CN116640204A - Heat-resistant fibronectin, preparation method and application thereof, nucleic acid, expression vector and strain - Google Patents
Heat-resistant fibronectin, preparation method and application thereof, nucleic acid, expression vector and strain Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses a heat-resistant fibronectin, a preparation method and application thereof, nucleic acid, an expression vector and a strain, wherein the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO. 6. According to the technical scheme, the heat-resistant fibronectin is obtained by utilizing a directed evolution method through a genetic engineering method, and has high heat stability, the biological activity of the heat-resistant fibronectin is not changed, and the application prospect of the heat-resistant fibronectin in the fields of cosmetic raw materials and the like is remarkably improved.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to heat-resistant fibronectin, and a preparation method and application thereof, nucleic acid, an expression vector and a strain.
Background
Fibronectin (Fn), a major non-collagenous glycoprotein present in the extracellular matrix and basement membrane, is involved in regulating cell polarity, differentiation and growth, and plays a key role in cell adhesion. Fibronectin is involved in many critical vital activities in the human body: 1) Is involved in migration, adhesion, proliferation, hemostasis and tissue repair of cells, and embryo development, and can promote ordered proliferation of cells and collagen; 2) Promote the fibroblast to secrete protease and decompose protein impurities; 3) Inducing the reconstruction and normal keratinization process of the basal lamina under the epidermis, accelerating the comprehensive repair of cells and tissues in structure and function. Therefore, fibronectin has been widely used in the fields of cosmetic skin care ingredients, medical wound dressings, in vivo treatments and the like, and has extremely high biological and medical application values.
Natural fibronectin is a dimeric glycoprotein, each monomer having a molecular weight of 230-250 kDa, with variable molecular conformation and splice variants. Fibronectin contains different functional regions and can bind to integrins, heparin, collagen, DNA and the like to play important physiological functions. Although fibronectin has multiple biological activities such as wound repair promotion and cell adhesion promotion, its low thermal stability greatly limits its application in the fields of cosmetics and in vitro medical devices. Wild fibronectin is very susceptible to protein denaturation during cosmetic emulsification, thereby losing its bioactivity and application value.
Disclosure of Invention
The invention mainly aims to provide a heat-resistant fibronectin, a preparation method and application thereof, nucleic acid, an expression vector and a strain, and aims to improve the thermal stability of fibronectin.
In order to achieve the aim of the invention, the following technical scheme is adopted.
A thermostable fibronectin comprising at least one of:
the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO.2, and SEQ ID NO.2:
PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPST；
the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO.4, and SEQ ID NO.4:
PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVRGD；
the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO.6, SEQ ID NO.6:
PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKKTDELPQLVRGD;
the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO.8, and SEQ ID NO.8:
SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL；
the amino acid sequence of the heat-resistant fibronectin is shown as SEQ ID NO.10, SEQ ID NO.10:
SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEPTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLPSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVPEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL。
the heat-resistant fibronectin has good heat stability, and Tm values are all over 50 ℃ and the highest Tm value is even over 80 ℃. This temperature is much higher than the lowest emulsification temperature of cosmetics by 75 ℃, so the heat-resistant fibronectin of the invention can be used for the preparation of cosmetics. Similarly, the heat-resistant fibronectin has excellent heat stability, so that the heat-resistant fibronectin is also suitable for the fields of preparing medicines, taking the medicines as auxiliary materials, preparing culture mediums and the like, which have requirements on the heat resistance of the fibronectin.
The invention also discloses a nucleic acid which codes the heat-resistant fibronectin.
Preferably, the nucleic acid is DNA.
More preferably, the sequence of the DNA is:
a sequence of the heat-resistant fibronectin with the encoded amino acid sequence shown as SEQ ID NO.2, SEQ ID NO. 1:
CCCACTGATCTAAGGTTTACAAATATAGGACCCGATACCATGCGTGTGACCTGGGCACCGCCTCCAAGCATCGACCTGACCAACTTCTTGGTACGTTACAGCCCGGTTAAAAACGAAGAGGACGTTGCGGAACTGAGCATCTCTCCTAGCGACAACGCGGTTGTTCTGACCAACCTCCTTCCGGGTACAGAGTATGTGGTGTCAGTGTCGTCCGTTTATGAGCAGCACGAAAGCACCCCGCTGCGTGGTCGCCAGAAGACCGGTCTTGATTCCCCGACAGGTATTGACTTCTCTGACATCACGGCTAACTCCTTTACGGTGCATTGGATTGCGCCGAGAGCTACCATCACCGGCTACCGCATTCGCCATCACCCGGAGCACTTTAGCGGTCGTCCGCGCGAGGATCGTGTTCCGCACAGCCGTAATAGCATCACCTTGACGAACCTGACCCCGGGCACCGAGTACGTTGTCAGCATTGTTGCATTGAATGGTCGGGAAGAATCTCCGTTGCTCATTGGTCAACAAAGCACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCACCCTGCCCCACCCGAATCTGCATGGTCCGGAAATTCTGGATGTGCCGTCAACCTAA；
a sequence encoding a thermostable fibronectin having an amino acid sequence as shown in SEQ ID NO.4, SEQ ID NO.3:
CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATTAA；
a sequence of the heat-resistant fibronectin with the encoded amino acid sequence shown as SEQ ID NO.6, SEQ ID NO.5:
CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTACTGAGTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGAAGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCCCGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGCCCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCCCACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCCCACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATTAA；
a sequence encoding a thermostable fibronectin having an amino acid sequence as shown in SEQ ID NO.8, SEQ ID NO. 7:
AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGTAA
a sequence encoding a thermostable fibronectin having an amino acid sequence as shown in SEQ ID NO.10, SEQ ID NO.9:
AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAACCGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCCCGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGCCTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGTAA。
the invention also discloses a preparation method of the heat-resistant fibronectin, which comprises the following steps:
designing recombinant fibronectin;
site-directed mutagenesis of amino acid residues is performed on recombinant fibronectin;
the heat-resistant fibronectin is obtained after expressing the protein subjected to site-directed mutagenesis.
More preferably, the specific procedure for the design of recombinant fibronectin steps is as follows:
recombinant fibronectin was designed based on sequence and active domain screening.
More preferably, the step of expressing recombinant fibronectin is performed as follows:
the recombinant fibronectin base sequence which is subjected to codon optimization and artificial synthesis is inserted into a plasmid vector to obtain an expression vector. Transferring the expression vector into escherichia coli, culturing in a shake flask to express recombinant fibronectin, and purifying the recombinant fibronectin.
Still more preferably, the plasmid vector is a pET28a vector, and the inserted cleavage site is NdeI/XhoI. The vector is not limited to pET28a, and other common plasmids pET22a, pET22b, pET22c, pET14, pET21, pET30 and pET42 in the escherichia coli can realize the expression of the recombinant fibronectin. The strain is not limited to Escherichia coli BL21 (DE 3), and other types of Escherichia coli, yeast or Bacillus subtilis can be used.
Still more preferably, the site-directed mutagenesis of an amino acid residue comprises at least one of the following amino acid residues:
g39, K140, T179, G251, a269, T335, N357, V170, E213, T226, K248, E369, V72, D73, I94, M151, M169, a206, V232, R245 and K375. Wherein the letter G, K, T, A, N, V, E, D, I, M, R is a single letter abbreviation for the amino acid, followed by a number indicating the position of the amino acid.
Still more preferably, site-directed mutagenesis of an amino acid residue comprises at least one of the following mutations:
G39P, K140, P, T, 179P, G251, P, A, P, T, 335, P, N, 357, 170, P, T, 226, P, K, 248, 4639, P, V, T, D, E, I, L, M, V, M, K, A, L, V, 232, I, R, 245V and K375V. Wherein, the letter G, K, T, A, N, V, E, D, I, M, R before the number is a single letter abbreviation of the amino acid, the number indicates the position of the amino acid, and the letter P, T, E, L, V, L, I, V after the number indicates the amino acid at the position after mutation.
Still more preferably, the mutant primer of G39 comprises G39P-F, G39P-R having the sequences SEQ ID NO.27 and SEQ ID NO.28, respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of K140 are SEQ ID NO.29 and SEQ ID NO.30 respectively; and/or the number of the groups of groups,
the sequences of the mutant primers of T179 are SEQ ID NO.31 and SEQ ID NO.32 respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of G251 are SEQ ID NO.33 and SEQ ID NO.34 respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of A269 are SEQ ID NO.35 and SEQ ID NO.36 respectively; and/or the number of the groups of groups,
the sequences of the mutant primers of T335 are SEQ ID NO.37 and SEQ ID NO.38 respectively; and/or the number of the groups of groups,
the sequence of the mutation primer of N357 is SEQ ID NO.39, SEQ ID NO.40 and/or,
the sequences of the mutation primers of V170 are SEQ ID NO.41, SEQ ID NO.42 and/or SEQ ID NO.42 respectively,
the sequences of the mutant primers of E213 are SEQ ID NO.43, SEQ ID NO.44 and/or,
the sequences of the mutant primers of T226 are SEQ ID NO.45, SEQ ID NO.46 and/or SEQ ID NO.46 respectively,
the sequences of the K248 mutation primers are SEQ ID NO.47, SEQ ID NO.48 and/or SEQ ID NO.48 respectively,
the sequences of the mutant primers of E369 are SEQ ID NO.49, SEQ ID NO.50 and/or SEQ ID NO.50 respectively,
the sequences of the V72 mutation primers are SEQ ID NO.51, SEQ ID NO.52 and/or SEQ ID NO.52 respectively,
the sequence of the mutant primer of D73 is SEQ ID NO.51, SEQ ID NO.52 and/or SEQ ID NO.52 respectively,
the sequences of the mutation primers of I94 are SEQ ID NO.53, SEQ ID NO.54 and/or SEQ ID NO.54 respectively,
the sequences of the mutation primers of M151 are SEQ ID NO.55, SEQ ID NO.56 and/or SEQ ID NO.56 respectively,
the sequences of the mutation primers of M169 are SEQ ID NO.57, SEQ ID NO.58 and/or SEQ ID NO.58 respectively,
the sequences of the mutation primers of A206 are SEQ ID NO.59, SEQ ID NO.60 and/or SEQ ID NO.60 respectively,
the sequences of the V232 mutation primers are SEQ ID NO.61, SEQ ID NO.62 and/or SEQ ID NO.62 respectively,
the sequences of the mutation primers of R245 are SEQ ID NO.63, SEQ ID NO.64 and/or SEQ ID NO.64 respectively,
the sequences of the mutant primers of K375 are SEQ ID NO.65 and SEQ ID NO.66 respectively.
More preferably, the specific manipulation of the site-directed mutagenesis step of amino acid residues on recombinant fibronectin comprises:
and (3) inputting a protein sequence to predict a tertiary structure by using the environment of an alpha fold2.2.0 structure prediction algorithm, and selecting a mutation site according to a prediction result to mutate the mutation site.
More preferably, the above method for preparing heat-resistant fibronectin comprises the steps of:
designing recombinant fibronectin;
recombining domains in the recombinant fibronectin;
site-directed mutagenesis of amino acid residues of the domain recombined recombinant fibronectin;
the heat-resistant fibronectin is obtained after expressing the protein subjected to site-directed mutagenesis.
Still further preferably, the above method for preparing heat-resistant fibronectin comprises the steps of:
designing recombinant fibronectin;
expressing recombinant fibronectin;
recombining domains in the recombinant fibronectin;
fibronectin expressing a recombination domain;
site-directed mutagenesis of amino acid residues of the domain recombined recombinant fibronectin;
the heat-resistant fibronectin is obtained after expressing the protein subjected to site-directed mutagenesis.
Still more preferably, the specific procedure for the step of recombining domains in recombinant fibronectin is as follows:
respectively expressing and purifying the structural domains contained in the recombinant fibronectin, and detecting the Tm value of the structural domains; different domains are combined to obtain the recombinant fibronectin with higher heat stability and improved heat resistance.
Fibronectin type I regions contain 12 highly similar repeat domain units, type II regions contain 2 repeat domain units, and type III regions contain 15 repeat domain units (Fn 1-Fn15 for short). Wherein the fibronectin type III region is functionally most important, comprises central binding domains Fn1-12 (the Fn10 domain of which comprises an arginine-glycine-aspartic acid sequence (Arg-Gly-Asp, RGD), recognizes and binds to cell surface integrins, thereby effecting cell adhesion, migration, etc.), and heparin binding domains Fn12-14. According to the structural characteristics of fibronectin, the fibronectin type III region is researched, so that the miniaturized recombinant fibronectin with high bioactivity and high stability is designed.
Still further preferred, the recombined domains comprise at least one of:
Fn8、Fn9、Fn10、Fn12、Fn13、Fn14。
more preferably, the specific procedure for site-directed mutagenesis of amino acid residues in recombinant fibronectin with a recombinant domain is as follows:
the structural calculation and three-dimensional modeling of the domain recombined recombinant fibronectin was performed using alphafold2.2.0. The domain recombined recombinant fibronectin is then subjected to molecular dynamics simulation and rational design, and potential unstable amino acid residues are selected and mutated. Constructing, purifying, screening mutation sites with improved heat stability, and combining to obtain the final high heat-resistant recombinant fibronectin variant.
The heat-resistant fibronectin prepared by the steps has good heat stability and extremely high biological activity. The test shows that rFN and Hythermfn have extremely high collagen generation activity, cell adhesion and cell migration promotion activity of human fibroblast I, and have good application prospect.
The invention also discloses an expression vector for preparing the heat-resistant fibronectin, which comprises nucleic acid for encoding the proteins shown in SEQ ID NO.2, 4, 6, 8 and 10.
Preferably, the expression vector comprises a nucleic acid as set forth in at least one of SEQ ID NO.1, 3, 5, 7, 9.
More preferably, the expression vector further comprises at least one pair of the above-described mutant primer sequences.
More preferably, the expression vector further comprises at least one pair of domain screening primers comprising SEQ ID NO.11-SEQ ID NO.26.
The invention also discloses an expression strain for preparing the heat-resistant fibronectin, which comprises the expression vector for preparing the heat-resistant fibronectin.
Preferably, the strain is at least one of escherichia coli, saccharomycetes and bacillus subtilis.
More preferably, the strain is E.coli BL21 (DE 3).
The invention also discloses application of the heat-resistant fibronectin, which comprises at least one of the following:
use in the preparation of cosmetics;
use in the manufacture of a medicament and/or delivery of a medicament;
use in the preparation of a cell culture medium.
Preferably, the thermostable fibronectin comprises the proteins shown in SEQ ID nos. 2, 4, 6, 8, 10.
More preferably, the thermostable fibronectin comprises the proteins shown in SEQ ID No.4, 6, 10.
Compared with the prior art, the invention has the beneficial effects that:
according to the technical scheme, the heat-resistant fibronectin is obtained by utilizing a directed evolution method through a genetic engineering method, the heat-resistant fibronectin has high heat stability, the biological activity of the heat-resistant fibronectin is not changed, and the application prospect of the fibronectin in the fields of cosmetic raw materials and the like is remarkably improved. The heat-resistant fibronectin has good heat stability, and the Tm values are all over 50 ℃ and the highest Tm value is even over 80 ℃. Has good thermal stability and extremely high biological activity at the same time of having good thermal stability. The test shows that rFN and Hythermfn have extremely high collagen generation activity, cell adhesion and cell migration promotion activity of human fibroblast I, and have good application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing SDS-PAGE results of rFN protein purification;
FIG. 2 is a predictive model of rFN protein structure;
FIG. 3 is a predictive model of rFN protein structure;
FIG. 4 is a flow chart of the fating G calculation of rFN;
FIG. 5 is a type III fibronectin domain sequence alignment;
FIG. 6 is a block diagram of the mutated amino acid residues of R245V and K375V;
FIG. 7 shows the results of a HythermFN-promoted human fibroblast adhesion assay;
FIG. 8 is a graph showing the comparison of the ability of HythermFN to promote human fibroblast adhesion before and after heating;
FIG. 9 is a comparison of the ability of HythermFN and wild type fibronectin to promote collagen production in human fibroblasts;
FIG. 10 is a diagram of rFn protein expression purification;
FIG. 11 is a predicted tertiary structure of rFn protein.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Preparation of recombinant fibronectin rFN1
1. Construction of rFN1 expression vector
The fibronectin type III Fn8-10 domain (cell binding domain, comprising RGD sequence) is linked to Fn12-14 domain (heparin binding domain) by Linker (amino acid sequence: KPSA) to form a novel recombinant fibronectin (rFN 1). Then, through codon optimization, the optimized rFN gene sequence is cloned between NdeI/XhoI restriction sites of the pET28a vector (vector construction is carried out by the Nanjing Jinsri biotechnology Co., ltd.) to obtain the pET28a-rFN1 vector. Wherein the base sequence corresponding to rFN protein is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO.2.
SEQ ID NO. 1：
CCCACTGATCTAAGGTTTACAAATATAGGACCCGATACCATGCGTGTGACCTGGGCACCGCCTCCAAGCATCGACCTGACCAACTTCTTGGTACGTTACAGCCCGGTTAAAAACGAAGAGGACGTTGCGGAACTGAGCATCTCTCCTAGCGACAACGCGGTTGTTCTGACCAACCTCCTTCCGGGTACAGAGTATGTGGTGTCAGTGTCGTCCGTTTATGAGCAGCACGAAAGCACCCCGCTGCGTGGTCGCCAGAAGACCGGTCTTGATTCCCCGACAGGTATTGACTTCTCTGACATCACGGCTAACTCCTTTACGGTGCATTGGATTGCGCCGAGAGCTACCATCACCGGCTACCGCATTCGCCATCACCCGGAGCACTTTAGCGGTCGTCCGCGCGAGGATCGTGTTCCGCACAGCCGTAATAGCATCACCTTGACGAACCTGACCCCGGGCACCGAGTACGTTGTCAGCATTGTTGCATTGAATGGTCGGGAAGAATCTCCGTTGCTCATTGGTCAACAAAGCACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCACCCTGCCCCACCCGAATCTGCATGGTCCGGAAATTCTGGATGTGCCGTCAACCTAA；
SEQ ID NO.2：
PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPST*。
2. Construction of rFN1 expression strain: pET28a-rFN1 was transformed into BL21 (DE 3) strain to give BL21/pET28a-rFN1 recombinant strain.
3. rFN1 protein expression: the recombinant strain was streaked on Km-resistant LB solid medium plates and cultured overnight at 37 ℃. The following day, the monoclonal colonies were selected and inoculated into 10 mL LB liquid medium (Km resistance), shake-cultured at 37℃at 230 rpm to grow approximately 6-8 h, 10 mL bacterial liquid was added to 1L large bottle LB liquid medium (Km resistance), shake-cultured at 37℃at 230 rpm to grow approximately 3 h, and OD600 was measured to be 0.5-0.6. IPTG solution is added into a large bottle of culture medium to obtain a final concentration of 500 mu M, and the culture is carried out on a shaking table at 18 ℃ and 230 rpm overnight.
4. rFN1 protein purification: (i) Culturing BL21 (DE 3) bacteria liquid expressing protein overnight, centrifuging at 4deg.C for 6000 and g for 10 min, and discarding supernatant; (ii) Adding 40 mL PBS buffer solution, and vortex shaking to resuspend the bacterial liquid until no obvious bacterial block exists; (iii) Using a high-pressure bacteria-breaking instrument to lyse escherichia coli released protein, and performing lysis for 8 min under the pressure of 80 MPa, wherein the high-pressure bacteria-breaking instrument is balanced by using a protein purification bacteria lysis buffer in advance and precooled to 4 ℃; (iv) The lysate of the last step is centrifuged at a high speed of 9500 g for 30min at 4 ℃; (v) Pouring the supernatant into a new sterile centrifuge tube, and filtering with a 0.45 mu m filter membrane to further remove impurities; (vi) All filtrate is loaded on Ni columns which are balanced by PBS buffer solution in advance; (vii) Washing the Ni column with a PBS buffer washing buffer containing 50 mM imidazole of 40 mL to remove the impurity proteins; (viii) The target protein was eluted from the Ni column using 250 mM imidazole PBS buffer of 30 mL; (ix) Transferring the protein solution to a protein concentration and ultrafiltration tube, centrifuging 5500 and g to 2 mL volumes, then adding PBS buffer solution without imidazole to 10 mL, and uniformly mixing; (x) Repeating step (ix) once, wherein the imidazole concentration in the protein solution is diluted to about 10 mM; (xi) The protein solution of the previous step 10 mL is fully loaded on a heparin column for deep purification, so that the protein purity is further improved, and the gel chromatographic column is balanced by PBS buffer solution in advance; (xii) Mixing conventional PBS buffer with PBS buffer containing 1M sodium chloride according to the program (PBS buffer containing 1M sodium chloride is increased linearly according to 0% to 100% content), and collecting proteins according to UV absorption peaks; (xiii) The protein solution was dialyzed, the protein buffer was replaced with conventional PBS buffer, and the protein was subsequently diluted to 1mg/mL and stored in a-80℃refrigerator for a long period of time. The rFn protein was subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed good purification of the protein, and the results are shown in FIG. 1.
5. rFN1 protein thermostability assay: the thermal stability of rFn1 protein was tested using the Protein Thermal Shift ™ kit (from ThermoFisher SCIENTIFIC). The thermal stability reaction system is 20 mu L (12.5 mu L protein solution (concentration 1 mg/mL), 2.5 mu L8 Xfluorescent dye and 5 mu L reaction buffer solution), and the thermal stability reaction system is uniformly mixed, a fluorescent PCR instrument is used for detecting a protein Tm value, and the whole sample adding process is operated on ice. Wherein the temperature rise program of the fluorescent PCR instrument is as follows: 25. incubating at the temperature of 2min, then uniformly raising the temperature to 99 ℃ at the temperature rising speed of 0.5 ℃/s (monitoring the fluorescence value in real time in the whole temperature rising process), incubating at the temperature of 99 ℃ for 1min, and analyzing the fluorescence data according to the internal program of a fluorescence PCR instrument after the reaction is finished to obtain the Tm value. The thermal stability experiment was repeated three times. The measurement result shows that the Tm value of rFn1 protein is 55.5 ℃ (Table 2) which is 75 ℃ lower than the lowest emulsification temperature of cosmetics.
Example 2
Preparation of recombinant fibronectin rFN2 No.2
1. Predicted rFn protein structure: the environment of the local alpha fold2.2.0 structure prediction algorithm is built, a rFn1 protein sequence is input to perform three-level structure calculation, and the prediction result is shown in fig. 2.
2. Expression and purification of the domains: according to the prediction result, 6 single domains of Fn8, fn9, fn10, fn12, fn13 and Fn14, and 2 triple domains of Fn8-10 and Fn12-14 are expressed and purified respectively. The construction of single domain expression plasmids, fn8-10 three domain expression plasmids and Fn12-14 three domain expression plasmids adopts a homologous recombination method to construct: PCR amplification was performed on the pET28a plasmid backbone and the target gene fragment, respectively, using the pET28a-rFN1 plasmid as a template (primers are shown in Table 1).
TABLE 1 primers for domain screening
The amplified product is subjected to agarose gel electrophoresis to detect a target band, and finally, a target gene fragment is cloned between NdeI/XhoI cleavage sites of the pET28a vector in a homologous recombination mode. The ligation product was transformed into E.coli DH 5. Alpha. Strain, and finally verified by sanger sequencing to construct the correct expression vector. Transforming the correctly constructed expression vector into BL21 (DE 3) strain to obtain expression strain, and using the expression strain to express; expression was followed by purification, and protein expression was consistent with the purification procedure described in example 1, rFN.
3. Domain thermal stability test: the experimental procedure was consistent with rFN protein. The results of the thermal stability experiments are shown in Table 2.
TABLE 2 domain thermal stability
| Domain (combination) | Tm（℃） |
| rFN1 | 55.5 |
| Fn8 | 63.7 |
| Fn9 | 49.4 |
| Fn10 | 82.5 |
| Fn12 | 77.2 |
| Fn13 | 66.3 |
| Fn14 | 70.9 |
| Fn8-10 | 54.0 |
| Fn12-14 | 72.6 |
| rFN2 | 73.0 |
From the thermal stability test results, it can be seen that: fn8 and Fn9 domains are relatively less thermostable than the other domains at 63.7℃and 49.4℃respectively. In addition, the thermostability (Tm: 54 ℃) was also much higher than that of Fn8-10 three domain protein (Tm: 72.6 ℃) compared to Fn12-14 three domain protein. Finally, fusion of Fn12-14 three domain protein with better thermal stability and Fn10 domain (comprising RGD sequence) to obtain rFN protein, wherein the corresponding base sequence is SEQ ID NO.3, and the amino acid sequence is SEQ ID NO.4;
SEQ ID NO.3：
CCGCACAGCCGTAATACCGTTAGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGCCGCAGCTGGTCCGTGGCGATTAA；
SEQ ID NO.4：
PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVRGD。
4. construction of rFN protein expression vector, protein expression and purification: the experimental procedure was consistent with the single domain proteins described above and the primers are shown in Table 1.
5. rFN2 protein thermostability assay: the experimental procedure was consistent with rFN protein. The results showed that the Tm of the rFN protein reached 73 ℃ (table 2), which is significantly better than the rFN protein.
Example 3
Directed evolution to improve rFN2 thermostability
Although the Tm value of rFN2 protein reaches 73 ℃, the Tm value is still lower than the lowest emulsification temperature of cosmetics by 75 ℃, so that the directed evolution is carried out based on rFN variant, the thermal stability of rFn protein is further improved, and the use requirement is met.
1. Predicted rFn protein structure: the rFN protein was structurally predicted using the local alphafold2.2.0 structure prediction algorithm, and the results are shown in fig. 3.
2. Determining mutation sites:
the fragile residues potentially affecting protein backbone stability at 368K73 ℃ were determined by rmsf calculations on protein backbone alpha carbon atoms using a gromacs kit to 800ns full atom molecular dynamics simulation of rFN protein sequences.
Aiming at rFN protein, a scheme (figure 4) for carrying out faterian father G high-throughput calculation in a Rosetta-MPI kit is established, and fatiguing residues obtained by the molecular dynamics simulation analysis are subjected to fatiguing G energy calculation, so that the influence of mutation specific residues at specific sites on the stability of a rFN structural skeleton is quantitatively predicted.
Through the calculation of the energy of the fatter G, the amino acid residues V170, E213, T226, K248 and E369 are respectively mutated into proline (P) so as to improve the stability of the protein.
Sequence conservation was analyzed by aligning different domains of fibronectin type III (fig. 5), and V72, D73, I94, M151, M169, a206, and V232, which were less conserved, were mutated into threonine (T), glutamic acid (E), leucine (L), valine (V), lysine (K), leucine (L), and isoleucine (I), respectively, which were more conserved at the corresponding sites.
Analysis of the rFN protein structure revealed that the side chains of the R245 and K375 amino acid residues deflect outwards, and that larger steric hindrance exists, which is detrimental to the stability of the domains (FIG. 6). Thus, we mutated the R245, K375 amino acid residues to valine (V) with less side chain steric hindrance, respectively.
3. Preparation of mutant proteins
According to the mutation sites obtained by the screening, 14 mutations of V170P, E213P, T226P, K P, E8238T, D73E, I94L, M151V, M169K, A206L, V232I, R45V and K375V are introduced into rFN protein in a single mutation or combined mutation mode; the mutations and primers required for construction of the expression vector are shown in Table 3. The expression vector is constructed by taking the pET28a-rFN2 vector as a PCR amplification template in a homologous recombination mode, and finally, the correct expression vector is constructed through sanger sequencing verification. The expression strain was obtained by transforming the constructed correct expression vector into BL21 (DE 3) strain, and the protein expression was consistent with the purification procedure and rFN purification procedure in example 1.
TABLE 3 primers required for amino acid point mutations
4. Thermal stability detection
Mutants containing a single mutation site and combinations of mutation sites were subjected to a thermostability experiment, and the results are shown in Table 4. Tm=tm (Mutant) -Tm (rFN 2) in table 4.
TABLE 4 mutant thermal stability
The thermal stability experiment shows that the HythermFN mutant containing 7 mutations (V72T/D73E/M169K/E213P/T226P/K248P/E369P) can raise the Tm value of rFN2 protein from 73 ℃ to 78.2 ℃, so that the thermal stability of fibronectin is obviously improved, and the application prospect of fibronectin is widened.
Example 4
Preparation of recombinant fibronectin rFN
1. Construction of rFN expression vector
Based on structural analysis, the FNIII subunit Fn10 domain containing RGD sequence is connected with Fn12-14 domain through Linker (amino acid sequence: KPSA) to form new recombinant fibronectin (rFn). And (3) cloning the optimized rFn sequence between NdeI/XhoI restriction sites of the pET28a vector (entrusting the construction of an expression vector by Nanjin St biotechnology Co., ltd.) through codon optimization to obtain the pET28a-rFn vector. Wherein the base sequence corresponding to the rFn protein is SEQ ID NO.7, and the amino acid sequence is SEQ ID NO.8;
SEQ ID NO. 7：
AGCGATGTCCCGCGCGATTTAGAGGTGGTTGCGGCAACGCCAACCAGCCTGCTGATCAGCTGGGATGCGCCAGCCGTCACTGTGCGCTATTATCGTATCACCTATGGTGAAACCGGCGGTAATAGTCCGGTGCAAGAATTCACCGTTCCGGGCTCGAAGAGCACGGCGACGATCTCTGGCTTGAAGCCAGGTGTTGATTACACCATTACGGTGTATGCCGTGACCGGTCGTGGCGATTCCCCGGCGAGCTCTAAGCCGATCTCTATTAACTATCGCACCGAGATTGATAAACCGTCCGCAATTCCAGCGCCGACTGACCTGAAGTTCACACAGGTTACCCCGACCTCGCTGTCTGCGCAGTGGACCCCGCCGAACGTGCAGCTGACTGGTTACCGCGTTCGTGTGACGCCGAAAGAAAAGACCGGACCGATGAAAGAGATCAATCTGGCTCCGGACAGCAGCTCCGTCGTGGTGTCTGGTCTGATGGTTGCCACCAAATACGAGGTTAGCGTCTACGCATTGAAGGATACCCTCACGAGCCGTCCGGCGCAAGGTGTGGTCACCACCTTGGAGAACGTATCCCCGCCGCGTCGTGCGCGTGTCACCGACGCCACCGAGACCACCATCACGATCTCCTGGCGTACGAAGACCGAGACCATTACTGGCTTTCAGGTGGACGCTGTGCCGGCGAACGGCCAAACCCCGATTCAGCGTACCATCAAACCGGACGTTCGCAGCTATACGATCACCGGTCTGCAACCGGGCACCGACTATAAAATCTACCTGTATACCCTGAATGATAACGCGCGTTCTAGCCCGGTTGTCATCGACGCTAGCACTGCGATTGACGCACCGTCCAATTTACGTTTTTTGGCGACCACCCCGAATTCGCTGCTGGTTTCCTGGCAGCCGCCACGCGCTCGTATCACCGGCTACATTATCAAGTACGAAAAACCAGGTAGCCCGCCGAGAGAGGTTGTACCTCGTCCTCGTCCGGGCGTGACTGAGGCAACCATCACTGGCCTGGAACCGGGCACTGAGTACACGATCTACGTGATTGCCCTGAAGAACAACCAAAAAAGCGAACCGCTTATCGGCCGTAAAAAAACGGATGAACTGTAA；
SEQ ID NO.8：
SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL。
2. construction of rFN expression strain: pET28a-rFn was transformed into BL21 (DE 3) strain to give BL21/pET28a-rFn recombinant strain.
3. rFN protein expression: the recombinant strain was streaked on Km-resistant LB solid medium plates and cultured overnight at 37 ℃. The following day, the monoclonal colonies were selected and inoculated into 10 mL LB liquid medium (Km resistance), shake-cultured at 37℃at 230 rpm to grow approximately 6-8 h, 10 mL bacterial liquid was added to 1L large bottle LB liquid medium (Km resistance), shake-cultured at 37℃at 230 rpm to grow approximately 3 h, and OD600 was measured to be 0.5-0.6. IPTG solution is added into a large bottle of culture medium to obtain a final concentration of 500 mu M, and the culture is carried out on a shaking table at 18 ℃ and 230 rpm overnight.
4. rFN protein purification: (i) Culturing BL21 (DE 3) bacteria liquid expressing protein overnight, centrifuging at 4deg.C for 6000 and g for 10 min, and discarding supernatant; (ii) Adding 40 mL PBS buffer solution, and vortex shaking to resuspend the bacterial liquid until no obvious bacterial block exists; (iii) Using a high-pressure bacteria-breaking instrument to lyse escherichia coli released protein, and performing lysis for 8 min under the pressure of 80 MPa, wherein the high-pressure bacteria-breaking instrument is balanced by using a protein purification bacteria lysis buffer in advance and precooled to 4 ℃; (iv) The lysate of the last step is centrifuged at a high speed of 9500 g for 30min at 4 ℃; (v) Pouring the supernatant into a new sterile centrifuge tube, and filtering with a 0.45 mu m filter membrane to further remove impurities; (vi) All filtrate is loaded on Ni columns which are balanced by PBS buffer solution in advance; (vii) Washing the Ni column with a PBS buffer washing buffer containing 50 mM imidazole of 40 mL to remove the impurity proteins; (viii) The target protein was eluted from the Ni column using 250 mM imidazole PBS buffer of 30 mL; (ix) Transferring the protein solution to a protein concentration and ultrafiltration tube, centrifuging 5500 and g to 2 mL volumes, then adding PBS buffer solution without imidazole to 10 mL, and uniformly mixing; (x) Repeating step (ix) once, wherein the imidazole concentration in the protein solution is diluted to about 10 mM; (xi) The protein solution of the previous step 10 mL is fully loaded on a heparin column for deep purification, so that the protein purity is further improved, and the gel chromatographic column is balanced by PBS buffer solution in advance; (xii) The proteins were collected according to UV absorbance peaks, the protein solution was transferred to a protein concentrate ultrafiltration tube, the proteins were concentrated to 1mg/mL and stored in a-80℃refrigerator for long periods. The rFn protein was subjected to denaturing polyacrylamide gel electrophoresis (SDS-PAGE), which showed good purification of the protein, and the results are shown in FIG. 10.
Example 5
1. Predicted rFn protein structure: the environment of the local alphafold2.2.0 structure prediction algorithm was constructed, and the rFn protein sequence was input to perform three-level structure prediction, and the prediction result was shown in fig. 11.
2. Determining mutation sites: based on the predicted results, 7 amino acid residues G39, K140, T179, G251, a269, T335 and N357 were selected for mutation to proline (P).
3. Preparation of mutant proteins
The mutants were amplified using pET28a-rFn plasmid as template, and the successfully constructed templates were transformed into BL21 (DE 3) strain and expressed the protein. The mutant expression and purification were identical to the method of expression and purification rFn in example 4. The mutant primers are shown in Table 5.
TABLE 5 primers for amino acid point mutations
4. Thermal stability detection
The detection kit is Protein Thermal Shift ™ kit (ThermoFisher SCIENTIFIC). The thermal stability reaction system is 20 mu L (12.5 mu L protein solution (concentration 1 mg/mL), 2.5 mu L8 Xfluorescent dye and 5 mu L reaction buffer solution), and the thermal stability reaction system is uniformly mixed, a fluorescent PCR instrument is used for detecting a protein Tm value, and the whole sample adding process is operated on ice. Wherein the temperature rise program of the fluorescent PCR instrument is as follows: 25. incubating at the temperature of 2min, then raising the quality of 99 ℃ at a constant speed at the temperature raising speed of 0.5 ℃/s (monitoring the fluorescence value in real time in the whole temperature raising process), incubating at the temperature of 99 ℃ for 1min, and analyzing the fluorescence data according to the internal program of a fluorescence PCR instrument after the reaction is finished to obtain the Tm value. Each mutant thermostability experiment was repeated three times and the experimental results are shown in table 6.
TABLE 6 mutant thermal stability
The results of the thermostability experiments showed that the mutations of K140P, N P357 and T335P monoamino acid residues increased their Tm to 74.5 ℃, 76.0 ℃ and 75.2 ℃ respectively, compared to the melting temperature of rFn protein 72.2 ℃. Finally, three mutations of K140P, N357P and T335P are introduced simultaneously, namely high heat-resistant ThrFn, and a thermal stability experiment shows that the Tm value reaches 81.0 ℃ and the heat resistance requirement in the cosmetic emulsification process is met.
Test example 1
Detection of thermotolerance and biological Activity of Hythermfn mutant
Fibronectin bioactivity appears in many ways, with promotion of cell adhesion and type I collagen production being important activity indicators. The biological activity of fibronectin was thus assessed by examining its adhesion to human fibroblasts and its effect on the secretion of type I collagen.
Sample treatment: filtering and sterilizing 1mg/ml fibronectin solution with 0.2 μm filter membrane; the appropriate amount of the solution is respectively placed in 70, 72 and 75 ℃ water baths for heating for 30min.
Cell adhesion detection: the fibronectin solution was diluted to 100 μg/ml, a proper amount of the solution was added to a 48-well plate to cover the surface, the plate was left at 4 ℃ overnight, the excess solution was sucked up, and a fibronectin coating layer was formed on the surface. Human Dermal Fibroblasts (HDF) P6 were cultured in complete medium (dmem+1% pen/strep+10% fbs), and when the cell density reached 70% -80%, cells were collected, inoculated in fibronectin-coated well plates, cultured for 6 hours, and after 6 hours, the cell adhesion state was observed by photographing with a microscope, and the cell adhesion rate was calculated with imageJ.
Collagen content detection: human Dermal Fibroblasts (HDF) P6 were cultured in complete medium (dmem+1% pen/strep+10% fbs), when the cell density reached 70% -80%, cells were collected, inoculated in 96-well plates for culturing cells, and after 24 hours, the new medium was replaced and fibronectin samples were added at the detection concentration. Culturing was continued in an incubator at 37 ℃. After 24 hours, cell supernatants were collected. The amount of collagen in the cell supernatant was measured by using human type I collagen (Col-1) ELISA Kit, and the fluorescence absorption intensity was measured by using an ELISA Kit.
Fig. 7 shows that the heated hythmfn still showed a significant effect of improving cell adhesion, fig. 8 shows that the hythmfn significantly promoted type I collagen production after heating at room temperature and 75 ℃ as compared to the blank, and fig. 9 shows that there was no significant difference in the effect of the hythmfn on promoting type I collagen production from wild-type WT fibronectin. The above results demonstrate that the hythmfn mutant exhibits extremely high thermostability while retaining the biological activity of the protein.
The foregoing description of the embodiments of the present invention is merely an optional embodiment of the present invention, and is not intended to limit the scope of the invention, and all equivalent structural modifications made by the present invention in the light of the present invention, the description of which and the accompanying drawings, or direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (10)
1. A thermostable fibronectin, characterized in that the amino acid sequence of the thermostable fibronectin comprises at least one of the following:
SEQ ID NO.2：
PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVTLPHPNLHGPEILDVPST；
SEQ ID NO.4：
PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDELPQLVRGD；
SEQ ID NO.6：
PHSRNTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGTEYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLKVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATPTTITISWRTKTEPITGFQVDAVPANGQTPIQRTIPPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSPPLIGRKKTDELPQLVRGD；
SEQ ID NO.8：
SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL；
SEQ ID NO.10：
SDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPSAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEPTGPMKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLPSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVPEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTDEL。
2. a nucleic acid encoding the thermostable fibronectin according to claim 1.
3. A method for preparing a thermostable fibronectin according to claim 1, comprising the steps of:
designing recombinant fibronectin;
site-directed mutagenesis of amino acid residues is performed on recombinant fibronectin;
the heat-resistant fibronectin is obtained after expressing the protein subjected to site-directed mutagenesis.
4. The method for producing heat-resistant fibronectin according to claim 3, wherein the site-directed mutation of the amino acid residue comprises mutation of at least one of the following amino acid residues:
g39, K140, T179, G251, a269, T335, N357, V170, E213, T226, K248, E369, V72, D73, I94, M151, M169, a206, V232, R245 and K375.
5. The method for preparing heat-resistant fibronectin according to claim 4, wherein the site-directed mutation of the amino acid residue comprises at least one of the following mutation modes:
G39P, K140, P, T, 179P, G251, P, A, P, T, 335, P, N, 357, 170, P, T, 226, P, K, 248, 4639, P, V, T, D, E, I, L, M, V, M, K, A, L, V, 232, I, R, 245V and K375V.
6. The method for preparing thermostable fibronectin according to claim 4, wherein the sequences of the mutant primers of G39 are SEQ ID No.27 and SEQ ID No.28, respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of K140 are SEQ ID NO.29 and SEQ ID NO.30 respectively; and/or the number of the groups of groups,
the sequences of the mutant primers of the T179 are SEQ ID NO.31 and SEQ ID NO.32 respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of G251 are SEQ ID NO.33 and SEQ ID NO.34 respectively; and/or the number of the groups of groups,
the sequences of the mutation primers of A269 are SEQ ID NO.35 and SEQ ID NO.36 respectively; and/or the number of the groups of groups,
the sequences of the mutant primers of the T335 are SEQ ID NO.37 and SEQ ID NO.38 respectively; and/or the number of the groups of groups,
the sequence of the mutation primer of N357 is SEQ ID NO.39, SEQ ID NO.40 and/or SEQ ID NO.40 respectively,
the sequences of the mutation primers of V170 are SEQ ID NO.41, SEQ ID NO.42 and/or SEQ ID NO.42 respectively,
the sequences of the mutation primers of the E213 are SEQ ID NO.43, SEQ ID NO.44 and/or SEQ ID NO.44 respectively,
the sequences of the mutation primers of the T226 are SEQ ID NO.45, SEQ ID NO.46 and/or SEQ ID NO.46 respectively,
the sequences of the K248 mutation primers are SEQ ID NO.47, SEQ ID NO.48 and/or SEQ ID NO.48 respectively,
the sequences of the mutation primers of E369 are SEQ ID NO.49, SEQ ID NO.50 and/or SEQ ID NO.50 respectively,
the sequences of the mutation primers of the V72 are SEQ ID NO.51, SEQ ID NO.52 and/or SEQ ID NO.52 respectively,
the sequences of the mutant primers of the D73 are SEQ ID NO.51, SEQ ID NO.52 and/or SEQ ID NO.52 respectively,
the sequences of the mutation primers of the I94 are SEQ ID NO.53, SEQ ID NO.54 and/or SEQ ID NO.54 respectively,
the sequences of the mutation primers of the M151 are SEQ ID NO.55, SEQ ID NO.56 and/or SEQ ID NO.56 respectively,
the sequences of the mutation primers of the M169 are SEQ ID NO.57, SEQ ID NO.58 and/or SEQ ID NO.58 respectively,
the sequences of the mutation primers of A206 are SEQ ID NO.59, SEQ ID NO.60 and/or SEQ ID NO.60 respectively,
the sequences of the V232 mutation primers are SEQ ID NO.61, SEQ ID NO.62 and/or SEQ ID NO.62 respectively,
the sequences of the mutation primers of R245 are SEQ ID NO.63, SEQ ID NO.64 and/or SEQ ID NO.64 respectively,
the sequences of the K375 mutant primers are SEQ ID NO.65 and SEQ ID NO.66 respectively.
7. A method for preparing thermostable fibronectin according to claim 3, comprising the steps of:
designing recombinant fibronectin;
recombining domains in the recombinant fibronectin;
site-directed mutagenesis of amino acid residues of the domain recombined recombinant fibronectin;
the heat-resistant fibronectin is obtained by expressing the protein after the site-directed mutagenesis;
wherein the domain comprises at least one of:
Fn8、Fn9、Fn10、Fn12、Fn13、Fn14。
8. an expression vector for producing thermostable fibronectin, characterized in that the expression vector comprises the nucleic acid of claim 2.
9. An expression strain for producing thermostable fibronectin, characterized in that the strain comprises the expression vector of claim 8.
10. Use of a thermostable fibronectin according to claim 1, characterized in that it comprises at least one of the following:
use in the preparation of cosmetics;
use in the manufacture of a medicament and/or delivery of a medicament;
use in the preparation of a cell culture medium.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117143224A (en) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | Recombinant human fibronectin truncated peptide and preparation method and application thereof |
| CN117466992A (en) * | 2023-12-27 | 2024-01-30 | 宝萃生物科技有限公司 | Fibronectin mutant and preparation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117143224A (en) * | 2023-10-26 | 2023-12-01 | 广州暨南大学医药生物技术研究开发中心有限公司 | Recombinant human fibronectin truncated peptide and preparation method and application thereof |
| CN117143224B (en) * | 2023-10-26 | 2024-01-30 | 广州暨南大学医药生物技术研究开发中心有限公司 | Recombinant human fibronectin truncated peptide and preparation method and application thereof |
| CN117466992A (en) * | 2023-12-27 | 2024-01-30 | 宝萃生物科技有限公司 | Fibronectin mutant and preparation and application thereof |
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