CN110960687A - Application of transthyretin for transferring fusion protein into eyes - Google Patents
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Abstract
The invention discloses an application of transthyretin in transferring fusion protein into eyes, belonging to the technical field of protein engineering. The invention provides a method for transporting exogenous protein into eyes through an eye barrier by the fusion expression of transthyretin shown in SEQ ID NO.1 and the exogenous protein. Animal experiments prove that the fusion protein prepared by the invention can be used for eye dropping treatment of rats and rabbits, and can quickly and effectively convey foreign protein to eyeground. The transthyretin used in the method is derived from human bodies, has good biocompatibility and safety in human bodies, and has important application prospect in the field of medicines as a substitute of injection medicines.
Description
Technical Field
The invention relates to an application of transthyretin in transferring fusion protein into eyes, in particular to an application of transthyretin carrying exogenous protein to be transferred into eyes through an eye barrier, belonging to the technical field of protein engineering.
Background
Treatment of eye diseases, requiring effective delivery of drugs to the eye, particularly age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, glaucoma-induced nerve changes, and the like; however, effective delivery of drugs to the eye is challenging due to the greater barrier presented to the eye. Accordingly, recently, ocular drug delivery systems have received a wide range of attention. At present, eye drops are the most used dosage form for clinically treating eye diseases, but the eye drops quickly flow out of the surface of eyes and the bioavailability is low. In order to deal with the situation, the developed dosage forms such as microspheres, liposomes, microemulsions and the like can improve the bioavailability of the medicament to a certain extent, but the medicament absorption is still greatly limited due to the existence of an ocular barrier.
Depending on the ocular anatomy, ocular barriers include primarily the tear barrier, the corneal/conjunctival barrier, and the blood-ocular barrier; the multiple barriers can effectively block the invasion of exogenous chemical and biological molecules while protecting eyes, but also bring great obstruction to the transmission of drugs. Currently, the common administration modes of the eye mainly comprise conventional eye drops, subconjunctival injection and scleral administration, and intravitreal injection administration. Wherein, the contact time of the conventional eye drops and the ocular surface is short, and due to the structure and the characteristics of the conventional eye drops, some exogenous drugs and proteins are difficult to effectively enter eyes; subconjunctival and intravitreal injections often cause some trauma. In recent years, there are reports of designing functional small peptides capable of effectively breaking through various ocular barriers to reach eyes, such as patents of zhengying, xu et al (a new class of small peptides inhibiting new vessels and applications thereof, ZL2013100527142), patents of suli et al (a class of small peptides inhibiting new vessels and applications thereof, ZL201310058978.9) and patents of yangjiu et al (a small molecule polypeptide preventing and/or treating inflammatory responses and applications thereof).
The researches have certain limitations, intraocular injection can bring certain trauma, the delivery efficiency of the functional small peptide is low, and the half-life period is short, so that a thought needs to be developed to find a safer and more stable delivery mode from a protein or peptide segment which has a transport function and can break through a physiological barrier and exists in a human body.
Disclosure of Invention
The present application provides a method for the fusion expression of transthyretin to deliver foreign proteins into the eye through the ocular barrier. The human transthyretin used in the method has better biocompatibility and safety in human bodies, and can effectively deliver exogenous protein into eyes.
The first purpose of the invention is to provide the application of transthyretin as a carrier of protein drugs entering into eyes through an ocular barrier; the transthyretin is as shown in (a) or (b):
(a) a protein consisting of amino acids represented by SEQ ID No. 1;
(b) and (b) the protein derived from the protein (a) and having the function of inhibiting angiogenesis, wherein the protein (a) is obtained by substituting, actually or adding one or more amino acids in the amino acid sequence in the protein (a).
In one embodiment, the use is the fusion of transthyretin to a proteinaceous drug.
In one embodiment, the fusion is of a proteinaceous drug to the N-terminus or C-terminus of the transthyretin.
In one embodiment, the drug is a protein having a molecular weight of no more than 45kDa, including but not limited to: lysozyme, egg white protein or green fluorescent protein.
In one embodiment, the lysozyme is egg white lysozyme having GenBank accession No. AAL 69327.1.
In one embodiment, the transthyretin is expressed by a microbial cell and purified.
In one embodiment, the vector used for expression is selected from the pET series plasmids, and the microorganism used for expression is Escherichia coli.
In one embodiment, the e.coli includes, but is not limited to, e.coli BL21, e.coli BL21(DE3), e.coli JM109, e.coli DH5 α, or e.coli TOP 10.
In one embodiment, the purification is by endotoxin adsorption column (Pierce)TMHigh CapacityEndotoxin Removal Spin Columns, ThermoFisher) through 0.22. mu.lRemoving residual bacteria by a filter membrane with the m-aperture.
In one embodiment, the fusion protein is prepared by: (1) connecting the encoding genes of transthyretin and drug protein to pET 21a (+) plasmid to obtain recombinant plasmid; (2) transforming the recombinant plasmid constructed in the step (1) into a host cell for expression; (3) using TB culture medium, fermenting at 30-40 deg.C and OD600nmInducing with 0.1-0.5mM IPTG for 8-16h when reaching 1.5-2.0; (4) purifying with nickel column affinity adsorption to obtain expressed target protein, and purifying with endotoxin adsorption column (Pierce)TMHigh Capacity endo toxin Removal SpinColums, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration.
In one embodiment, the purified TTR fusion protein is applied to the eye by eye dropping, wherein the frequency of dropping is 1-3 times per day, and the dropping amount is 0.6-0.8nmol protein per eye.
The second object of the present invention is to provide drops containing transthyretin or a fusion protein of transthyretin and a protein having a pharmaceutical effect; the transthyretin is as shown in (a) or (b):
(a) a protein consisting of amino acids represented by SEQ ID No. 1;
(b) and (b) the protein derived from the protein (a) and having the function of inhibiting angiogenesis, wherein the protein (a) is obtained by substituting, actually or adding one or more amino acids in the amino acid sequence in the protein (a).
In one embodiment, the drops further comprise physiological saline.
The beneficial technical effects of the invention are as follows:
the invention provides a new application of transthyretin shown in SEQ ID NO.1 in fusion expression of protein drugs and serving as a drug carrier to carry drugs to eyes through an ocular barrier, and animal experiments prove that the prepared fusion protein is used for eye drop treatment of rats and rabbits, and can effectively convey foreign proteins to eyeground. The transthyretin used in the method is derived from human bodies, has good biocompatibility and safety in human bodies, and has important application prospect in the field of medicines as a substitute of injection medicines.
Drawings
FIG. 1 shows a plasmid map of pET 21a (+) -TTR-X. The front end of the gene sequence is fused and expressed with a His-tag sequence and is connected into a plasmid through NdeI and EcoRI restriction enzyme cutting sites; "X" represents a protein fused to TTR.
Fig. 2 is an electrophoresis pattern diagram of E.coli BL21(DE3) expression human transthyretin, fusion protein purified product and green fluorescent protein, egg white lysozyme and egg white albumin standard.
FIG. 3 shows a western-blot of a target protein in a vitreous chamber of a human transthyretin and a fusion protein thereof, wherein the left eye is a dropping eye and the right eye is a control eye, after the human transthyretin and the fusion protein thereof are dropped into rats and rabbits for two weeks. Due to the fact that human source/rat source/rabbit source TTR has high homology, after TTR is dropped into eyes, a vitreous body sample is detected to have a background TTR positive signal by using an anti-TTR antibody; when the anti-His-tag antibody is used for detection, the dropwise addition of the intraocular positive signal is obviously increased, which indicates that the humanized TTR can effectively enter the eye to reach the vitreous body; TTR can effectively enter eyes after being fused and expressed with exogenous proteins such as GFP, Lysozyme, Ovalbumin and the like, but the proteins which are not fused and expressed cannot enter eyes.
FIG. 4 shows that the sequence similarity of human/rat/rabbit transthyretin reaches nearly 95% after homology comparison, and it can be seen that the bulk TTR positive signal in FIG. 3A is the intraocular homologous protein signal.
Detailed Description
Example 1:
preparing transthyretin: the method comprises the following steps:
(1) construction of recombinant plasmid pET 21a (+) -TTR: the nucleotide sequence shown in SEQ ID NO.2 was synthesized, ligated to pET 21a (+) with Nde I and EcoR I enzymes, and verified by sequencing, the construction was successful.
(2) Expression and purification of recombinant TTR: transforming the pET 21a (+) -TTR plasmid constructed in the step (1) into E.coli BL21(DE3) cells, culturing the recombinant E.coli BL21(DE3) in LB medium, preparing seed solution, and inoculating 5% of the seed solutionInoculating into 5L TB medium, culturing at 37 deg.C and stirring paddle rotation speed of 150rpm to OD6001.5-2.0; induction was carried out for 8-16h with the addition of 0.1-0.5mM IPTG (Table 1). And (3) performing high-pressure homogenization to break bacteria, and performing affinity adsorption on the supernatant through a nickel column to obtain the TTR. The obtained protein is adsorbed by endotoxin adsorption column (Pierce)TMHigh Capacity endo toxin Removal SpinColums, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration. TTR protein production was measured and the results are shown in Table 1. At OD600When the concentration is 1.5-1.8, inducing with 0.3-0.5 mM IPTG for 12-14 h, and the protein yield is not less than 17mg/g wet thallus.
TABLE 1 expression of TTR under different Induction conditions
Example 2:
transthyretin-green fluorescent protein fusion protein (TTR-GFP) was prepared as follows:
(1) construction of recombinant plasmid pET 21a (+) -TTR-GFP: the TTR-GFP sequence shown in SEQ ID NO.3 was synthesized, ligated to pET 21a (+) with Nde I and EcoR I enzymes, and verified by sequencing, the construction was successful.
(2) Expression of TTR-GFP fusion protein: e.coli BL21(DE3) cells transformed with the recombinant plasmid constructed in the step (1) into pET 21a (+) -TTR-GFP plasmid were inoculated into 5L TB medium at 37 ℃ and the rotating speed of a stirring paddle is 150rpm, and OD is cultured600nmTo 1.5-2.0; adding 0.1-0.5mM IPTG to induce for 8-16 h; the TTR-GFP fusion protein is prepared by high-pressure homogeneous bacteria breaking and the supernatant fluid is subjected to affinity adsorption through a nickel column (Table 2). The obtained protein is adsorbed by endotoxin adsorption column (Pierce)TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration. The production of TTR-GFP protein was measured and the results are shown in Table 2. At OD600When the concentration is 1.5-1.9, inducing with 0.3-0.5 mM IPTG for 12h, and the protein yield is not less than 10mg/g wet thallus.
TABLE 2 expression of TTR-GFP under different Induction conditions
Example 3:
a method of fusion expressing transthyretin for delivery of a foreign protein into the eye through the ocular barrier, the method comprising the steps of:
(1) construction of the recombinant plasmid pET 21a (+) -TTR-Lysozyme: the TTR-Lysozyme sequence shown in SEQ ID NO.4 was synthesized, ligated with pET 21a (+) using Nde I and EcoR I enzymes, and verified by sequencing, the construction was successful.
(2) Transforming pET 21a (+) -TTR-Lysozyme constructed in the step (1) into E.coli BL21(DE3) cells at an inoculation amount of 5%, inoculating 5L TB medium at 37 ℃, rotating speed of a stirring paddle of 150rpm, and culturing OD600nmTo 1.5-2.0; adding 0.1-0.5mM IPTG to induce for 8-16 h; the TTR-Lysozyme fusion protein (Table 3) is prepared by high-pressure homogenization for bacteria breaking and passing the supernatant through a nickel column for affinity adsorption. The obtained protein is adsorbed by endotoxin adsorption column (Pierce)TMHigh hcapacity Endotoxin Removal of Endotoxin from Spin Columns, thermo fisher) and Removal of residual bacteria through a 0.22 μm pore size filter.
TABLE 3 expression of TTR-Lysozyme under different Induction conditions
Example 4:
(1) construction of recombinant plasmid pET 21a (+) -TTR-Ovalbumin: the TTR-Ovalbumin sequence shown in SEQ ID NO.5 is synthesized, and is connected with pET 21a (+) by Nde I and EcoR I enzymes, and the construction is successful after sequencing verification.
(2) The pET 21a (+) -TTR-Ovalbumin plasmid constructed in the step (1) is transformed into E.coli BL21(DE3) cells, and the E.coli BL21(DE3) cells are inoculated into 5L of TB medium at the temperature of 37 DEG CCulture OD at 150rpm of the paddle600nmTo 1.5-2.0; adding 0.1-0.5mM IPTG to induce for 8-16 h; the TTR-Ovalbumin fusion protein (Table 4) is prepared by high-pressure homogenization for bacteria breaking and nickel column affinity adsorption of the supernatant. The obtained protein is adsorbed by endotoxin adsorption column (Pierce)TMHigh Capacity endo toxin Removal Spin Columns, ThermoFisher) and the residual bacteria were removed by 0.22 μm pore size filtration.
TABLE 4 expression of TTR-Ovalbumin under different Induction conditions
Example 5:
the purified TTR protein (concentration: 4. mu. mol/L) obtained in example 1 after removal of endotoxin and bacteria was treated by eye-dropping in healthy SD rats (rats norgeicus) (6 weeks old) and New Zealand big ear rabbits (Orycolagus cuniculus) (2 months old, 2.5kg), with the protein sample eye added to the left eye and physiological saline added to the right eye as a blank. The dropping times are 1-3 times per day, and the dropping amount is 0.4-0.8nmol each time. After two weeks, the eyeballs are picked and separated to obtain a vitreous body sample, the fact that TTR can enter the vitreous cavity from the ocular surface is preliminarily verified by a western-blot method (figure 3A), and the western-blot result with Anti-His tag antibody as a primary antibody shows that the signal intensity of the exogenous TTR in the vitreous body of the left eye of the SD rat is 28.7 times that of the right eye, and the signal intensity of the exogenous TTR in the vitreous body of the left eye of the New Zealand big ear rabbit is 35.6 times that of the right eye; the amount of TTR in the vitreous body samples was determined by ELISA using Anti-His tag antibody as a primary antibody (tables 5, 6). The results show that after being added dropwise 2 times daily at a concentration of 0.6nmol, SD rats and New Zealand big ear rabbits detected intravitreal higher values of exogenous TTR content.
TABLE 5 Effect of TTR in reaching SD rat eyes under different treatment modes
TABLE 6 Effect of TTR in reaching eyes of New Zealand big ear rabbits with different treatment modes
Example 6:
the purified TTR-GFP protein (concentration 4. mu. mol/L) obtained in example 2 after removal of endotoxin and bacteria was treated by eye-dropping with healthy SD rats (rats norgeicus (6 weeks old) and New Zealand big ear rabbits (Orycola plus mice) (2 months old,. about.2.5 kg), the left eye was a protein sample eye, the right eye was a white control eye to which physiological saline was added dropwise at a rate of 1 to 3 times per day in an amount of 0.4 to 0.8 nmol.two weeks later, the eyeball was extracted and a vitreous body was isolated to obtain a vitreous body sample, and it was preliminarily verified by the western-method (FIG. 3B) that TTR-GFP could enter the vitreous cavity from the ocular surface, the result of the western-blot using Anti-GFP antibody as primary antibody showed that the signal intensity of exogenous GFP in the glass of SD rat was 62.3 times that in the left eye was 47.6 times that the signal intensity of the exogenous GFP in the left eye of the New Zealand big ear rabbits was 47.6 times that the Anti-His antibody The signal intensity of the exogenous GFP in vivo is stronger, but no signal exists in the right eye, and the signal intensity of the exogenous GFP in the vitreous body of the left eye of the New Zealand big ear rabbit is 45.4 times that of the right eye. The amount of TTR-GFP in the vitreous samples was determined by ELISA using Anti-His tag antibody as a primary antibody (tables 7, 8). The results show that after 2 drops at 0.6nmol daily, higher values of exogenous GFP were detected in the vitreous of SD rats and New Zealand big ear rabbits.
TABLE 7 Effect of TTR-GFP in different treatment modalities on reaching the eyes of SD rats
TABLE 8 Effect of TTR-GFP in reaching eyes of New Zealand big ear rabbits with different treatment modalities
Example 7:
the purified prepared TTR-Lysozyme protein (concentration: 4. mu. mol/L) obtained in example 3 after removal of endotoxin and bacteria was treated by eye-dropping in healthy SD rats (6-week-old) and New Zealand big ear rabbits (Orycolagus cuniculus) (2-month-old, 2.5kg), with the protein sample eye dropped in the left eye and physiological saline solution dropped in the right eye as a blank. The dropping times are 1-3 times per day, and the dropping amount is 0.4-0.8nmol each time. After two weeks, the eyeballs are picked and separated to obtain a vitreous body sample, the TTR-Lysozyme can enter the vitreous cavity from the ocular surface through preliminary verification (figure 3C) by a western-blot method, and the result of the western-blot with Anti-Lysozyme antibody as a primary antibody shows that the signal intensity of the exogenous Lysozyme in the vitreous body of the left eye of the SD rat is 34.6 times that of the right eye, and the signal intensity of the exogenous Lysozyme in the vitreous body of the left eye of the New Zealand big ear rabbit is stronger and no signal exists in the right eye; the result of western-blot using Anti-His tag antibody as primary antibody showed that the signal intensity of exogenous Lysozyme in the left eye vitreous body of SD rat was 30.2 times that of the right eye, and the signal intensity of exogenous Lysozyme in the left eye vitreous body of New Zealand big ear rabbit was 46.3 times that of the right eye. The amount of TTR-Lysozyme in the vitreous body samples was determined by ELISA using Anti-His tag antibody as a primary antibody (tables 9, 10). The results show that after being added dropwise 2 times at a content of 0.6nmol per day, SD rats and New Zealand big ear rabbits have higher exogenous Lysozyme content detected in the vitreous.
TABLE 9 Effect of TTR-Lysozyme on reaching eyes of SD rats in different treatment modes
TABLE 10 Effect of TTR-Lysozyme on reaching eyes of New Zealand big ear rabbits by different treatment modes
Example 8:
after endotoxin and bacteria were removed in example 4, the prepared TTR-Ovalbumin protein (concentration: 4. mu. mol/L) was purified, and healthy SD rats (rats norgeicus) (6 weeks old) and New Zealand big ear rabbits (Orycolagus cuniculus) (2 months old, 2.5kg) were treated by eye dropping, in the left eye, a protein sample eye was dropped, and in the right eye, a physiological saline solution was dropped as a blank. The dropping times are 1-3 times per day, and the dropping amount is 0.4-0.8nmol each time. After two weeks, the eyeballs are picked and separated to obtain a vitreous body sample, the TTR-Ovalbumin can enter the vitreous cavity from the ocular surface through preliminary verification (figure 3B) by a western-blot method, and the western-blot result with Anti-Ovalbumin antibody as a primary antibody shows that the signal intensity of the exogenous Ovalbumin in the vitreous body of the left eye of the SD rat is 25.3 times of that of the right eye, and the signal of the exogenous Ovalbumin the vitreous body of the left eye of the New Zealand big ear rabbit is stronger and no signal exists in the right eye; the result of western-blot using Anti-His tag antibody as primary antibody shows that the signal intensity of the exogenous Ovalbumin in the left eye vitreous body of SD rat is 37.8 times of that of the right eye, and the signal intensity of the exogenous Ovalbumin in the left eye vitreous body of New Zealand big ear rabbit is stronger and no signal is generated in the right eye. The amount of TTR-Ovalbumin in the vitreous body samples was measured by ELISA using Anti-His tag antibody as a primary antibody (tables 11, 12). The results show that after being added dropwise 2 times at a content of 0.6nmol per day, SD rats and New Zealand big ear rabbits detected in the vitreous body higher values of exogenous Ovalbumin content.
TABLE 11 Effect of TTR-Ovalbumin reaching the eyes of SD rats in different treatment modes
TABLE 12 Effect of TTR-Ovalbumin on New Zealand big ear eyes under different treatment modes
Comparative example 1:
the present embodiment is the same as example 6, except that the GFP protein not fused to TTR (Genbank accession No. QAA95705.1) was expressed according to the method of example 2, and the eye drop test of SD rats and New Zealand big ear rabbits was carried out using the same procedure as in example 6 with respect to the GFP protein not fused to TTR, and it was revealed that GFP did not enter the vitreous body of SD rats and New Zealand big ear rabbits (tables 13 and 14)
TABLE 13 GFP content 0.6nmol each time, effect of GFP reaching SD rat eyes after different times of dropping
Wherein "-" indicates no detection.
TABLE 14 Effect of GFP concentration 0.6nmol per drop and GFP reaching the eyes of New Zealand big ears after different times of drops
Wherein "-" indicates no detection.
Comparative example 2:
the specific embodiment is the same as example 7 except that the Lysozyme protein not fused with TTR (Genbank accession No.: AAL69327.1) was expressed according to the method of example 3, and the eye drop test of the unfused Lysozyme protein was performed on SD rat and New Zealand big ear rabbit according to the same procedure as example 7, and it was revealed that Lysozyme did not enter into the vitreous of SD rat and New Zealand big ear rabbit (tables 15 and 16)
TABLE 15 Lysozyme content 0.6nmol per drop, Effect of Lysozyme reaching SD rat eyes after different times of drop
TABLE 16 Effect of Lysozyme reaching the eyes of New Zealand big ears after different treatments after different times of adding 0.6nmol of Lysozyme per time
Comparative example 3:
the present embodiment is the same as example 8, except that the method of example 4 was used to express the Avubmin protein not fused to TTR (Uniprot accession number P01012), and the same procedures as in example 9 were used to perform the eye drop test on SD rats and New Zealand big ear rabbits, which revealed that Avubmin did not enter the vitreous body of SD rats and New Zealand big ear rabbits (tables 17 and 18)
TABLE 17 Effect of Ovalbumin reaching SD rat eyes after Ovalbumin is added to SD rat eyes at different times every day with the Ovalbumin content of 0.6nmol
TABLE 18 Effect of Ovalbumin content of 0.6nmol added dropwise each time and reaching the eye of New Zealand big ear after different times of daily addition
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> Shanghai Kanji biological medicine science and technology Co., Ltd
<120> use of transthyretin for the transport of fusion proteins into the eye
<160>5
<170>PatentIn version 3.3
<210>1
<211>127
<212>PRT
<213>Homo sapiens (human)
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Gly Pro Thr Gly Thr Gly Glu Ser Lys Cys Pro Leu Met Val Lys Val
1 5 10 15
Leu Asp Ala Val Arg Gly Ser Pro Ala Ile Asn Val Ala Val His Val
20 25 30
Phe Arg Lys Ala Ala Asp Asp Thr Trp Glu Pro Phe Ala Ser Gly Lys
35 40 45
Thr Ser Glu Ser Gly Glu Leu His Gly Leu Thr Thr Glu Glu Glu Phe
50 55 60
Val Glu Gly Ile Tyr Lys Val Glu Ile Asp Thr Lys Ser Tyr Trp Lys
65 70 75 80
Ala Leu Gly Ile Ser Pro Phe His Glu His Ala Glu Val Val Phe Thr
85 90 95
Ala Asn Asp Ser Gly Pro Arg Arg Tyr Thr Ile Ala Ala Leu Leu Ser
100 105 110
Pro Tyr Ser Tyr Ser Thr Thr Ala Val Val Thr Asn Pro Lys Glu
115 120 125
<210>2
<211>420
<212>DNA
<213> Artificial sequence
<400>2
atgagaggat cgcatcatca tcatcatcat ggatccggtc cgaccggtac cggtgaatct 60
aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120
gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180
acctctgaat ctggtgaact gcacggtctg accaccgaag aagaattcgt tgaaggtatc 240
tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300
gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360
gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaatga 420
<210>3
<211>1137
<212>DNA
<213> Artificial sequence
<400>3
atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60
aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120
gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180
acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240
tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300
gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360
gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420
tctatgtcta aaggtgaaga actgttcacc ggtgttgttc cgatcctggt tgaactggac 480
ggtgacgtta acggtcacaa attctctgtt tctggtgaag gtgaaggtga cgctacctac 540
ggtaaactga ccctgaaatt catctgcacc accggtaaac tgccggttcc gtggccgacc 600
ctggttacca ccttcaccta cggtgttcag tgcttctctc gttacccgga ccacatgaaa 660
cagcacgact tcttcaaatc tgctatgccg gaaggttacg ttcaggaacg taccatcttc 720
ttcaaagacg acggtaacta caaaacccgt gctgaagtta aattcgaagg tgacaccctg 780
gttaaccgta tcgaactgaa aggtatcgac ttcaaagaag acggtaacat cctgggtcac 840
aaactggaat acaactacaa ctctcacaac gtttacatca tggctgacaa acagaaaaac 900
ggtatcaaag ttaacttcaa aatccgtcac aacatcgaag acggttctgt tcagctggct 960
gaccactacc agcagaacac cccgatcggt gacggtccgg ttctgctgcc ggacaaccac 1020
tacctgtcta cccagtctgc tctgtctaaa gacccgaacg aaaaacgtga ccacatggtt 1080
ctgctggaat ttgttaccgc tgctggtatc acccacggta tggacgaact gtacaaa 1137
<210>4
<211>864
<212>DNA
<213> Artificial sequence
<400>4
atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60
aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120
gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180
acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240
tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300
gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360
gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420
tctatgcgtt ctctgctgat cctggttctg tgcttcctgc cgctggctgc tctgggtaaa 480
gttttcggtc gttgcgaact ggctgctgct atgaaacgtc acggtctgga caactaccgt 540
ggttactctc tgggtaactg ggtttgcgct gctaaattcg aatctaactt caacacccag 600
gctaccaacc gtaacaccga cggttctacc gactacggta tcctgcagat caactctcgt 660
tggtggtgca acgacggtcg taccccgggt tctcgtaacc tgtgcaacat cccgtgctct 720
gctctgctgt cttctgacat caccgcttct gttaactgcg ctaaaaaaat cgtttctgac 780
ggtaacggta tgaacgcttg ggttgcttgg cgtaaccgtt gcaaaggtac cgacgttcag 840
gcttggatcc gtggttgccg tctg 864
<210>5
<211>1581
<212>DNA
<213> Artificial sequence
<400>5
atgcgtggtt ctcaccacca ccaccaccac ggttctggtc cgaccggtac cggtgaatct 60
aaatgcccgc tgatggttaa agttctggac gctgttcgtg gttctccggc tatcaacgtt 120
gctgttcacg ttttccgtaa agctgctgac gacacctggg aaccgttcgc ttctggtaaa 180
acctctgaat ctggtgaact gcacggtctg accaccgaag aagaatttgt tgaaggtatc 240
tacaaagttg aaatcgacac caaatcttac tggaaagctc tgggtatctc tccgttccac 300
gaacacgctg aagttgtttt caccgctaac gactctggtc cgcgtcgtta caccatcgct 360
gctctgctgt ctccgtactc ttactctacc accgctgttg ttaccaaccc gaaagaaggt 420
tctatgggtt ctatcggtgc tgcttctatg gaattttgct tcgacgtttt caaagaactg 480
aaagttcacc acgctaacga aaacatcttc tactgcccga tcgctatcat gtctgctctg 540
gctatggttt acctgggtgc taaagactct acccgtaccc agatcaacaa agttgttcgt 600
ttcgacaaac tgccgggttt cggtgactct atcgaagctc agtgcggtac ctctgttaac 660
gttcactctt ctctgcgtga catcctgaac cagatcacca aaccgaacga cgtttactct 720
ttctctctgg cttctcgtct gtacgctgaa gaacgttacc cgatcctgcc ggaatacctg 780
cagtgcgtta aagaactgta ccgtggtggt ctggaaccga tcaacttcca gaccgctgct 840
gaccaggctc gtgaactgat caactcttgg gttgaatctc agaccaacgg tatcatccgt 900
aacgttctgc agccgtcttc tgttgactct cagaccgcta tggttctggt taacgctatc 960
gttttcaaag gtctgtggga aaaagctttc aaagacgaag acacccaggc tatgccgttc 1020
cgtgttaccg aacaggaatc taaaccggtt cagatgatgt accagatcgg tctgttccgt 1080
gttgcttcta tggcttctga aaaaatgaaa atcctggaac tgccgttcgc ttctggtacc 1140
atgtctatgc tggttctgct gccggacgaa gtttctggtc tggaacagct ggaatctatc 1200
atcaacttcg aaaaactgac cgaatggacc tcttctaacg ttatggaaga acgtaaaatc 1260
aaagtttacc tgccgcgtat gaaaatggaa gaaaaataca acctgacctc tgttctgatg 1320
gctatgggta tcaccgacgt tttctcttct tctgctaacc tgtctggtat ctcttctgct 1380
gaatctctga aaatctctca ggctgttcac gctgctcacg ctgaaatcaa cgaagctggt 1440
cgtgaagttg ttggttctgc tgaagctggt gttgacgctg cttctgtttc tgaagaattt 1500
cgtgctgacc acccgttcct gttctgcatc aaacacatcg ctaccaacgc tgttctgttc 1560
ttcggtcgtt gcgtttctcc g 1581
Claims (10)
1. The transthyretin is used as a carrier for protein drugs to enter eyes through an ocular barrier; the transthyretin is as shown in (a) or (b):
(a) a protein consisting of amino acids represented by SEQ ID No. 1;
(b) and (b) the protein derived from the protein (a) and having the function of inhibiting angiogenesis, wherein the protein (a) is obtained by substituting, actually or adding one or more amino acids in the amino acid sequence in the protein (a).
2. The use of claim 1, wherein the transthyretin is expressed as a fusion with a proteinaceous drug.
3. The use of claim 2, wherein the fusion is of a proteinaceous drug to the N-terminus or C-terminus of the transthyretin.
4. The use according to any one of claims 1 to 3, wherein the proteinaceous drugs include, but are not limited to: lysozyme and EGFR antibody, and the molecular weight is not more than 45 kDa.
5. Use according to claim 4, wherein the lysozyme is egg white lysozyme having GenBank accession No. AAL 69327.1.
6. The use of claim 2, wherein the transthyretin is expressed in microbial cells and purified when fused to a proteinaceous agent.
7. The use of claim 6, wherein the purification comprises removing endotoxin by an endotoxin-adsorbing column and removing residual bacteria by a 0.22 μm pore size filter.
8. A drop, which is characterized by comprising transthyretin or a fusion protein of transthyretin and a protein drug; the transthyretin is as shown in (a) or (b):
(a) a protein consisting of amino acids represented by SEQ ID No. 1;
(b) and (b) the protein derived from the protein (a) and having the function of inhibiting angiogenesis, wherein the protein (a) is obtained by substituting, actually or adding one or more amino acids in the amino acid sequence in the protein (a).
9. Drops according to claim 8, wherein the content of said fusion protein is greater than or equal to 4 μmol/L.
10. Drops according to claim 8, characterised in that they also contain physiological saline.
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CN201911302937.3A CN110960687A (en) | 2019-12-17 | 2019-12-17 | Application of transthyretin for transferring fusion protein into eyes |
CN202010506950.7A CN111437398B (en) | 2019-12-17 | 2020-06-05 | Transthyretin for insertion into the eye and use thereof in the preparation of drops |
EP20903323.2A EP3988122A4 (en) | 2019-12-17 | 2020-11-13 | Application of transthyretin in entering eye and preparing drop |
PCT/CN2020/128588 WO2021120937A1 (en) | 2019-12-17 | 2020-11-13 | Application of transthyretin in entering eye and preparing drop |
US17/579,510 US20220168434A1 (en) | 2019-12-17 | 2022-01-19 | Application of transthyretin in entering eye and preparing drop |
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CN111920940A (en) * | 2020-09-16 | 2020-11-13 | 童妍(上海)医疗器械有限公司 | Eye preparation and preparation method and application thereof |
CN114058636A (en) * | 2021-11-16 | 2022-02-18 | 大连润生康泰医学检验实验室有限公司 | Method for cloning, expressing and purifying transthyretin gene |
CN114558112A (en) * | 2020-09-16 | 2022-05-31 | 易舟(上海)生物医药有限公司 | Eye preparation and preparation method and application thereof |
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CN111920940A (en) * | 2020-09-16 | 2020-11-13 | 童妍(上海)医疗器械有限公司 | Eye preparation and preparation method and application thereof |
CN114558112A (en) * | 2020-09-16 | 2022-05-31 | 易舟(上海)生物医药有限公司 | Eye preparation and preparation method and application thereof |
CN114058636A (en) * | 2021-11-16 | 2022-02-18 | 大连润生康泰医学检验实验室有限公司 | Method for cloning, expressing and purifying transthyretin gene |
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CN111437398A (en) | 2020-07-24 |
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