CN113444708B - Hyaluronidase mutant for subcutaneous injection preparation of medicine - Google Patents
Hyaluronidase mutant for subcutaneous injection preparation of medicine Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses a hyaluronidase mutant for a medicine subcutaneous injection preparation, and belongs to the technical field of genetic engineering. The invention carries out amino acid site-directed mutagenesis on human hyaluronidase PH20 to construct a hyaluronidase mutant which is modified by low glycosylation and has improved catalytic activity; according to the invention, four species with different sources are selected, comparison of hyaluronidase protein sequences of the four species is carried out by using BIOXM, 11 important amino acid sites influencing the activity of hyaluronidase are determined, according to the distribution positions of the sites on the prediction of the secondary structure of the hyaluronidase and the properties of amino acids of the sites, a mutant sequence and activity analysis are finally determined, and N1-N4 total 4 hyaluronidase mutants which are free of glycosylation modification and retain catalytic activity are obtained, wherein the mutant can retain activity under the condition of reducing glycosylation sites; the mutant is favorable for the diffusion of the medicine in subcutaneous tissues.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a hyaluronidase mutant for a medicine subcutaneous injection preparation.
Background
Hyaluronic acid is a kind of viscous polysaccharide, it is a straight-chain high molecular polysaccharide formed by repeatedly connecting disaccharide units, its disaccharide units are formed from N-acetylglucosamine and D-glucuronic acid, and are connected by beta-1, 3 glycosidic bond, and these two monosaccharides are formed according to the mole ratio of 1:1, and between disaccharide units are connected by beta-1, 4 glycosidic bond, and are extensively existed in the extracellular matrix of animal connective tissue. It is a component of the tissue matrix that limits the diffusion of moisture and other extracellular substances, and hyaluronic acid can hinder the diffusion of drug molecules when performing drug therapy. Hyaluronidase (HAase) is a generic term for enzymes that can lower the molecular weight of hyaluronic acid, and which can reduce the activity and viscosity of hyaluronic acid and increase the fluid permeability in tissues. HAase is widely used as an osmotic agent (diffusion factor) of drugs in clinic at present, and can promote drug absorption by combining with the drugs.
The hyaluronidase in the current market is mainly obtained by three methods of extracting from bovine and sheep tissues, namely, recombinant human hyaluronidase expressed by yeast expression and CHO expression systems, the purity of the hyaluronidase extracted from the animal tissues is lower, the immunogenicity is higher, the yeast expression amount is lower, the cost of the CHO expression systems is higher, and a low-cost prokaryotic expression system is lacking at present, mainly because the hyaluronidase needs to be subjected to N-glycosylation to have activity, and the glycosylation process can only be completed in a eukaryotic expression system, and the prokaryotic expression system cannot be subjected to glycosylation modification process.
Hyaluronidase has been used in various fields, and the U.S. FDA has approved hyaluronidase as a dispersing agent for promoting the diffusion and absorption of drugs, and has been used as an anesthetic aid, often in combination with anesthetic drugs, to enhance anesthetic effects. Hyaluronidase is also used for rapid absorption of injection, and besides, because tumor cells can secrete HA and HAase simultaneously, and secretion is enhanced with metastasis of tumor cells, hyaluronidase is also used as an ideal marker for tumor detection. Hyaluronidase can promote metabolism, relieve vascular damage and edema of cells and tissues, and increase side circulation, so that hyaluronidase can also be used for relieving ischemia injury and reducing myocardial infarction probability. Most of the HAases used in the market today are extracted from mammalian testes, and have limited sources, low yield and high cost, and lack of effective prokaryotic expression systems due to glycosylation limitations, and are subject to immunogenicity and purity limitations, allergic reactions and rapid elimination reactions in clinical applications, and are greatly limited in clinical applications.
Disclosure of Invention
The invention aims to provide a hyaluronidase mutant for a medicine subcutaneous injection preparation, which solves the problems in the prior art, and the invention selects a mammalian cell 293T cell to express human hyaluronidase PH20, selects an amino acid site with great influence on the activity of the hyaluronidase through analyzing the primary structure and the secondary structure prediction, constructs the hyaluronidase mutant through an amino acid site-directed mutagenesis technology, and carries out transient transfer of a recombinant expression plasmid of the hyaluronidase mutant protein into the cell through a PEI transfection reagent to carry out protein expression. Finally, comparing the activity change condition of each mutant of the hyaluronidase through activity analysis, and finally obtaining the hyaluronidase which has activity without glycosylation.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a hyaluronidase mutant for a medicine subcutaneous injection preparation, which is prepared by carrying out amino acid site-directed mutation on human hyaluronidase PH20, and the sequence of the mutation is as follows: N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S; wherein the hyaluronan mutant has a reduced amount of glycosylation and retains catalytic activity.
Preferably, the hyaluronidase mutants are respectively:
N1：PH20-N47S/N131D/N200S/N219K/Q234L/I228V
N2：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T
N3：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I
N4：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S
preferably, the target gene amplification primers of N1-N4 are shown in SEQ ID NO. 5-26.
The invention also provides application of the hyaluronidase mutant, and the hyaluronidase mutant can be used for preparing a pharmaceutical composition for treating diseases or symptoms of hyaluronidase substrate accumulation.
Preferably, the pharmaceutical composition is formulated for oral administration, or Intravenous (IV), subcutaneous, intramuscular, intratumoral, intradermal, topical, transdermal, rectal or topical subcutaneous injection.
Preferably, the pharmaceutical composition comprises an Immunoglobulin (IG), a recombinant protein, a synthetic polypeptide, RNA, DNA or a chemical drug.
Preferably, the pharmaceutical composition is formulated for subcutaneous administration in an amount sufficient to render the pharmaceutical composition therapeutically effective.
The invention discloses the following technical effects:
according to the invention, four species with different sources are selected, comparison of the hyaluronidase protein sequences of the four species is performed by using BIOXM, 11 important amino acid sites influencing the activity of the hyaluronidase are determined, and total 4 hyaluronidase mutants of N1-N4 are finally obtained according to the distribution positions of the sites on the prediction of the secondary structure of the hyaluronidase and the properties of the amino acids of the sites.
The molecular transformation of hyaluronidase is mainly realized by applying the amino acid site-directed mutagenesis technology. Firstly amplifying a human hyaluronidase sequence by PCR, designing primers according to amino acid sites to be mutated, and sequentially constructing each hyaluronidase mutant by using a point mutation technology. The double enzyme cutting PTT5 plasmid is connected with target gene through homologous recombination to construct mutant protein recombinant expression vector, the constructed recombinant expression vector plasmid is instantaneously transferred into 293T cells for continuous subculture, the cultured cells are collected, PBS is used for proper resuspension, after ultrasonic disruption, the disrupted supernatant is purified through His column, and finally the mutant protein is obtained. The activity change of the mutant protein was measured by DNS. Reducing sugar in the HA product of the degradation of DNS by hyaluronidase in alkaline solution is reduced into amino compound, a color reaction can occur after boiling water bath, the equivalent of reducing sugar in enzymatic reaction liquid can be measured by measuring the absorbance value at A540, and the hyaluronidase activity is measured by the method. And further screening out amino acid sites with great influence on the activity of the hyaluronidase, and finally obtaining the human hyaluronidase which has activity without glycosylation.
Drawings
FIG. 1 is a scheme of a hyaluronidase molecular engineering technique;
FIG. 2 is a hyaluronidase secondary structure analysis, A hyaluronidase glycosylation site distribution, B four species secondary structure alignment, C four species evolutionary tree construction, D four species sequence alignment equivalence analysis;
FIG. 3 shows the results of sequence alignment of hyaluronidases of four species;
FIG. 4 is a hyaluronidase homology modeling secondary structure prediction;
FIG. 5 shows the amino acids (D111, E113, E249) C of the active center site of the protein α An atomic trajectory change situation map;
FIG. 6 is a graph showing statistics of changes in active pockets of each mutant;
FIG. 7 shows the charge change of mutant proteins;
FIG. 8 shows a construction scheme for hyaluronidase and its mutants;
FIG. 9 is a schematic representation of each mutant;
FIG. 10 shows the PCR results of colony constructed by PH20 protein recombinant expression vector, wherein lanes 1-5 are the results of agarose gel separation of colony constructed by PH20 protein recombinant expression vector;
FIG. 11 shows hyaluronidase and mutant activity thereof.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
The reagents and equipment used in the present invention are, unless otherwise specified, commercially available products of ordinary use or available to those skilled in the art by the disclosed route.
In order to obtain the active hyaluronidase without glycosylation expressed by a prokaryotic expression system, FIG. 1 is a technical route of the invention, and the sequence of the human hyaluronidase is obtained from a Genbank database and is used as a template to carry out primary sequence alignment with the hyaluronidase sequence of cattle and bees which do not need glycosylation and are active, 4 glycosylation sites and 7 amino acid sites related to the activity are successfully found, and the mutation sequence of the 11 amino acid sites is finally determined by analyzing the properties of amino acids of the mutation sites and consulting literature: N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S, the hyaluronidase was molecularly engineered according to the order of mutation.
The molecular modification of hyaluronidase mainly uses amino acid site-directed mutagenesis technology. Firstly amplifying a human hyaluronidase sequence by PCR, designing primers according to amino acid sites to be mutated, and sequentially constructing each hyaluronidase mutant by using a point mutation technology. The double enzyme cutting PTT5 plasmid is connected with target gene through homologous recombination to construct mutant protein recombinant expression vector, the constructed recombinant expression vector plasmid is instantaneously transferred into 293T cells for continuous subculture, the cultured cells are collected, PBS is used for proper resuspension, after ultrasonic disruption, the disrupted supernatant is purified through His column, and finally the mutant protein is obtained. The activity change of the mutant protein was measured by DNS. Reducing sugar in the HA product of the degradation of DNS by hyaluronidase in alkaline solution is reduced into amino compound, a color reaction can occur after boiling water bath, the equivalent of reducing sugar in enzymatic reaction liquid can be measured by measuring the absorbance value at A540, and the hyaluronidase activity is measured by the method. And further screening out amino acid sites with great influence on the activity of the hyaluronidase, and finally obtaining the human hyaluronidase which has activity without glycosylation.
Example 1 selection of hyaluronidase mutation sites
1. Determination of mutation sites
According to the known literature, it is known that hyaluronidase contains 6 glycosylation sites (Asn 47, 131, 200, 219, 333, 358), these six glycosylation sites are greatly different in the type and proportion of glycosylation modification, and the positions of these six glycosylation sites on the secondary structure of hyaluronidase are shown in fig. 2A below, wherein the glycosylation modification sites at Asn333 and Asn358 are located at the C-terminal end of hyaluronidase, and the proportion of high-sugar-exposure modification is high. The invention selects cattle (> AAP 5571.1), hornet (> CBY 83816.1), bees (> AAAP 55713.1) and three hyaluronidase species sequences with higher activity are compared with human PH20 (> NP-001167516.1) sequences, and a evolutionary tree (figure 2C) is constructed for homology analysis.
The results of the sequence alignment of the hyaluronidases of the four species are shown in FIG. 3. According to the comparison result of the hyaluronidase sequences of four different species, the dark gray parts are the positions of six glycosylation sites of the hyaluronidase active site amino acid and the hyaluronidase, the glycosylation sites are required to be mutated in order to finish the process that the hyaluronidase is not glycosylated, and according to the analysis result of the homology of the evolutionary tree, the homology of the human and the bovine hyaluronidase is higher, so that the mutation site amino acid is selected by taking the bovine hyaluronidase sequence with higher homology as a main part, and taking the hyaluronidase sequences of the bees and the wasps as an auxiliary part to carry out the design of the glycosylation site mutant. The N47, 131, 219 mutant amino acids were selected to correspond to the amino acid at this site in bovine hyaluronic acid, and the N47 mutation was S, N to D, N219 to K. The N200 mutant amino acid is selected to be identical to the amino acid at the site of bee hyaluronic acid, and N200 is mutated to S. In addition, the glycosylation sites at N333 and 358 are positioned at the tail end of the human hyaluronidase sequence and at the back of the active pocket, so that the two glycosylation sites are discarded, and finally the four glycosylation sites of the mutation are determined as N47S, N131D, N200S, N219K.
The amino acid in gray and high light is the screened sites which have possible influence on the hyaluronidase activity, the sites have high homology on the hyaluronidase sequences of cattle, bees and wasps, and the sites are close to the catalytic pocket in secondary structure. By structural analysis of the homologous modeling model of each mutant of the hyaluronidase, the active pockets of each mutant are different in change, and the possibility of high activity exists.
The secondary structures of the hyaluronidases of the four species were aligned, as in FIG. 2B, with a larger portion of overlap at the active pocket, and sequence identity E<10 -5 As shown in FIG. 2D, four species are shown to have higher homology, and where human and bovine homology is highest, it is believed that amino acid positions selected to potentially increase hyaluronidase are based on sequence alignment.
2. Determination of the sequence of mutations
As shown in fig. 4, according to the homology modeling secondary structure prediction analysis (blue is an alpha helix structure and green is a beta sheet structure), the hyaluronidase activity pocket is formed by random coil formed by 111-118 amino acid peptide fragments and alpha helix formed by 240-245 peptide fragments and beta sheet layer folds nearby the alpha helix, and the probability of influencing the hyaluronidase activity after mutation of the amino acid position nearer to the activity pocket is higher, and besides, the property change of the amino acid at the mutation position also influences the activity of the hyaluronidase mutant protein. According to the prediction of the secondary structure of the hyaluronidase, glycosylation sites 47, 131, 200 and 219 are defined as primary mutation sites, two amino acid sites 228 and 234 are the sites with highest weight for influencing the catalytic reaction domain-alpha helical folding, the two amino acid sites are defined as secondary mutation sites, and 53, 279, 293, 302 and 306 are amino acid sites with higher weight for influencing the folding of the beta sheet structure of the active pocket and are defined as tertiary mutation sites. In addition to this, human-carried hyaluronidase sequences have a high degree of homology in bovine, bee and hornet sequences, such as I83, S84, E285, M323, which are all located in the random coil structure between the alpha helix and the beta sheet that form the active pocket, so that, irrespective of these sites, the three-stage mutation sequence was initially determined as follows, depending on the nature of the amino acids, hydrophilicity and hydrophobicity, charge conditions and review of the relevant literature: T293L-K279T-V53I-V302I-T306S.
The following four mutants were established:
N1：PH20-N47S/N131D/N200S/N219K/Q234L/I228V
N2：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T
N3：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I
N4：PH20-N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S
3. mutant active pocket change analysis
Human PH20 gene sequence is found from NCBI, the signal peptide part of the 35 amino acid sequence at the N end is removed, the C end glycosyl phosphatidylinositol modification site is removed, and 447 amino acids in the 35 th amino acid to 482 th amino acid intermediate sequence are selected as target protein expression sequences.
The PH20 nucleotide sequence is shown as SEQ ID NO. 1.
Inputting 4 mutant protein sequences into https:// swissmodel. Expasy. Org/interactive website for model construction according to mutation sequence, opening a PDB format model file by using PDB-VIEWEER, and analyzing amino acids (D111, E113, E249) at active center sites of each protein α The change of the atomic trajectory is shown in fig. 5.
The above 4 glycosylation site mutant protein activity pocket changes were counted as shown in FIG. 6. According to analysis, the change of the activity pocket of each mutant is different, and the change of the protein activity is unpredictable. The change in hyaluronidase activity was verified by experiments.
4. Analysis of charge changes in mutant proteins
As shown in FIG. 7, the charge change of the mutant protein was measured by inputting the mutant protein into Discovery, the change of the surface charges of PH20 and N1 proteins was large (red represents positive charge, blue represents negative charge), the isoelectric points of PH20 and N1 proteins were not changed by 8.6, but the electrostatic potential was changed, N0:1710.43 N1:1780.52 potential docking of substrate to the active center of hyaluronidase, increased protein potential, higher probability of binding to substrate, and the rest of mutant docking were also analyzed as described above, depending on the change in mutant surface charge. The construction of the above 4 glycosylation site mutants was finally determined.
EXAMPLE 2 construction, expression and Activity studies of hyaluronidase and its mutants
1. Experimental materials
And (3) cells: 293T cells are supplied by this laboratory; and (3) strain: e.coli TG1 laboratory supplies; plasmid: PTT5 plasmid, vast organism purchase; the gene of interest: the gene PH20 was synthesized by Kirschner.
2. Experimental method
2.1 construction of hyaluronidase and mutant proteins thereof
The invention mainly constructs the recombinant expression vector of the hyaluronidase through homologous recombination connection, and verifies through colony PCR and sequencing, and the technical route of the construction of the hyaluronidase and mutant proteins thereof is shown in figure 8.
Construction of 2.2PH20 protein recombinant expression vector
Optimization and synthesis of 2.2.1PH20 gene
The N-terminal signal peptide part and the C-terminal GPI site are removed from the PH20 gene sequence downloaded from NCBI, and the luciferase signal peptide is added to the N-terminal, and the amino acid sequence is MGVKVLFALICIAVAEA. His tag is added at the C end, so that the subsequent protein expression detection and purification operation are facilitated. The PH20 gene is codon optimized according to the codon preference of the mammalian cells, and the amino acid of the optimized sequence is unchanged. And (3) sending the optimized target gene sequence to Nanjing Jinsri biological company for gene synthesis, and constructing the target gene on a PUC-57 plasmid vector, wherein the optimized target gene sequence is shown as SEQ ID NO. 2.
The invention constructs the hyaluronidase expression vector by utilizing homologous recombination connection, the homologous recombination connection is a DNA directional cloning technology, and the PCR recovery product of the inserted fragment can be directionally cloned to any position of any vector by utilizing the directional cloning technology, so that the method is simple, quick and efficient.
(5) The activity of the obtained human hyaluronidase mutant protein was measured by the DNS method.
The method comprises the following specific steps:
( 1) A gene fragment encoding human hyaluronidase was obtained, the gene sequence (SEQ ID No.1, genBankAccession number: NP-001167516.1 )
(2) And (3) connecting the GLU signal peptide sequence to the 5 'end of the human hyaluronidase sequence, removing the GPI site at the 3' end, connecting the obtained SEQ ID No.2 gene sequence to a PTT5 (SEQ ID No. 3) carrier, and transferring the obtained product into 293T cells for expression to obtain the human hyaluronidase protein.
(3) The gene sequence of SEQ ID NO.2 is modified through site-directed mutagenesis to obtain a glycosylation complete deletion mutant (SEQ ID NO. 4), which is connected to a PTT5 carrier and transferred into 293T cells for expression, thus obtaining the human hyaluronidase mutant protein.
(4) On the basis of the SEQ ID NO.4 gene sequence, modifying amino acid at a specific site to obtain a mutant strain N1-N4 gene sequence, connecting the mutant strain N1-N4 gene sequence to a PTT5 carrier, and transferring the mutant strain into 293T cells for expression to obtain the human hyaluronidase mutant protein.
(5) The activity of the obtained human hyaluronidase mutant protein was measured by the DNS method.
Example 3 construction of plasmid PTT5-PH20
The plasmid PTT5 is purchased from vast Propioneer, the gene expressing human hyaluronidase is subjected to PCR amplification, and the amplified product is incorporated between EcoRI/HindIII sites on the PTT5 plasmid to obtain the plasmid PTT5-PH20. The constructed sequence is sent to the biological engineering limited company for sequencing verification.
The construction process is as follows:
1. the human hyaluronidase gene fragment is obtained by amplification by using the primers PH20-F (SEQ ID NO. 5) and PH20-R (SEQ ID NO. 6) as primers and the human hyaluronidase sequence SEQ ID NO.2 as a template.
PCR cycling procedure (KOD-Plus-neo enzyme):
Step1：94℃2min
Step2：98℃10s
Step3：63℃30s
step4:68 ℃ for 45s (to step2, 40 cycles)
Step5：68℃5min
Detection of PCR products: the PCR product was taken in an amount of 3. Mu.L, and the result of agarose gel electrophoresis detection (as shown in FIG. 10) showed successful amplification of the human hyaluronidase gene fragment.
2. The PCR product obtained above is connected to PTT5 carrier by homologous recombinase and transferred into TG1 competent cells, and the specific operation method is as follows:
the PCR amplified SEQ ID NO.2 sequence was ligated to the double digested PTT5 vector using EcoRI and HindIII double digested plasmid PTT5 using homologous recombination enzymes to give PTT5-PH20, 10. Mu.L of the ligation product was added to 200mLTG1 competent cells, ice-bath was performed for 30min, heat shock was performed at 42℃for 90s, ice-bath was performed for 2min after heat shock was completed, 800. Mu.LLB liquid medium (no resistance) was added, 37℃was recovered by resuscitating at 140rpm for 45min,5000rpm was centrifuged to collect the cells, 900. Mu.L of the medium supernatant was discarded, the cells were resuspended and then plated (ampicillin resistance, 100 ng/. Mu.L) and recombinant bacteria were selected by colony PCR, and sequencing verification was performed.
3. The colony with correct sequencing is selected and cultured in 5mL LB liquid medium (ampicillin resistance, 100 ng/. Mu.L) at 37 ℃ for 15h at 180rpm, plasmid extraction is carried out by using a endotoxin removal kit, and the extracted plasmid is transiently transferred into 293T cells by using lip2000 for protein expression, and the specific operation is as follows:
adding 20 μg of plasmid and 20 μl of lip2000 into 48 μl of culture medium (DMEM high sugar culture medium) respectively, mixing, standing for 20min, dripping the mixture into a cell culture dish, continuously culturing for 3 days, collecting cells, performing ultrasonic disruption treatment, collecting cell disruption supernatant, vacuum filtering with 0.22 μm membrane, purifying with His affinity chromatography column, dialyzing, lyophilizing, etc. to obtain human hyaluronidase protein with purity of above 90%.
4. The activity of the obtained human hyaluronidase protein is measured, the human hyaluronidase activity is measured by adopting a DNS method, and the specific operation is as follows:
a standard curve was made using a 2mg/L glucose solution. The glucose solution volumes were respectively: 0.5, 7.5, 10, 12.5, 15, 17.5, 20. Mu.L, and adding to 1.5mL centrifuge tubes as requiredAdding glucose solution, supplementing 100 μL with PBS buffer solution, mixing, adding 200 μL DNS reagent into centrifuge tube, mixing, boiling for 10min, adding 700 μL ultrapure water, mixing, adding 200 μL mixed solution into 96-well plate, and measuring A with multifunctional enzyme-labeled instrument 540 Absorbance values.
The obtained human hyaluronidase protein was diluted with PBS buffer, 20. Mu.L of a predetermined amount of the protein dilution was taken, 20. Mu.L of a 0.5% HA solution was added thereto, the mixture was made up to 100. Mu.L with PBS, and the control was replaced with PBS. After being fully and uniformly mixed, the mixture is reacted for 10min at 37 ℃. After the reaction, the enzymatic reaction solution is placed on a metal bath at 100 ℃ for 5min, and the protein is deactivated. Immediately adding 200 mu L of DNS reagent into the enzymatic reaction solution, heating for 10min at 100 ℃ after uniformly mixing, adding 700 mu L of ultrapure water, fully and uniformly mixing, absorbing 200 mu L of mixed solution, adding into a 96-well plate, and measuring A by a multifunctional enzyme-labeled instrument 540 Absorbance values.
Glucose concentration is taken as abscissa, A 540 The absorbance at the position is taken as an ordinate, and a standard glucose amount calibration curve is drawn. According to A 540 The absorbance was calculated as the equivalent product of reducing sugar in the enzymatic reaction solution, and the hyaluronidase activity was measured.
EXAMPLE 4 construction and Activity determination of glycosylation site Total deletion mutant
Introducing amino acid mutation into PH20 target gene (SEQ ID NO. 2) by utilizing a site-directed mutagenesis technology, amplifying a full-length target gene template of mutant protein by overlap extension PCR, and constructing a recombinant expression vector. Glycosylation site variant construction the required primers were synthesized by Shanghai Biotechnology Co., ltd:
primer design: 47-F (SEQ ID NO. 7), 47-R (SEQ ID NO. 8), 131-F (SEQ ID NO. 9), 131-R (SEQ ID NO. 10), 200-F (SEQ ID NO. 11), 200-R (SEQ ID NO. 12), 219-F (SEQ ID NO. 13), 219-R (SEQ ID NO. 14).
The PCR reaction was performed using SEQ ID NO.2 as a template and the above primers to obtain SEQ ID NO.4, and the PCR cycling process and subsequent construction, purification, and activity measurement were performed as in example 3.
EXAMPLE 3 construction of Activity revertant mutants and Activity test
Seven-site mutation is respectively introduced into SEQ ID NO.4 sequence by utilizing a site-directed mutagenesis technology, and the full-length target gene template amplification of the mutant protein is carried out by overlap extension PCR, so as to construct a recombinant expression vector. The primers required for mutant construction were synthesized by Shanghai Biotechnology Co., ltd:
primer design: 234-228-F (SEQ ID NO. 15), 234-228-R293-F (SEQ ID NO. 16), 293-F (SEQ ID NO. 17), 293-R (SEQ ID NO. 18), 279-F (SEQ ID NO. 19), 279-R (SEQ ID NO. 20), 53-F (SEQ ID NO. 21), 53-R (SEQ ID NO. 22), 302-F (SEQ ID NO. 23), 302-R (SEQ ID NO. 24), 306-F (SEQ ID NO. 25), 306-R (SEQ ID NO. 26).
PCR reaction is carried out by using SEQ ID NO.4 as a template and the primers to obtain N1-N4 protein nucleotide sequences (shown as SEQ ID NO. 27-30), and the construction schematic diagram of each mutant is shown in FIG. 9. The PCR cycling process and subsequent construction, purification and activity determination procedures were the same as in example 3. The activities of the mutants are shown in FIG. 11 and Table 1, and the activity recovery mutant N3 activity was measured to be 103.5U/mg at the highest and reached 70% of the activity of the wild-type human hyaluronidase.
TABLE 1 hyaluronidase and the respective mutant enzyme activities
Example 4 comparative studies of the effect of the biological permeation enhancer human hyaluronidase on enhancing tissue drug absorption using radio-tracer imaging.
The human hyaluronidase mutant N3 and a negative control physiological saline are respectively injected into healthy ICR mice through veins, the injection is carried out for 2 hours, a 99 mTc-marked tracer methoxyisobutyl isonitrile (99 mTc-semibi) is injected subcutaneously at the left or right thigh part, a gamma camera special for high resolution small animals is used for obtaining the whole body imaging of the living mice, and the absorption speed and the whole body tissue distribution of the radioactive marked drug simulated compound after the subcutaneous injection are observed.
The results show that the radioactivity absorption distribution of the human hyaluronidase mutant N3 animal (A) absorbed by the subcutaneous injection site of 99 mTc-semibi shows that the radioactivity distribution and the main organ radioactivity level of the human hyaluronidase mutant N3 treated mice are obviously higher than those of the normal saline control animals.
The present invention provides a human hyaluronidase PH20 mutant strain, and by way of example, the skilled person can carry out the present technology by appropriately modifying the methods described herein within the context and scope of the present invention.
Sequence listing
<110> Henan Saima biotechnology Co., ltd
<120> hyaluronidase mutant for subcutaneous injection of drug preparation
<160> 30
<170> SIPOSequenceListing 1.0
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atgggcaagt gggtgaaggt gctgtttgcc ctgatctgca tcgccgtggc ctacagcctg 60
aactttagag ccccccctgt gatccccaac gtgcctttcc tgtgggcctg gaatgccccc 120
tctgagttct gtctgggcaa gtttgacgag cctctggata tgtctctgtt cagctttatc 180
ggcagcccca gaatcaatgc caccggccag ggcgtgacaa tcttttacgt ggacaggctg 240
ggctactatc catatatcga tagcatcacc ggagtgacag tgaacggagg aatcccacag 300
aagatctccc tgcaggacca cctggataag gccaagaagg atatcacctt ctacatgcct 360
gtggacaatc tgggcatggc cgtgatcgat tgggaggagt ggaggccaac atgggcaagg 420
aactggaagc ccaaggacgt gtataagaat aggtccatcg agctggtgca gcagcagaac 480
gtgcagctgt ctctgaccga ggccacagag aaggccaagc aggagttcga gaaggccggc 540
aaggactttc tggtggagac catcaagctg ggcaagctgc tgcgccctaa ccacctgtgg 600
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acagccctgt acccttctat ctatctgaac acccagcagt ctccagtggc cgccacactg 780
tatgtgcgga atagagtgag ggaggccatc cgcgtgagca agatcccaga cgccaagtcc 840
cctctgccag tgttcgccta cacccggatc gtgtttacag accaggtgct gaagttcctg 900
tcccaggatg agctggtgta taccttcggc gagacagtgg ccctgggagc atctggcatc 960
gtgatctggg gcaccctgag catcatgcgg tccatgaagt cttgcctgct gctggataat 1020
tacatggaga ccatcctgaa cccctatatc atcaatgtga cactggccgc caagatgtgc 1080
agccaggtgc tgtgccagga gcagggcgtg tgcatcagaa agaactggaa tagctccgat 1140
tacctgcacc tgaaccctga caattttgcc atccagctgg agaagggcgg caagttcacc 1200
gtgaggggca agccaacact ggaggacctg gagcagttct ccgagaagtt ttactgcagc 1260
tgttattcca ccctgtcttg caaggagaag gccgatgtga aggacacaga tgccgtggac 1320
gtgtgcatcg ccgatggcgt gtgcatcgac gcctttctga agccacccat ggagaccgag 1380
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atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
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ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatca atgccaccgg ccagggcgtg acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa gaataggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctacaa cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaatga gtccacagcc 720
ctgtaccctt ctatctatct gaacacccag cagtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgaagtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagaca gtggccctgg gagcatctgg catcgtgatc 960
tggggcaccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac tgatga 1416
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<213> Artificial sequence (Artificial Sequence)
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gtacatttat attggctcat gtccaatatg accgccatgt tgacattgat tattgactag 60
ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 120
tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 180
gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 240
ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 300
tccgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 360
gaccttacgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 420
ggtgatgcgg ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt 480
tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 540
ctttccaaaa tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg 600
gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcctca ctctcttccg 660
catcgctgtc tgcgagggcc agctgttggg ctcgcggttg aggacaaact cttcgcggtc 720
tttccagtac tcttggatcg gaaacccgtc ggcctccgaa cggtactccg ccaccgaggg 780
acctgagcga gtccgcatcg accggatcgg aaaacctctc gagaaaggcg tctaaccagt 840
cacagtcgca aggtaggctg agcaccgtgg cgggcggcag cgggtggcgg tcggggttgt 900
ttctggcgga ggtgctgctg atgatgtaat taaagtaggc ggtcttgaga cggcggatgg 960
tcgaggtgag gtgtggcagg cttgagatcc agctgttggg gtgagtactc cctctcaaaa 1020
gcgggcatta cttctgcgct aagattgtca gtttccaaaa acgaggagga tttgatattc 1080
acctggcccg atctggccat acacttgagt gacaatgaca tccactttgc ctttctctcc 1140
acaggtgtcc actcccaggt ccaagtttaa acggatctct agcgaattcc ctctagaggg 1200
cccgtttctg ctagcaagct tgctagcggc cgctcgaggc cggcaaggcc ggatcccccg 1260
acctcgacct ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt 1320
ttgtgtctct cactcggaag gacatatggg agggcaaatc atttggtcga gatccctcgg 1380
agatctctag ctagaggatc gatccccgcc ccggacgaac taaacctgac tacgacatct 1440
ctgccccttc ttcgcggggc agtgcatgta atcccttcag ttggttggta caacttgcca 1500
actgaaccct aaacgggtag catatgcttc ccgggtagta gtatatacta tccagactaa 1560
ccctaattca atagcatatg ttacccaacg ggaagcatat gctatcgaat tagggttagt 1620
aaaagggtcc taaggaacag cgatgtaggt gggcgggcca agataggggc gcgattgctg 1680
cgatctggag gacaaattac acacacttgc gcctgagcgc caagcacagg gttgttggtc 1740
ctcatattca cgaggtcgct gagagcacgg tgggctaatg ttgccatggg tagcatatac 1800
tacccaaata tctggatagc atatgctatc ctaatctata tctgggtagc ataggctatc 1860
ctaatctata tctgggtagc atatgctatc ctaatctata tctgggtagt atatgctatc 1920
ctaatttata tctgggtagc ataggctatc ctaatctata tctgggtagc atatgctatc 1980
ctaatctata tctgggtagt atatgctatc ctaatctgta tccgggtagc atatgctatc 2040
ctaatagaga ttagggtagt atatgctatc ctaatttata tctgggtagc atatactacc 2100
caaatatctg gatagcatat gctatcctaa tctatatctg ggtagcatat gctatcctaa 2160
tctatatctg ggtagcatag gctatcctaa tctatatctg ggtagcatat gctatcctaa 2220
tctatatctg ggtagtatat gctatcctaa tttatatctg ggtagcatag gctatcctaa 2280
tctatatctg ggtagcatat gctatcctaa tctatatctg ggtagtatat gctatcctaa 2340
tctgtatccg ggtagcatat gctatcctca tgataagctg tcaaacatga gaattaattc 2400
ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 2460
ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 2520
atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 2580
tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 2640
cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 2700
agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 2760
taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 2820
tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg 2880
catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 2940
ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 3000
ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 3060
catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 3120
aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt 3180
aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 3240
taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 3300
atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 3360
gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 3420
tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 3480
ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 3540
gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 3600
agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 3660
aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 3720
agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 3780
tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 3840
atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 3900
taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 3960
gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 4020
gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 4080
aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 4140
tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 4200
gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 4260
cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 4320
ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 4380
cgagtcagtg agcgaggaag c 4401
<210> 4
<211> 1410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120
ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatct ccgccaccgg ccagggcgtg acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa ggacaggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctactc cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaagga gtccacagcc 720
ctgtaccctt ctatctatct gaacacccag cagtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgaagtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagaca gtggccctgg gagcatctgg catcgtgatc 960
tggggcaccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac 1410
<210> 5
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gtttaaacgg atctctagcg aattcatggg cgtgaaggtg ctgttcgca 49
<210> 6
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
ggccgctagc aagctttcat cagtggtggt ggtggtggtg gtagaag 47
<210> 7
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
ttcagcttta tcggcagccc cagaatctcc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
ggagattctg gggctgccga taaagctgaa 30
<210> 9
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
caaggacgtg tataaggaca ggtccat 27
<210> 10
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
atggacctgt ccttatacac gtccttg 27
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
ctactccggc tcctgtttca atg 23
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
cattgaaaca ggagccggag tag 23
<210> 13
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctgtggaagg agtccac 17
<210> 14
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
gtggactcct tccacag 17
<210> 15
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
gtacccttct gtctatctga acacccagct gtctccagtg gccgccaca 49
<210> 16
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tgtggcggcc actggagaca gctgggtgtt cagatagaca gaagggtac 49
<210> 17
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 17
gtataccttc ggcgagatag tgg 23
<210> 18
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 18
ccactatctc gccgaaggta tac 23
<210> 19
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 19
acagaccagg tgctgacgtt cctgtc 26
<210> 20
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gacaggaacg tcagcacctg gtctgt 26
<210> 21
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 21
gccagggcat tacaatcttt tacgtgg 27
<210> 22
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 22
ccacgtaaaa gattgtaatg ccctggc 27
<210> 23
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 23
gcatctggca tcattatctg ggg 23
<210> 24
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 24
ccccagataa tgatgccaga tgc 23
<210> 25
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 25
cattatctgg ggctccctga gcatc 25
<210> 26
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 26
gatgctcagg gagccccaga taatg 25
<210> 27
<211> 1410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 27
atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120
ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatct ccgccaccgg ccagggcgtg acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa ggacaggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctactc cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaagga gtccacagcc 720
ctgtaccctt ctgtgtatct gaacacccag ctgtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgaagtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagaca gtggccctgg gagcatctgg catcgtgatc 960
tggggcaccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac 1410
<210> 28
<211> 1410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120
ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatct ccgccaccgg ccagggcgtg acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa ggacaggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctactc cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaagga gtccacagcc 720
ctgtaccctt ctgtgtatct gaacacccag ctgtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgacgtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagata gtggccctgg gagcatctgg catcgtgatc 960
tggggcaccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac 1410
<210> 29
<211> 1410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120
ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatct ccgccaccgg ccagggcatt acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa ggacaggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctactc cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaagga gtccacagcc 720
ctgtaccctt ctgtgtatct gaacacccag ctgtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgacgtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagata gtggccctgg gagcatctgg catcgtgatc 960
tggggcaccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac 1410
<210> 30
<211> 1410
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60
agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120
ttctgtctgg gcaagtttga cgagcctctg gatatgtctc tgttcagctt tatcggcagc 180
cccagaatct ccgccaccgg ccagggcatt acaatctttt acgtggacag gctgggctac 240
tatccatata tcgatagcat caccggagtg acagtgaacg gaggaatccc acagaagatc 300
tccctgcagg accacctgga taaggccaag aaggatatca ccttctacat gcctgtggac 360
aatctgggca tggccgtgat cgattgggag gagtggaggc caacatgggc aaggaactgg 420
aagcccaagg acgtgtataa ggacaggtcc atcgagctgg tgcagcagca gaacgtgcag 480
ctgtctctga ccgaggccac agagaaggcc aagcaggagt tcgagaaggc cggcaaggac 540
tttctggtgg agaccatcaa gctgggcaag ctgctgcgcc ctaaccacct gtggggctac 600
tatctgtttc cagattgcta caatcaccac tataagaagc ccggctactc cggctcctgt 660
ttcaatgtgg agatcaagcg gaacgacgat ctgagctggc tgtggaagga gtccacagcc 720
ctgtaccctt ctgtgtatct gaacacccag ctgtctccag tggccgccac actgtatgtg 780
cggaatagag tgagggaggc catccgcgtg agcaagatcc cagacgccaa gtcccctctg 840
ccagtgttcg cctacacccg gatcgtgttt acagaccagg tgctgacgtt cctgtcccag 900
gatgagctgg tgtatacctt cggcgagata gtggccctgg gagcatctgg catcattatc 960
tggggctccc tgagcatcat gcggtccatg aagtcttgcc tgctgctgga taattacatg 1020
gagaccatcc tgaaccccta tatcatcaat gtgacactgg ccgccaagat gtgcagccag 1080
gtgctgtgcc aggagcaggg cgtgtgcatc agaaagaact ggaatagctc cgattacctg 1140
cacctgaacc ctgacaattt tgccatccag ctggagaagg gcggcaagtt caccgtgagg 1200
ggcaagccaa cactggagga cctggagcag ttctccgaga agttttactg cagctgttat 1260
tccaccctgt cttgcaagga gaaggccgat gtgaaggaca cagatgccgt ggacgtgtgc 1320
atcgccgatg gcgtgtgcat cgacgccttt ctgaagccac ccatggagac cgaggagcct 1380
cagatcttct accaccacca ccaccaccac 1410
Claims (2)
1. A hyaluronidase mutant for subcutaneous injection of a pharmaceutical formulation, characterized in that amino acid site-directed mutagenesis is performed on human hyaluronidase PH20 to construct a hyaluronidase mutant, wherein the number of glycosylation of the hyaluronidase mutant is reduced and catalytic activity is retained;
the hyaluronidase mutant is N1-N4, wherein the nucleotide sequence of the N1 is SEQ ID NO:27, wherein the nucleotide sequence of N2 is SEQ ID NO:28, the nucleotide sequence of the N3 is SEQ ID NO:29, the nucleotide sequence of N4 is SEQ ID NO: shown at 30.
2. The hyaluronidase mutant according to claim 1, wherein the target gene amplification primers of N1-N4 are shown in SEQ ID nos. 5-26.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110813358.6A CN113444708B (en) | 2021-07-19 | 2021-07-19 | Hyaluronidase mutant for subcutaneous injection preparation of medicine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110813358.6A CN113444708B (en) | 2021-07-19 | 2021-07-19 | Hyaluronidase mutant for subcutaneous injection preparation of medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113444708A CN113444708A (en) | 2021-09-28 |
| CN113444708B true CN113444708B (en) | 2024-02-13 |
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| CN102065886A (en) * | 2008-04-14 | 2011-05-18 | 哈洛齐梅公司 | Modified hyaluronidases and uses in treating hyaluronan-associated diseases and conditions |
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| CN102065886A (en) * | 2008-04-14 | 2011-05-18 | 哈洛齐梅公司 | Modified hyaluronidases and uses in treating hyaluronan-associated diseases and conditions |
Non-Patent Citations (2)
| Title |
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| A recombinant human hyaluronidase sustained release gel for the treatment of post-surgical edema;Tara Nekoroski, BA等;《The International Society of Dermatology》;第53卷(第6期);第777–785页 * |
| 重组长效透明质酸酶的构建及其在皮下给药与肿瘤治疗中的应用研究;刘珊;《中国博士论文全文数据库》(第2016年11期期);第E079-5页 * |
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