosts. The nucleotide sequence designed by the present invention can be chemically synthesized or obtained by PCR. The pET series expression vectors that are efficiently expressed in Escherichia coli, the pPIC9k expression vectors that are expressed in yeast, and the pcDNA3.1 expression vectors that are expressed in CHO cells are selected.

[0042] The specific preparation method is: culturing the engineered bacteria, collecting the bacteria by centrifugation, breaking the bacteria, collecting the inclusion bodies, washing, denaturing and renaturing the inclusion bodies, and purifying them using a cationic chromatography medium and an anionic chromatography medium respectively to obtain a sample with a purity of more than 95%.

[0043] The third aspect of the present invention provides the application of the above fusion protein. The fusion protein prepared by the present invention can be used for the preparation of various growth hormone drugs, and can also be used for the treatment and prevention of growth hormone deficiency diseases.

[0044] Preferred growth hormone deficiency diseases are childhood short stature or dwarfism;

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] The fusion protein hGHmutF31Q / V90E-(AP)5-HSA provided by the present invention can improve the metabolic stability of hGH, prolong the half-life of hGH, and enhance the administration effect of hGH, and has a broad application prospect in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1: Electrophoresis of recombinant plasmid, where band 1 is hGH-(AP)5-HSA and band 2 is hGHmutF31Q / V90E-(AP)5-HSA.

[0048] Figure 2 : SDS-PAGE image of fusion protein, wherein band 1 is hGH-(AP)5-HSA and band 2 is hGHmutF31Q / V90E-(AP)5-HSA.

[0049] Figure 3 : Changes of serum growth hormone levels in mice under each group of drug administration.

[0050] Figure 4 : Changes in body weight of mice under each group of drug administration. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0052] In the following examples, various processes and methods not described in detail are conventional methods known in the art. The sources, trade names and components of the reagents used are indicated when they first appear, and the same reagents used thereafter are the same as the first indicated unless otherwise specified; the reagents and materials involved are all obtained from commercial sources unless otherwise specified.

[0053] Example 1 Construction of recombinant plasmid hGH-(AP)5-HSA

[0054] According to the amino acid map, a group of amino acids that may affect the spatial structure of hGH enzyme were screened and mutated, and the nucleotide sequence of hGHmutF31Q / V90E was obtained by site-directed mutagenesis technology.

[0055] Sangon Biotechnology (Shanghai) Co., Ltd. fully synthesized hGH-(AP)5-HSA and hGHmutF31Q / V90E-(AP)5-HSA, wherein the nucleic acid sequence of hGH is shown in SEQ ID NO:3; the nucleotide sequence of hGHmutF31Q / V90E is shown in SEQ ID NO:4; the nucleotide sequence encoding HSA is shown in SEQ ID NO:6; (AP)5 is a connecting peptide, and its nucleic acid sequence is shown in SEQ ID NO:7.

[0056] The synthesized gene fragment was linked to the E. coli plasmid PET30a(+) plasmid by double enzyme post-digestion overnight and then analyzed by 2% agarose gel electrophoresis. Figure 1A band of about 7.8 kb in size was visible, indicating that the recombinant expression plasmid PET30a(+)-hGH-(AP)5-HSA and hGHmutF31Q / V90E-(AP)5-HSA recombinant plasmid were successfully constructed.

[0057] Example 2 Expanding the Culture of Escherichia coli Expressing hGH-(AP)5-HSA

[0058] The freshly transformed recombinant Escherichia coli BL21 was transferred to solid LB medium (agar powder 15 g / L, tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.4) with 50 μg / mL kanamycin and cultured overnight at 37°C. Then, a single colony after overnight culture was transferred to 5 ml of liquid LB medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.4) containing 50 μg / mL kanamycin and cultured overnight in an orbital shaker at 37°C and 200 rpm. Subsequently, 3 mL of the overnight cultured Escherichia coli BL21 was inoculated into 300 mL of liquid LB medium containing 50 μg / mL kanamycin and grown at 37°C, 200 rpm for 3 hours or until the OD600 of the bacteria reached between 0.4 and 0.6.

[0059] The cell culture fluid was centrifuged to collect the supernatant, and the residual cell debris was removed by filtering with a 0.22 μm filter membrane. The collected cell culture fluid was purified using a Protein A chromatography column, and the target peak was collected. It was further purified by anion exchange chromatography, and the protein was finally eluted and collected with 0.02 M PBS. The sample was tested by 12% SDS-PAGE electrophoresis, and the electrophoresis pattern showed a single band, such as Figure 2 As shown, the size is about 88KDa, which is basically consistent with the theoretical molecular weight, indicating that both fusion proteins were successfully expressed in the E. coli expression system.

[0060] Example 3 Fusion protein effect verification

[0061] Within 2 days, young rats (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were surgically removed from the pituitary gland via the parapharyngeal approach under sterile conditions. Each rat was marked and weighed, and qualified animals were screened according to the Chinese Pharmacopoeia method 2 weeks later. Chinese Pharmacopoeia method: Compared with the preoperative body weight, the body weight change of pituitary-removed rats within 2 weeks (0-14 days) after surgery was less than 10% of that of healthy rats.

[0062] Qualified pituitary-ectomized rats screened by the above method were randomly divided into a blank control (normal saline) group, an hGH experimental group (1 mg / kg), an hGH-(AP)5-HSA group (1 mg / kg), and an hGHmutF31Q / V90E-(AP)5-HSA group (1 mg / kg), with 20 rats in each group.

[0063] The specific experimental steps are:

[0064] The rats were fasted from food and water on the morning before the experiment. After 24 hours, the rats in each group were given the corresponding drugs at a dosage of 1 mg / kg. The injection site was the quadriceps muscle of the left hind limb of the rats, and the injections were continued for 3 consecutive days. The rats were weighed at the same time after 3 administrations, and the weight gain of the rats was calculated compared with the weight before administration, and the growth curve was drawn. At the same time, 5 rats were randomly selected from each group on the 1st, 5th, 10th, 15th and 20th days after the 3 injections, and blood was drained from the eyeballs. The serum was separated and placed at -20℃ for testing.

[0065] The growth hormone content in serum was measured by growth hormone assay kit. Figure 3-Figure 4 shown.

[0066] Depend on Figure 3 It can be seen that the rate of growth hormone reduction in mice treated with hGHmutF31Q / V90E-(AP)5-HSA was significantly lower than that in other groups, indicating that it can significantly increase the half-life of hGH. At the same time, the weight gain of mice treated with hGHmutF31Q / V90E-(AP)5-HSA from 0 to 20 days was significantly higher than that in other groups (such as Figure 4 ), indicating that the fusion protein mutant significantly enhanced the administration effect of growth hormone.

[0067] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A growth hormone fusion protein, characterized in that: Its structure is hGHmutF31Q / V90E-linker-HSA, the amino acid sequence of HSA is shown in SEQ ID NO:5, and the amino acid sequence of hGHmutF31Q / V90E is shown in SEQ ID NO:

2.

2. The fusion protein according to claim 1, wherein the linker is (EAAAK)3, (AP)5, (GGGGS)3, S3N 10 Any of . The fusion protein according to claim 2 , wherein the linker is (AP)5.

4. The fusion protein according to claim 1, wherein the nucleotide sequence of HSA is shown in SEQ ID NO:

6.

5. The fusion protein according to claim 1, wherein the nucleotide sequence of the linker is shown in SEQ ID NO:

7.

6. The method for preparing the fusion protein according to claim 1, comprising the following steps: (1) Connecting the human growth hormone mutant to the linker and HSA in sequence to form the hGHmutF31Q / V90E-linker-HSA fusion plasmid; (2) introducing the fusion plasmid into the host expression system and expanding the culture in the culture medium; (3) Isolate and purify the target fusion protein.

7. The preparation method according to claim 6, wherein the expression system is any one of CHO cells, Escherichia coli or yeast.

8. A growth hormone drug, comprising the fusion protein according to any one of claims 1 to 5 or the fusion protein prepared by the method according to any one of claims 6 to 7.

9. The drug according to claim 8, further comprising a pharmaceutically acceptable auxiliary material and / or carrier.

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