Hyaluronidase mutant for subcutaneous injection preparation of medicine

文档序号：44710 发布日期：2021-09-28 浏览：50次中文

阅读说明：本技术 一种用于药物皮下注射制剂的透明质酸酶突变体 (Hyaluronidase mutant for subcutaneous injection preparation of medicine ) 是由张守涛于双营方惠敏田庆南郭亚楠马强于 2021-07-19 设计创作，主要内容包括：本发明公开了一种用于药物皮下注射制剂的透明质酸酶突变体,属于基因工程技术领域。本发明对人透明质酸酶PH20进行氨基酸定点突变,构建低糖基化修饰且催化活性提高的透明质酸酶突变体；本发明选取了来源不同的四个物种,运用BIOXM进行四个物种透明质酸酶蛋白序列的比较,确定了11个影响透明质酸酶活性的重要氨基酸位点,根据这些位点在透明质酸酶二级结构预测上的分布位置,和这些位点氨基酸的性质,最终确定突变序列及活性分析,并获得N1-N4一共4个无糖基化修饰且保留催化活性的透明质质酸酶突变体,该突变体能够在减少糖基化位点情况下保留活性；该突变体有利于药物在皮下组织的扩散。(The invention discloses a hyaluronidase mutant for a drug hypodermic injection preparation, belonging to the technical field of genetic engineering. The invention carries out amino acid site-directed mutagenesis on the human hyaluronidase PH20 to construct a hyaluronidase mutant with low glycosylation modification and improved catalytic activity; according to the invention, four species with different sources are selected, BIOXM is used for comparing hyaluronidase protein sequences of the four species, 11 important amino acid sites influencing the activity of hyaluronidase are determined, according to the distribution positions of the sites on the hyaluronidase secondary structure prediction and the properties of the amino acids of the sites, a mutation sequence and activity analysis are finally determined, and 4 total non-glycosylation modified hyaluronidase mutants with reserved catalytic activity of N1-N4 are obtained, and the mutants can reserve activity under the condition of reducing glycosylation sites; the mutant facilitates the diffusion of the drug in the subcutaneous tissue.)

1. A hyaluronidase mutant for use in a subcutaneous formulation of a drug, comprising performing site-directed mutagenesis of an amino acid of human hyaluronidase PH20 to construct a hyaluronidase mutant, wherein the hyaluronidase mutant has reduced glycosylation and retains catalytic activity.

2. The hyaluronidase mutant of claim 1, wherein the hyaluronidase mutant is characterized by:

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. the hyaluronidase mutant of claim 2, wherein the target gene amplification primer of N1-N4 is shown as SEQ ID nos. 5-26.

4. Use of the hyaluronidase mutant according to any of claims 1-3, for the preparation of a pharmaceutical composition for the treatment of diseases or conditions in which the hyaluronidase substrate accumulates.

5. The use of the hyaluronidase mutant of claim 4, wherein the pharmaceutical composition is formulated for oral administration, or intravenous, subcutaneous, intramuscular, intratumoral, intradermal, topical, transdermal, rectal, or subcutaneous injection.

6. The use of the hyaluronidase mutant of claim 5, wherein the pharmaceutical composition comprises an immunoglobulin, a recombinant protein, a synthetic polypeptide, RNA, DNA, or a chemical.

7. The use of the hyaluronidase mutant of claim 5, wherein the pharmaceutical composition is formulated for subcutaneous administration and the amount of the hyaluronidase mutant in the pharmaceutical composition is sufficient to render the pharmaceutical composition therapeutically effective.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a hyaluronidase mutant for a drug hypodermic injection preparation.

Background

Hyaluronic acid is a viscous polysaccharide, which is a linear high molecular polysaccharide formed by repeatedly connecting disaccharide units, wherein the disaccharide units are formed by N-acetylglucosamine and D-glucuronic acid and are connected by beta-1, 3 glycosidic bonds, the two monosaccharides are formed according to a molar ratio of 1:1, and the disaccharide units are connected by the beta-1, 4 glycosidic bonds and widely exist in extracellular matrixes of animal connective tissues. It is a component of the tissue matrix that limits the diffusion of water and other extracellular substances, and hyaluronic acid blocks the diffusion of drug molecules during drug therapy. Hyaluronidase (HAase) is a generic term for enzymes that produce a low molecular weight effect on hyaluronic acid, and it reduces the activity and viscosity of hyaluronic acid and increases the ability of fluid penetration in tissues. Currently, HAase is widely used as a penetrating agent (diffusion factor) of drugs in clinic, and can promote drug absorption under the combined action of the HAase and the drugs.

The hyaluronidase in the market is mainly obtained by three methods, namely, the hyaluronidase is extracted from bovine and ovine tissues and is expressed by a yeast expression system and a recombinant human hyaluronidase expressed by a CHO expression system, the hyaluronidase extracted from animal tissues has low purity, high immunogenicity and low yeast expression amount, the CHO expression system has high cost, and a low-cost prokaryotic expression system is absent at present, mainly because the hyaluronidase can have activity only by N-glycosylation, the glycosylation process can only be completed in the eukaryotic expression system, and the prokaryotic expression system cannot perform the glycosylation modification process.

Hyaluronidase has been used in a number of areas, and FDA in the united states has approved hyaluronidase as a dispersant that can be used to promote the diffusion and absorption of drugs, and hyaluronidase has been used in anesthetic aids, often in combination with anesthetic drugs, to enhance anesthetic effects. Hyaluronidase is also used for rapid absorption of injections, and in addition, hyaluronidase is also used as an ideal marker for tumor detection because tumor cells can secrete HA and HAase simultaneously and secretion is enhanced as tumor cells metastasize. Hyaluronidase can promote metabolism, relieve vascular damage and cell and tissue edema, and increase side circulation, so hyaluronidase can also be used for relieving ischemic injury and reducing myocardial infarction probability. Most of the HAases currently used in the market are extracted from mammalian testis, have limited sources, low yield and high cost, lack an effective prokaryotic expression system due to the limitation of glycosylation, and have the limitations of immunogenicity and purity, so that allergic reaction and rapid clearance reaction occur in clinical application, and the clinical application is greatly limited.

Disclosure of Invention

The invention aims to provide a hyaluronidase mutant for a drug hypodermic injection preparation, which aims to solve the problems in the prior art, selects a mammal cell 293T cell to express human hyaluronidase PH20, selects an amino acid site which has a large influence on the activity of the hyaluronidase by analyzing the primary structure and the secondary structure of the cell and predicting, constructs the hyaluronidase mutant by an amino acid site-directed mutagenesis technology, and transiently transfers a hyaluronidase mutant protein recombination expression plasmid into the cell by a PEI transfection reagent to express the protein. Finally, by activity analysis, the activity change conditions among various mutants of the hyaluronidase are compared, and the hyaluronidase which is active without glycosylation is finally obtained.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a hyaluronidase mutant for a medicament subcutaneous injection preparation, which is constructed by carrying out amino acid site-specific mutagenesis on human hyaluronidase PH20, wherein the mutagenesis sequence is as follows: N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S; wherein the hyaluronic acid mutant has a reduced amount of glycosylation and retains catalytic activity.

Preferably, the hyaluronidase mutants are:

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 as SEQ ID NO. 5-26.

The invention also provides application of the hyaluronidase mutant, which can be used for preparing a pharmaceutical composition for treating diseases or symptoms caused by accumulation of hyaluronidase substrates.

Preferably, the pharmaceutical composition is formulated for oral, or Intravenous (IV), subcutaneous, intramuscular, intratumoral, intradermal, topical, transdermal, rectal or subcutaneous injection administration.

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, BIOXM is used for comparing hyaluronidase protein sequences of the four species, 11 important amino acid sites influencing the activity of hyaluronidase are determined, and according to the distribution positions of the sites on the secondary structure prediction of the hyaluronidase and the properties of the amino acids of the sites, 4 hyaluronidase mutants of N1-N4 are finally obtained.

The molecular modification of hyaluronidase mainly adopts amino acid site-directed mutagenesis technology. Firstly, amplifying a human hyaluronidase sequence by PCR, designing a primer according to an amino acid site to be mutated, and sequentially constructing each hyaluronidase mutant by using a point mutation technology. Carrying out homologous recombination and connection on a double-restriction enzyme PTT5 plasmid and a target gene to construct a mutant protein recombinant expression vector, transiently transferring the constructed recombinant expression vector plasmid into 293T cells for continuous subculture, collecting the cultured cells, carrying out appropriate resuspension on the cells by PBS (phosphate buffer solution), carrying out ultrasonic disruption, and purifying the disrupted supernatant by a His (His-through-His) column to finally obtain the mutant protein. The DNS method is used for detecting the activity change of the mutant protein. Reducing the reducing sugar in the HA product degraded by the hyaluronidase in the alkaline solution of the DNS into an amino compound, generating a color reaction after boiling water bath, measuring the light absorption value at A540 to measure the equivalent weight of the reducing sugar in the enzymatic reaction solution, and measuring the activity of the hyaluronidase by using the method. And then screening out amino acid sites which have great influence on the activity of the hyaluronidase, and finally obtaining the human hyaluronidase which does not need glycosylation and has activity.

Drawings

FIG. 1 is a technical roadmap for hyaluronidase molecular engineering;

FIG. 2 is a diagram of 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 equality analysis;

FIG. 3 is an alignment of hyaluronidase sequences from four species;

FIG. 4 is hyaluronidase homology modeling secondary structure prediction;

FIG. 5 shows the active center site amino acids (D111, E113, E249) C of the protein_αAn atom track change situation diagram;

FIG. 6 shows statistics of activity pocket changes of each mutant;

FIG. 7 shows the charge change of mutant proteins;

FIG. 8 is a technical route for construction of hyaluronidase and its mutants;

FIG. 9 is a schematic diagram of each mutant;

FIG. 10 shows the PCR results of the colony constructed by the recombinant expression vector of PH20 protein, wherein lanes 1-5 show the PCR agarose gel separation results of the colony constructed by the recombinant expression vector of PH20 protein;

FIG. 11 shows hyaluronidase and its mutant activity.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

The reagents and equipment used in the present invention are, unless otherwise specified, either commonly available products or available to those skilled in the art through published routes.

The invention mainly aims to obtain hyaluronidase which is expressed by a prokaryotic expression system and is active without glycosylation, and figure 1 is a technical route of the invention, a sequence of human hyaluronidase is obtained from a Genbank database, the sequence is used as a template, primary sequence comparison is carried out on the sequence and hyaluronidase sequences of cattle and bees which are active without glycosylation, 4 glycosylation sites and 7 amino acid sites related to activity are successfully found, and the mutation sequence of the 11 amino acid sites is finally determined by analyzing the properties of the amino acids of the mutation sites and consulting in literature: N47S/N131D/N200S/N219K/Q234L/I228V/T293L/K279T/V53I/V302I/T306S, and performing molecular modification on hyaluronidase according to a mutation sequence.

The molecular modification of hyaluronidase mainly uses amino acid site-directed mutagenesis technology. Firstly, amplifying a human hyaluronidase sequence by PCR, designing a primer according to an amino acid site to be mutated, and sequentially constructing each hyaluronidase mutant by using a point mutation technology. Carrying out homologous recombination and connection on a double-restriction enzyme PTT5 plasmid and a target gene to construct a mutant protein recombinant expression vector, transiently transferring the constructed recombinant expression vector plasmid into 293T cells for continuous subculture, collecting the cultured cells, carrying out appropriate resuspension on the cells by PBS (phosphate buffer solution), carrying out ultrasonic disruption, and purifying the disrupted supernatant by a His (His-through-His) column to finally obtain the mutant protein. The DNS method is used for detecting the activity change of the mutant protein. Reducing the reducing sugar in the HA product degraded by the hyaluronidase in the alkaline solution of the DNS into an amino compound, generating a color reaction after boiling water bath, measuring the light absorption value at A540 to measure the equivalent weight of the reducing sugar in the enzymatic reaction solution, and measuring the activity of the hyaluronidase by using the method. And then screening out amino acid sites which have great influence on the activity of the hyaluronidase, and finally obtaining the human hyaluronidase which does not need glycosylation and has activity.

Example 1 selection of hyaluronidase mutation sites

1. Determination of the site of mutation

According to the known literature, the hyaluronidase is known to contain 6 glycosylation sites (Asn47, 131, 200, 219, 333, 358), which have great difference in the type and proportion of glycosylation modification, and the six glycosylation sites are located on the secondary structure diagram of the hyaluronidase as shown in FIG. 2A, wherein the glycosylation modification sites at Asn333 and Asn358 are located at the C-terminal end of the hyaluronidase, and the proportion of high-mannose modification is high. The invention selects three hyaluronidase species sequences with high activity of cow (> AAP5571.1), wasp (> CBY83816.1) and bee (> AAAP55713.1) to compare with human PH20(> NP-001167516.1) and constructs an evolutionary tree (figure 2C) for homology analysis.

The alignment of the four species hyaluronidase sequences is shown in figure 3. According to the comparison results of hyaluronidase sequences of four different species, the dark gray part is the positions of six glycosylation sites of hyaluronidase active site amino acid and hyaluronidase, and the glycosylation sites need to be mutated to complete the process that the hyaluronidase does not carry out glycosylation. Therefore, the N47, 131, 219 mutant amino acids were selected to be identical to the amino acid at this site of bovine hyaluronic acid, and the N47 mutation to S, N131 mutation to D, N219 mutation to K. The N200 mutation amino acid is selected to be consistent with the amino acid at the position of the hyaluronic acid of the bee, and the N200 is mutated into 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 mutated glycosylation sites are determined to be four sites of N47S, N131D, N200S and N219K.

The amino acids in the gray highlight region are screened sites which may have influence on the activity of hyaluronidase, the sites have higher homology on hyaluronidase sequences of cattle, bees and wasps, and the sites are close to a catalytic pocket in secondary structure. Through structural analysis of a homology modeling model of each mutant of hyaluronidase, the activity pockets of the mutants have different change conditions, and the possibility of high activity exists.

Four species hyaluronidase secondary structures were aligned, as in fig. 2B, with a larger overlap at the active pocket and alignment identity E<10^-5As shown in fig. 2D, it is suggested that four species have higher homology, among which the homology between human and bovine is the highest, and it is believed that selecting amino acid sites that may increase hyaluronidase based on sequence alignment.

2. Determination of the order of mutations

As shown in FIG. 4, according to the analysis of the homology modeling secondary structure prediction (blue is alpha helix structure, green is beta sheet structure), the hyaluronidase activity pocket is formed by random coil formed by the 111-position 118 amino acid peptide segment, alpha helix formed by the 240-position 245 peptide segment and beta sheet folding nearby the alpha helix, the closer the amino acid site to the activity pocket is, the higher the possibility of influencing the hyaluronidase activity is, and besides, the change of the properties of the amino acid at the mutation site also influences the activity of the hyaluronidase mutant protein. According to the hyaluronidase secondary structure prediction, glycosylation sites 47, 131, 200 and 219 are defined as primary mutation sites, two amino acid sites 228 and 234 are sites with the highest weight influencing the catalytic reaction domain-alpha helical folding and are defined as secondary mutation sites, and amino acid sites 53, 279, 293, 302 and 306 are sites with higher weight influencing the beta sheet structure folding of an active pocket and are defined as tertiary mutation sites. Besides, some amino acid sites on the sequence of the human hyaluronidase have high homology on the sequences of cattle, bees and wasps, such as I83, S84, E285 and M323, and the sites are all positioned on a random coil structure between an alpha helix and a beta sheet which form an active pocket, so that the sites are not considered, and finally, according to the properties of amino acid, hydrophilicity and hydrophobicity, the charge condition and the analysis of relevant literature, the mutation sequence of the tertiary mutation site is preliminarily determined as follows: 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. analysis of active pocket Change of mutant

The sequence of human PH20 gene is found from NCBI, a signal peptide part of 35 amino acid sequences at the N terminal is removed, a glycosyl phosphatidylinositol modification site at the C terminal is removed, and 447 amino acids in the sequence from the 35 th amino acid to the 482 th amino acid are selected as the expression sequence of the target protein.

The PH20 nucleotide sequence is shown in SEQ ID NO. 1.

Inputting 4 mutant protein sequences into https:// swisssmall.expasy.org/interactive website for model construction according to mutation sequence, opening a PDB format model file by using PDB-VIEWER, and analyzing amino acid (D111, E113, E249) C of active center sites of each protein_αThe atomic trajectory variation is shown in fig. 5.

The change of the activity pocket of the mutant protein with the above 4 glycosylation sites is counted as shown in the following FIG. 6. Through analysis, the change conditions of the activity pockets of the mutants are different, and the change conditions of the protein activity are unpredictable. The change of the hyaluronidase activity is verified through experiments.

4. Analysis of mutant proteins for Charge Change

Analysis of charge changes of mutant proteins by inputting mutant proteins into Discovery as shown in fig. 7, the surface charges of the proteins PH20 and N1 are changed greatly (red represents positive charge and blue represents negative charge), the isoelectric points of the proteins PH20 and N1 are not changed and are both 8.6, but the electrostatic potential is changed, N0: 1710.43, N1: the 1780.52 potential docked the substrate to the hyaluronidase active center, the protein potential increased and the probability of binding to the substrate was higher, and the rest of mutant docks were also analyzed as described above, based on the change in mutant surface charge. Finally, the construction of the above 4 glycosylation site mutants is determined.

Example 2 construction, expression and Activity Studies of Hyaluronidase and its mutants

1. Experimental Material

Cell: 293T cells provided in the laboratory; strain: coli TG1 laboratory supplies; plasmid: PTT5 plasmid, purchased from vast organisms; the target gene is as follows: kinsery synthesizes human PH20 gene.

2. Experimental methods

2.1 construction of hyaluronidase and its mutant protein

The hyaluronidase recombinant expression vector is constructed mainly through homologous recombination and connection, and is verified through colony PCR and sequencing, and the technical route for constructing the hyaluronidase and the mutant protein thereof is shown in FIG. 8.

2.2 construction of recombinant expression vector for PH20 protein

2.2.1 optimization and Synthesis of PH20 Gene

The PH20 gene sequence downloaded from NCBI has its N-terminal signal peptide part and C-terminal GPI site removed and luciferase signal peptide added to the N end, and its amino acid sequence is MGVKVLFALICIAVAEA. And the His label is added at the end of C, so that the subsequent protein expression detection and purification operation is facilitated. The PH20 gene is subjected to codon optimization according to the codon bias of mammalian cells, and the sequence amino acids are not changed after optimization. And (3) sending the optimized target gene sequence to Nanjing Kingsry biology 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 hyaluronidase expression vector is constructed by homologous recombination and connection, the homologous recombination and connection is a DNA directional cloning technology, and the PCR recovery product of the insert can be directionally cloned to any position of any vector by using the directional cloning technology, so that the method is simple, rapid 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) obtaining a gene fragment for coding human hyaluronidase, a gene sequence (SEQ ID NO.1, GenBank accession number: > NP-001167516.1)

(2) Connecting a GLU signal peptide sequence to the 5 'end of a human hyaluronidase sequence, removing a GPI site at the 3' end to obtain an SEQ ID No.2 gene sequence, connecting the gene sequence to a PTT5(SEQ ID No.3) vector, and transferring the gene sequence into 293T cells for expression to obtain the human hyaluronidase protein.

(3) The gene sequence of SEQ ID NO.2 is modified by 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 to obtain the human hyaluronidase mutant protein.

(4) On the basis of the gene sequence of SEQ ID No.4, the amino acid of a specific site is modified to obtain a mutant N1-N4 gene sequence, the mutant is connected to a PTT5 carrier and is transferred 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 the plasmid PTT5-PH20

The used plasmid PTT5 is purchased from vast Ling biology company, a gene expressing human hyaluronidase is subjected to PCR amplification, and an amplification product is doped between EcoRI/HindIII sites on a PTT5 plasmid to obtain a plasmid PTT5-PH 20. The constructed sequence is sent to engineering bioengineering limited company for sequencing verification.

The construction process is as follows:

1. the primers PH20-F (SEQ ID NO.5) and PH20-R (SEQ ID NO.6) are used as primers, and the human hyaluronidase sequence SEQ ID NO.2 is used as a template to amplify and obtain the human hyaluronidase gene segment.

PCR cycling procedure (KOD-Plus-neo enzyme):

Step1：94℃2min

Step2：98℃10s

Step3：63℃30s

step 4: 68 ℃ for 45s (to step2, 40 cycles)

Step5：68℃5min

Detection of PCR products: and 3 mu.L of PCR product is taken and detected by agarose gel electrophoresis (as shown in figure 10), and the result shows that the human hyaluronidase gene fragment is successfully amplified.

2. The PCR product obtained above is connected to PTT5 vector by homologous recombinase, and transferred into TG1 competent cell, the specific operation method is as follows:

the plasmid PTT5 is subjected to double enzyme digestion by EcoRI and Hind III, the sequence of SEQ ID NO.2 amplified by PCR is connected to a PTT5 vector subjected to double enzyme digestion by homologous recombinase to obtain PTT5-PH20, 10 mu L of enzyme linked product is taken and added into 200mLTG1 competent cells, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, ice bath is carried out for 2min after heat shock is finished, 800 mu LLB liquid culture medium (without resistance) is added, 37 ℃, recovery is carried out at 140rpm for 45min, centrifugation is carried out at 5000rpm to collect thalli, 900 mu L of culture medium supernatant is discarded, after thalli are resuspended, a plate is coated (ampicillin resistance, 100 ng/mu L) is carried out, recombinant bacteria are selected by colony PCR, and sequencing verification is carried out.

3. The colony with the correct sequencing is picked up and cultured in 5mL LB liquid culture medium (ampicillin resistance, 100 ng/. mu.L) at 37 ℃ for 15h at 180rpm, plasmid extraction is carried out by using an 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:

respectively adding 20 mu g of plasmid and 20 mu L of lip2000 into 48 mu L of culture medium (DMEM high-sugar culture medium) to be uniformly mixed, standing for 20min, dripping the mixed solution into a cell culture dish, continuously culturing for 3 days, collecting cells, carrying out ultrasonic disruption treatment, collecting cell disruption supernatant, carrying out suction filtration by using a 0.22 mu m membrane, purifying by using a His affinity chromatography column, dialyzing the obtained protein, freeze-drying and the like to obtain the human hyaluronidase protein with the purity of more than 90%.

4. The activity of the obtained human hyaluronidase protein is measured, the invention adopts a DNS method to measure the activity of the human hyaluronidase, and the specific operation is as follows:

a standard curve was prepared using 2mg/L glucose solution. The volumes of the glucose solutions were: 0.5, 7.5, 10, 12.5, 15, 17.5 and 20 mu L, adding a glucose solution into a 1.5mL centrifuge tube according to requirements, supplementing the glucose solution to 100 mu L by using PBS buffer solution, mixing the solutions uniformly, adding 200 mu L DNS reagent into the centrifuge tube respectively, mixing the solutions uniformly, boiling for 10min, adding 700 mu L ultrapure water, mixing the solutions uniformly, absorbing 200 mu L of mixed solution respectively, adding the mixed solution into a 96-well plate, and measuring A by using a multifunctional microplate reader₅₄₀And (4) light absorption value.

The obtained human hyaluronidase protein was diluted with PBS buffer, and a certain amount of 20. mu.L of protein diluent was added thereto with 20. mu.L of 0.5% HA solution, and the amount of 100. mu.L of HA solution was made up with PBS, and the enzyme diluent was replaced with PBS as a control. After mixing well, the mixture is reacted at 37 ℃ for 10 min. After the reaction, the enzymatic reaction solution was placed in a metal bath at 100 ℃ for 5min to inactivate the protein. Immediately adding 200 μ L DNS reagent into the enzymatic reaction solution, mixing, heating at 100 deg.C for 10min, adding 700 μ L ultrapure water, mixing, sucking 200 μ L mixed solution, adding into 96-well plate, and measuring A with multifunctional enzyme-labeling instrument₅₄₀And (4) light absorption value.

Glucose concentration as abscissa, A₅₄₀The light absorption value is the ordinate, and a standard glucose calibration curve is drawn. According to A₅₄₀And calculating the equivalent product of reducing sugar in the enzymatic reaction solution by using the light absorption value, and further measuring the activity of the hyaluronidase.

Example 4 construction and Activity measurement of glycosylation site Total deletion mutant

The site-directed mutagenesis technology is utilized to introduce amino acid mutation into PH20 target gene (SEQ ID NO.2), the full-length target gene template amplification of mutant protein is carried out through overlap extension PCR, and a recombinant expression vector is constructed. Glycosylation site variant construction primers required were synthesized by Shanghai Biotechnology Limited:

designing a primer: 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 cycle and subsequent construction, purification and activity determination operations were the same as in example 3, except that the primers described above were used to perform PCR reaction using SEQ ID NO.2 as the template to obtain SEQ ID NO. 4.

Example 3 construction of Activity Return mutants and Activity test

Respectively introducing seven-site mutation into the SEQ ID NO.4 sequence by utilizing a site-specific mutagenesis technology, carrying out amplification on a full-length target gene template of the mutant protein by overlapping extension PCR, and constructing a recombinant expression vector. Primers required for mutant construction were synthesized by Shanghai Biotechnology Ltd:

designing a primer: 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).

The primers are used for PCR reaction by taking SEQ ID NO.4 as a template 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 cycle and subsequent construction, purification and activity determination were as in example 3. The activity of each mutant is shown in figure 11 and table 1, and the activity of the activity reversion mutant N3 is detected to be up to 103.5U/mg, which reaches 70% of the activity of wild type human hyaluronidase.

TABLE 1 Hyaluronidase and respective mutant enzyme activities

Example 4 comparative study of the effect of the bio-permeation enhancer human hyaluronidase on enhancing tissue drug uptake using radiotracer imaging.

The human hyaluronidase mutant N3 and negative control normal saline are respectively injected into a healthy ICR mouse through veins, 99 mTc-labeled tracer methoxy isobutyl isonitrile (99 mTc-sesamibi) is injected into the left or right thigh part subcutaneously 2 hours after injection, a gamma camera special for a high-resolution small animal is used for obtaining the whole body imaging of the living mouse, and the absorption speed and the whole body tissue distribution of a radioactive labeled drug simulation compound after subcutaneous injection are observed.

The results show that the radioactive absorption distribution of the 99 mTc-sesami absorbed by the subcutaneous injection site of the animal (A) injected with the human hyaluronidase mutant N3 shows that the radioactive distribution and the main organ radioactive level of the 99 mTc-sesami in the mice of the human hyaluronidase mutant N3 treatment group are obviously higher than those of the animals of the normal saline control group.

The present invention provides a mutant strain of human hyaluronidase PH20, which is described in detail by way of example, and those skilled in the art can make appropriate changes to the methods described herein within the scope and content of the present invention to achieve the technology of the present invention.

Sequence listing

<110> Henan Saiper Biotech Co., Ltd

<120> a hyaluronidase mutant for use in a subcutaneous injection preparation of a drug

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1398

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

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

ggctactatc tgtttccaga ttgctacaat caccactata agaagcccgg ctacaacggc 660

tcctgtttca atgtggagat caagcggaac gacgatctga gctggctgtg gaatgagtcc 720

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

gagcctcaga tcttctac 1398

<210> 2

<211> 1416

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggcgtga aggtgctgtt cgcactgatc tgcatcgcag tggcagaggc actgaacttt 60

agagcccccc ctgtgatccc caacgtgcct ttcctgtggg cctggaatgc cccctctgag 120

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

<210> 3

<211> 4401

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

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