CN116693697A - Eye drop for relieving asthenopia - Google Patents
Eye drop for relieving asthenopia Download PDFInfo
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- CN116693697A CN116693697A CN202310651792.8A CN202310651792A CN116693697A CN 116693697 A CN116693697 A CN 116693697A CN 202310651792 A CN202310651792 A CN 202310651792A CN 116693697 A CN116693697 A CN 116693697A
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- Prior art keywords
- eye drop
- eye
- protein
- recombinant
- defensin
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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Abstract
The invention provides a protein with a sequence shown as SEQ ID NO. 1, namely recombinant biological defensin (fusion factor), which can improve the secretion of tears. Also provided are eye drops containing the protein, which may further comprise a pH adjuster and an osmotic pressure adjuster. The eye drop can obviously improve the tear secretion, and can be used for relieving eye discomfort of asthenopia people.
Description
Technical Field
The invention relates to medical equipment and medicines, in particular to eye drops.
Background
Asthenopia is a discomfort of the eye, usually caused by prolonged concentration on visual activity. Such as long-term use of electronic devices (e.g., cell phones, computers, other electronic screens, etc.), reading, writing, or performing fine work. These activities require the eye to maintain focus on the screen or object for a long period of time, resulting in excessive use of the eye muscles. Other factors that may lead to visual fatigue include incorrect working posture, poor lighting conditions, dry room air, smoking, lack of rest and sleep, etc.
Visual fatigue may manifest as a range of uncomfortable symptoms including eye fatigue, dry eyes, blurred vision, eye stinging, double vision, eye redness and swelling, and the like. These symptoms may reduce the concentration and work efficiency of the individual, affecting daily life and work.
Visual fatigue is often associated with decreased tear secretion. Tears secreted by the lacrimal glands can form a lubricating film on the surface of the eye, helping to keep the eye moist. According to the american eye society, humans typically blink around 15 times per minute, and as more and more stares at electronic screens, our blinking frequency decreases to about 4 times per minute, which increases tear development, leading to dryness and fatigue of the eyes.
Visual fatigue may trigger an inflammatory response in the eye, thereby affecting the structure and function of the lacrimal gland and reducing tear secretion. Long-term tear loss may in turn lead to chronic ocular surface inflammation, further affecting tear secretion and eye health.
Some methods may alleviate asthenopia.
For example, stopping the work at hand every 20 minutes, visualizing the distance for 20 seconds, gives the eyes time to relax, which helps to alleviate the tiredness of the eyes. The device keeps a good working posture, keeps a correct sitting posture, and adjusts the height, angle and distance of a screen so as to lighten the burden of eyes. The brightness and contrast of the screen are adjusted to be coordinated with the surrounding environment. Ensuring proper illumination of the working area, avoiding an excessively bright or dark environment, natural light being the best choice. Eye exercises can also be performed, which help relax eye muscles and relieve fatigue. Simple eye exercises such as eye rotation, eye massage, and eye closure for a few seconds can be attempted.
Another common approach to relieving asthenopia is the use of eye drops, such as artificial tears. The common artificial tears containing polyethylene glycol, sodium hyaluronate or methylcellulose in the market can form a protective film layer on the surface of eyes to reduce the evaporation of water and maintain the moist state of eyes through the characteristics of hydrophilicity and film forming property of the components, namely, reduce tear evaporation or supplement tear, thereby reducing asthenopia. For example, non-prescription drugs such as artificial lacrimal eyes containing polyethylene glycol are approved for alleviating symptoms such as asthenopia and dry eyes in japan and australia.
However, many artificial tear products on the market at present have poor effect of improving the tear secretion, and many products contain bacteriostats such as benzalkonium chloride and phenylmercuric acetate, so that the side effects are remarkable and the artificial tear products cannot be used for a long time. The eye drops also contain viscosity enhancers such as carbomer, which can cause transient blurred vision and eye allergy. Thus, there is an urgent need for an effective, safe product that increases tear secretion.
Disclosure of Invention
In a first aspect the invention provides a protein comprising the amino acid sequence shown in SEQ ID NO. 1. Alternatively, the protein may increase tear secretion.
The invention provides a protein for improving the secretion amount of tears, which comprises an amino acid sequence shown as SEQ ID NO. 1.
In some embodiments of the invention, the amino acid sequence of the protein is shown in SEQ ID NO. 1.
The inventor refers to a protein with a designed sequence of SEQ ID NO. 1 as recombinant biological defensin (also called PHPV fusion factor), which is obtained by fusing a fragment of human epidermal growth factor (SEQ ID NO. 2) with a fragment of a folding promoting domain and an antibacterial peptide (namely biological defensin, the amino acid sequence of which is SEQ ID NO. 3), and then introducing a plurality of single point mutations (comprising mutating a plurality of amino acid residues into positively charged amino acid residues at specific proper positions).
Experiments prove that the recombinant biological defensin with the sequence of SEQ ID NO. 1 has certain antibacterial activity, can obviously improve the lacrimal secretion, and has better effect than the epidermal growth factor and the antibacterial peptide. Therefore, the recombinant biological defensin can be used for relieving eye discomfort of asthenopia people.
The sequence of the epidermal growth factor is as follows:
MKETWWETWWTEWSQPKKKRKVGSNSDSECPLSHDGYCLHDGVCMYIEALDK YACNCVVGYIGERCQYRDLKWWELR(SEQ ID NO:2)。
the sequence of the antibacterial peptide (namely, the biological defensin) is as follows:
MKLFVPALLSLGALGLCLAAPRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAIAEKKADAVTLDGGMVFEAGRDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLGRSAGWIIPMGILRPYLSWTESLEPLQGAVAKFFSASCVPCIDRQAYPNLCQLCKGEGENQCACSSREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQEKFGKNKSRSFQLFGSPPGQRDLLFKDSALGFLRIPSKVDSALYLGSRYLTTLKNLRETAEEVKARYTRVVWCAVGPEEQKKCQQWSQQSGQNVTCATASTTDDCIALVLKGEADALNLDGGYIYTAGKCGLVPVLAENRKSSKHSSLDCVLRPTEGYLAVAVVKKANEGLTWNSLKDKKSCHTAVDRTAGWNIPMGLIVNQTGSCAFDEFFSQSCAPGADPKSRLCALCAGDDQGLDKCVPNTKEKYYGYNGAFRCLAEDVGDVAFVKNDTVWENTNGESTADWAKNLNREDFRLLCLDGTRKPVTEAQSCHLAVAPNHAVVSRSDRAAHVEQVLLHQQALFGKNGKNCPDKFCLFKSETKNLLFNDNTECLAKLGGRPTYEEYLGTEYVTAIANLKKCSTSPLLEACAFLTR(SEQ ID NO:3)。
without being bound by theory, the inventors speculate that the recombinant biodefense of the invention may increase tear secretion by:
a. the mutant form of the EGF fragment in the fusion protein can play the function of the EGF to a certain extent, and mainly participates in and promotes tear secretion;
b. the fusion protein is positively charged and has proper strength and distribution, and adheres to the surface of the negatively charged eye through charge interaction to form a physical antibacterial barrier, and simultaneously slows down the evaporation of water and wets the eye.
In a second aspect the present invention provides an eye drop comprising a protein according to the first aspect. Alternatively, the eye drops may be used to increase tear secretion. Alternatively, the eye drops may be used to relieve asthenopia.
The present invention provides an eye drop for increasing the amount of lacrimal secretion, the eye drop comprising the protein of the first aspect.
The invention provides an eye drop for relieving asthenopia, which comprises the protein in the first aspect.
In some embodiments of the invention, the eye drops comprise the recombinant bio-defensin of the first aspect.
In some embodiments of the invention, the eye drops comprise recombinant biodefense and a pharmaceutically acceptable adjuvant.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, a pH regulator, and an osmolality regulator. In some embodiments of the invention, the eye drops consist of recombinant bio-defensin, a pH adjustor and an osmotic pressure adjustor.
In some embodiments of the invention, the pH adjustor is optionally selected from boric acid, borax, dipotassium phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
In some embodiments of the invention, the osmolality adjusting agent is optionally selected from sodium chloride, mannitol, boric acid, glucose, potassium chloride, glycerol, or any combination thereof.
In some embodiments of the invention, the eye drops further comprise a viscosity increasing agent.
In some embodiments of the invention, the adhesion promoter is optionally selected from methylcellulose, polyvinyl alcohol, carbomers, and povidone.
In some embodiments of the invention, the eye drops have a pH of 6.0 to 7.5. In some embodiments of the invention, the eye drops have a pH of 6.5 to 7.0.
In some embodiments of the invention, the eye drops have an osmotic pressure of 290-305mOsm/L. In some embodiments of the invention, the eye drops have an osmotic pressure of 300mOsm/L.
In some embodiments of the invention, the eye drops comprise 0.01% (w/v, g/mL) of recombinant bio-defensin.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium chloride.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride.
In some embodiments of the invention, the recombinant bio-defensin eye drops are prepared by: dissolving recombinant biological defensin in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v, mass/volume, g/ml); adding a pH regulator, and regulating the pH to 6.5-7.0; adding osmotic pressure regulator, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
In some embodiments of the invention, the recombinant bio-defensin eye drops are prepared by: dissolving recombinant biological defensin in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v); adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
In a third aspect, the invention provides the use of a recombinant biodefense of the first aspect in the manufacture of a medical device or medicament for increasing tear secretion or alleviating asthenopia.
The invention provides an application of the recombinant biological defensin in the first aspect in preparing a medicament for relieving asthenopia.
The present invention provides the use of a recombinant biodefense of the first aspect in the manufacture of a medicament for increasing tear secretion.
The invention provides the use of the recombinant biodefense of the first aspect in the manufacture of a medical device for alleviating asthenopia.
The present invention provides the use of a recombinant biodefense of the first aspect in the manufacture of a medical device for increasing lacrimal secretion.
In some embodiments of the invention, the medical device or drug is an eye drop. In some embodiments of the invention, the medical device is an eye drop. In some embodiments of the invention, the medicament is an eye drop.
In a fourth aspect, the present invention provides the use of an eye drop according to the second aspect for the preparation of a medical device or medicament for increasing tear secretion or alleviating asthenopia.
The invention provides the use of the eye drop according to the second aspect for the manufacture of a medicament for increasing lacrimal secretion.
The invention provides application of the eye drops in the second aspect in preparing a medicament for relieving asthenopia.
The invention provides the use of the eye drop according to the second aspect for the manufacture of a medical device for increasing lacrimal secretion.
The invention provides application of the eye drops in the second aspect in preparing medical equipment for relieving asthenopia.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, a pH regulator, and an osmolality regulator. In some embodiments of the invention, the eye drops consist of recombinant bio-defensin, a pH adjustor and an osmotic pressure adjustor.
In some embodiments of the invention, the pH adjustor is optionally selected from boric acid, borax, dipotassium phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
In some embodiments of the invention, the osmolality adjusting agent is optionally selected from sodium chloride, mannitol, boric acid, glucose, potassium chloride, glycerol, or any combination thereof.
In some embodiments of the invention, the eye drops further comprise a viscosity increasing agent.
In some embodiments of the invention, the adhesion promoter is optionally selected from methylcellulose, polyvinyl alcohol, carbomers, and povidone.
In some embodiments of the invention, the eye drops have a pH of 6.0 to 7.5. In some embodiments of the invention, the eye drops have a pH of 6.5 to 7.0.
In some embodiments of the invention, the eye drops have an osmotic pressure of 290-305mOsm/L. In some embodiments of the invention, the eye drops have an osmotic pressure of 300mOsm/L.
In some embodiments of the invention, the eye drops comprise 0.01% (w/v) recombinant biodefense.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium chloride.
In some embodiments of the invention, the eye drops comprise recombinant biodefense, sodium dihydrogen phosphate, disodium hydrogen phosphate, and potassium chloride
In some embodiments of the invention, the recombinant bio-defensin eye drops are prepared by: dissolving recombinant biological defensin in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v); adding a pH regulator, and regulating the pH to 6.5-7.0; adding osmotic pressure regulator, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
In some embodiments of the invention, the recombinant bio-defensin eye drops are prepared by: dissolving recombinant biological defensin in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v); adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
The invention has the beneficial effects that:
(1) The recombinant biological defensin designed by the invention can obviously improve the tear secretion and can be used for relieving eye discomfort of asthenopia people.
(2) In terms of improving the lacrimal secretion, the recombinant biological defensin is obviously superior to the corresponding epidermal growth factor and antibacterial peptide, and unexpected technical effects are obtained.
(3) The eye drops containing the recombinant biological defensin can obviously improve the lacrimal secretion and can be used for relieving the eye discomfort of the asthenopia crowd.
(4) The recombinant biological defensin has certain antibacterial activity, and the eye drops containing the recombinant biological defensin can be free of antibacterial agents and can reduce side effects caused by the antibacterial agents during long-term administration.
(5) The recombinant biological defensin-containing eye drops of the invention can be free of adhesion promoters and can avoid side effects caused by adhesion promoters, such as blurred vision and eye allergy.
(6) The eye drops containing the recombinant biological defensin provided by the invention are simple to prepare and low in cost.
Detailed Description
The present disclosure is further illustrated by way of specific embodiments, and is not thereby limited in scope by the embodiments described. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Examples
EXAMPLE one preparation and confirmation of recombinant Biodefensin
The inventor fuses the human epidermal growth factor fragment with the fold promoting domain and the antibacterial peptide fragment, and then introduces a plurality of single point mutations to finally obtain the recombinant biological defensin, and the amino acid sequence of the recombinant biological defensin is shown as SEQ ID NO. 1.
MGSSHHHHHHKETWWETWWTEWSQPKKKRKVGGGGSNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRGGGGSMTMSLEVFEKLEAKVQQAIDTITLLQMEIEELKEKNNSLSQEVQNAQHQREELERENNHLKEQQNGWQKRLQALLGRMRRVGGGGSMKLFVPALLSLGALGLCLAAPRKNVRWCTISQPEWFKCRRWQWRMKKLGAPSITCVRRAFALECIRAIAEKKADAVTLDGGMVFEAGRDPYKLRPVAAEIYGTKESPQTHYYAVAVVKKGSNFQLDQLQGRKSCHTGLGRSAGWIIPMGILRPYLSWTRSLEPLQGAVAKFFSASCVPCIKRQAYPNLCQLCKGEGENQCACSSREPYFGYSGAFKCLQDGAGDVAFVKETTVFENLPEKADRDQYELLCLNNSRAPVDAFKECHLAQVPSHAVVARSVDGKEDLIWKLLSKAQRKFGKNKSRSFQLFGSPPGQRDLLFKDSALGFLRIPSK(SEQ ID NO:1)。
The DNA fragment containing the coding sequence of the recombinant biological defensin (online codon optimization is carried out by an ExpOptimer under default parameters) is synthesized outside the delegation, then is digested with pET28a (+) vector by BlpI and NcoI respectively, and the digested products are connected by T4 ligase to obtain the recombinant biological defensin expression vector plasmid (SEQ ID NO: 4). The reaction solution is transformed into stbl3, cultured overnight and positive clones are selected for sequencing and identification.
The recombinant biological defensin expression vector plasmid sequence (SEQ ID NO: 4) is:
tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaa
tttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttcttt
ccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcga
gacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaat
attttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacg
gataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctaccttt
gccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcccatt
tatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcataacaccccttgtatt
actgtttatgtaagcagacagttttattgttcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagat
caaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggat
caagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggcca
ccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttac
cgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg
aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc
ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgc
cacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcc
tggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgc
tcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctg
tgcggtatttcacaccgcatatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgt
gactgggtcatggctgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagac
aagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatca
gcgtggtcgtgaagcgattcacagatgtctgcctgttcatccgcgtccagctcgttgagtttctccagaagcgttaatgtctggcttctgata
aagcgggccatgttaagggcggttttttcctgtttggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatg
aaacgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacgttgtgagggtaaacaactggcggtatg
gatgcggcgggaccagagaaaaatcactcagggtcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcag
catcctgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagactttacgaaacacggaaaccgaagaccatt
catgttgttgctcaggtcgcagacgttttgcagcagcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaa
ccccgccagcctagccgggtcctcaacgacaggagcacgatcatgcgcacccgtggggccgccatgccggcgataatggcctgcttc
tcgccgaaacgtttggtggcgggaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatc
atcgtcgcgctccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaaga
cagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgag
atcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatt
aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctg
attgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggtt
aacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatg
gcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaa
aaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcag
acgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgta
ccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttcca
cagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacagg
cttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacg
gcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgta
attcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataa
gagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccata
ccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtagg
ttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccac
catacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagca
accgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcac
tataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatataccatgGGCTCTTCTCA
TCATCACCATCACCACAAAGAAACCTGGTGGGAAACCTGGTGGACCGAGTGGTCTC
AGCCGAAAAAAAAACGTAAAGTCGGCGGTGGTGGCTCTAACTCCGATTCCGAGTG
CCCGCTGTCTCATGATGGTTATTGCCTGCACGATGGCGTTTGTATGTACATTGAGGCG
CTGGACAAATACGCATGCAACTGTGTCGTCGGTTACATCGGCGAGCGTTGTCAATAC
CGTGATCTGAAATGGTGGGAACTGCGTGGTGGCGGCGGTAGCATGACCATGTCTCT
GGAAGTGTTCGAAAAACTGGAAGCTAAAGTCCAGCAGGCCATCGATACTATCACTC
TGCTGCAGATGGAAATCGAAGAACTGAAAGAAAAAAACAACTCCCTGTCCCAGGA
AGTCCAGAATGCACAGCACCAGCGTGAAGAACTGGAACGTGAGAACAACCACCTG
AAAGAACAGCAGAACGGTTGGCAAAAGCGTCTGCAGGCTCTGCTGGGTCGTATGC
GTCGTGTTGGTGGTGGCGGCTCTATGAAACTGTTCGTACCAGCTCTGCTGTCCCTGG
GTGCACTGGGTCTGTGCCTGGCGGCTCCGCGTAAAAACGTTCGTTGGTGTACTATCT
CTCAGCCGGAATGGTTTAAGTGTCGTCGTTGGCAGTGGCGCATGAAAAAACTGGGC
GCGCCGAGCATCACCTGTGTACGCCGTGCGTTCGCACTGGAATGCATTCGCGCAATC
GCAGAAAAAAAAGCGGATGCTGTCACTCTGGATGGTGGCATGGTTTTCGAGGCTGG
CCGTGACCCGTATAAGCTGCGCCCTGTTGCTGCCGAAATCTACGGTACCAAAGAATC
TCCGCAGACTCATTATTATGCGGTTGCGGTCGTAAAAAAAGGCTCTAACTTTCAACT
GGATCAGCTGCAGGGCCGTAAAAGCTGCCACACTGGTCTGGGTCGTTCTGCGGGCT
GGATTATCCCGATGGGTATCCTGCGCCCTTATCTGTCTTGGACGCGTAGCCTGGAGC
CGCTGCAAGGTGCTGTTGCGAAATTCTTCAGCGCCTCCTGTGTGCCATGTATTAAGC
GTCAGGCCTACCCGAACCTGTGCCAGCTGTGCAAAGGTGAAGGCGAAAACCAGTG
CGCATGCTCTTCTCGCGAGCCGTACTTCGGTTACTCCGGCGCATTCAAATGTCTGCA
GGATGGCGCTGGTGACGTAGCGTTCGTAAAAGAAACGACCGTGTTCGAAAACCTG
CCGGAAAAAGCAGACCGTGATCAGTATGAGCTGCTGTGTCTGAACAACTCTCGTGC
GCCGGTTGATGCCTTCAAAGAATGTCACCTGGCACAGGTACCAAGCCACGCAGTTG
TGGCGCGTTCTGTCGACGGCAAAGAGGACCTGATCTGGAAACTGCTGAGCAAAGC
TCAGCGCAAATTCGGCAAAAACAAGTCTCGTAGCTTCCAGCTGTTCGGTAGCCCGC
CGGGCCAGCGTGATCTGCTGTTCAAAGATTCTGCTCTGGGTTTCCTGCGCATCCCGT
CCAAAtaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacg
ggtcttgaggggttttttgctgaaaggaggaactatatccggat
after positive clone culture with correct sequencing, plasmid and transformation expression strain E.coli BL21 (DE 3) are extracted. Cultured overnight at 37℃in LB medium containing kanamycin. IPTG was added to 0.2mM and induction was carried out at 16℃for 24 hours. And centrifugally collecting thalli, adding the lysate, performing ultrasonic disruption, and centrifuging to obtain supernatant containing the fusion protein. Filtering with 0.45um filter membrane, purifying with nickel column chromatography, purifying with ion chromatography, and lyophilizing eluate to obtain recombinant biological defensin with purity of 90.4%. It exhibits a band in the range of 35kDa to 70kDa in SDS-PAGE analysis.
Example two preparation of eye drops
The proteins in Table 1 below were prepared using the procedure of example one and purified using chromatography with a purity of 90.2% -91.1%.
TABLE 1 control proteins
Preparation of recombinant biological defensin eye drops 1: the recombinant biodefense prepared in the first example was dissolved in water for injection to prepare an aqueous solution having a concentration of 0.01% (w/v, g/mL, the same applies hereinafter). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of recombinant biological defensin eye drops 2: the recombinant biological defensin prepared in the first example was dissolved in water for injection to prepare an aqueous solution having a concentration of 0.01% (w/v). Adding sodium dihydrogen phosphate and disodium hydrogen phosphate, and regulating the pH to 6.5; adding potassium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of recombinant biological defensin eye drops 3: the recombinant biological defensin prepared in the first example was dissolved in water for injection to prepare an aqueous solution having a concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH value to 7.0; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C1 eye drops: the prepared control protein C1 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C1-2 eye drops: the prepared control protein C1-2 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C2 eye drops: the prepared control protein C2 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C2-2 eye drops: the prepared control protein C2-2 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C3 eye drops: the prepared control protein C3 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein C4 eye drops: the prepared control protein C4 is dissolved in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v). Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein c1+c2 eye drops: the prepared control proteins C1 and C2 were dissolved together in water for injection to prepare an aqueous solution having a concentration of 0.005% (w/v) of both control proteins C1 and C2. Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Preparation of control protein c3+c4 eye drops: the prepared control proteins C3 and C4 were dissolved together in water for injection to prepare an aqueous solution having a concentration of 0.005% (w/v) of both control proteins C3 and C4. Adding monopotassium phosphate and dipotassium phosphate, and adjusting the pH to 6.5; adding sodium chloride, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, packaging under aseptic condition, and inspecting.
Example three, tear secretion test
Taking normal male Japanese white rabbits with weight of 4.1-4.7kg. 1.5mL (5 mg/mL) scopolamine was subcutaneously injected 2 times daily for 10 consecutive days. The test of tear secretion is carried out by adopting a Schirmer test on the 11 th day, one end of a tear detection filter strip is reversely folded for 5mm, the tear detection filter strip is placed in a conjunctival sac on the outer side of a lower eyelid, the tear detection filter strip is taken out after 5min, the wetted length of tear is read, the test is carried out for 3 times continuously, and the average value is obtained. Animals with tear wet-out lengths below 5mm were models of reduced tear secretion for subsequent testing.
Model rabbits were randomly grouped, 6 animals each, and the same volume of eye drops of example two (equivalent physiological saline was administered to the negative control group) were administered 2 drops each time, 4 times daily. After 4 days and 8 days of continuous administration, the amount of lacrimal secretion was measured in terms of the test paper wet length. The results are shown in Table 2.
The recombinant biodefense eye drops 1, 2 and 3 significantly increased tear secretion (P < 0.01) after 4 and 8 days of administration compared to the negative control group, and the increase in tear secretion was significantly greater than that of each control protein group (P < 0.01). Control proteins C1 and C1-2 increased tear secretion but were significantly weaker than recombinant biodefense eye drops. No synergy was produced in the control protein c1+c2 group. The control protein C3+C4 group had a certain effect of improving the amount of lacrimal secretion, but had limited effect.
Therefore, the fusion protein of the recombinant biological defensin has a similar synergistic effect, and the recombinant biological defensin eye drops have unexpected technical effects.
In addition, after 8 days of administration, the symptoms such as obvious abnormality of eyes of animals in the 1-3 groups of recombinant biological defensin eye drops and the control protein C1 and C1-2 eye drops are observed, and obvious congestion, edema and the like of cornea, iris and conjunctiva of the eyes are avoided, so that the recombinant biological defensin eye drops disclosed by the invention have no irritation to eyes and have very good biocompatibility and safety. Can be used for relieving asthenopia.
TABLE 2 lacrimal secretion test
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Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A protein comprising the amino acid sequence shown in SEQ ID NO. 1.
2. An eye drop comprising the protein of claim 1.
3. The eye drop according to claim 2, which can increase the amount of lacrimal secretion or relieve asthenopia.
4. The eye drops of claim 2 comprising the protein of claim 1, a pH adjuster and an osmolality adjuster.
5. The eye drop of claim 4, wherein said pH adjustor is selected from the group consisting of boric acid, borax, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate.
6. The eye drop of claim 4, wherein said osmolality adjusting agent is optionally selected from sodium chloride, mannitol, boric acid, glucose, potassium chloride, glycerol, or any combination thereof.
7. The eye drop of claim 4, wherein the eye drop has a pH of 6.0-7.5 and an osmotic pressure of 290-305mOsm/L.
8. The eye drop of claim 4, comprising 0.01% recombinant biodefense.
9. The eye drop according to claim 4, which is prepared by the following steps: dissolving recombinant biological defensin in water for injection to prepare an aqueous solution with the concentration of 0.01% (w/v); adding a pH regulator, and regulating the pH to 6.5-7.0; adding osmotic pressure regulator, regulating osmotic pressure to 300mOsm/L, mixing, filtering with 0.22 μm microporous membrane, sterilizing, and packaging in single dose.
10. Use of a protein according to claim 1 for the manufacture of a medical device or medicament for increasing tear secretion or alleviating asthenopia.
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CN110448678A (en) * | 2018-05-07 | 2019-11-15 | 易金阳 | A kind of fusion factor containing PHPV is used to alleviate the component and Preparing Use of the eye drops of visual fatigue |
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