CN114163533A - Phycocyanin/chimeric calcitonin fusion gene, fusion protein, recombinant plasmid, transgenic saccharomyces cerevisiae and application - Google Patents
Phycocyanin/chimeric calcitonin fusion gene, fusion protein, recombinant plasmid, transgenic saccharomyces cerevisiae and application Download PDFInfo
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- CN114163533A CN114163533A CN202111040295.1A CN202111040295A CN114163533A CN 114163533 A CN114163533 A CN 114163533A CN 202111040295 A CN202111040295 A CN 202111040295A CN 114163533 A CN114163533 A CN 114163533A
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- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/14—Yeasts or derivatives thereof
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C07K2319/00—Fusion polypeptide
Abstract
The invention discloses a phycocyanin/chimeric calcitonin fusion gene, a fusion protein, a recombinant plasmid, transgenic saccharomyces cerevisiae and application. The invention constructs the obtained linear rDNA2-leu2-Ppgkthe-cpCB/5 hsCT-rDNA1 fragment is mediated and integrated into leucine-deficient saccharomyces cerevisiae BY taking rDNA as homologous recombination sites to obtain transgenic yeast BY4742-LPB5, so that the high-efficiency and stable expression of exogenous genes in saccharomyces cerevisiae cells is realized, the expression stability of calcitonin is greatly improved, and the problem of adverse influence of environmental pollution or food safety caused BY selection markers such as antibiotics on later-stage industrial production is solved. The production period of the saccharomyces cerevisiae is short, the fermentation technology is mature, the safety is good, the transgenic saccharomyces cerevisiae can be directly orally taken without complex processes such as protein extraction, cutting processing and purification, and the industrial production cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of biology, and particularly relates to a phycocyanin/chimeric calcitonin fusion gene, a fusion protein, a recombinant plasmid, transgenic saccharomyces cerevisiae and application thereof.
Background
Calcitonin (CT) is a polypeptide hormone consisting of 32 amino acid residues and secreted by the hind-chin of vertebrates such as fish and birds or by the thyroid parafollicular cells (C cells) of mammals. Since the first discovery of calcitonin by Copp et al in 1961, extensive studies have been made on the molecular structure and mechanism of action. Calcitonin amino acid sequences from different sources are different, but calcitonin from different organisms has activity to human, and the activity of calcitonin from the posterior gill body is higher than that of calcitonin from the thyroid gland. At present, the activity of salmon calcitonin (sCT) is the highest clinically, and is 20-40 times of that of human calcitonin (hCT). Calcitonin has been used clinically for many years, mainly for the prevention and treatment of senile osteoporosis, Paget's syndrome, hypercalcemia, etc., and as an adjuvant treatment drug for diabetes, gastric ulcer, acute gastroenteritis, etc. At present, calcitonin has been developed into two dosage forms of injection and nasal spray, no oral dosage form is available, and the most clinically used is salmon calcitonin injection (such as mirgainflue, gehrin and the like) synthesized chemically. Due to the short biological half-life of calcitonin, the clinical administration time is as long as more than 12 months, so that the administration of calcitonin to patients is extremely inconvenient and is accompanied by additional pain; and the content of calcitonin in organisms is very little, and chemically synthesized calcitonin has low yield and high cost and is easy to cause environmental pollution, thereby greatly limiting the wide application of calcitonin.
With the rise and development of genetic engineering technology, it becomes possible to prepare cheap calcitonin by using efficient biological expression systems (prokaryotic expression system and eukaryotic expression system). In the last two decades, various calcitonin expression modes have been obtained through bioengineering technology at home and abroad. The exogenous protein in a prokaryotic expression system has high yield and short production period, but cannot be modified after transcription, the activity of recombinant expression calcitonin is directly influenced by the amidation process level, bacteria easily generate heat sources and endotoxin and are not easy to remove, the protein purification process is complex, and the production cost is very high. The eukaryotic expression system can perform amidation modification on the exogenous protein, so that the production cost is saved, but the expression quantity of the exogenous gene is generally low, the production period is long, and the protein extraction and purification process is more complex, so that the application of the exogenous gene in production is limited. Besides, calcitonin molecules are too small to be stable in solution, and the eukaryotic expression system still cannot avoid the complicated subsequent work of protein extraction, cutting, purification and the like, thereby seriously affecting the yield of target protein. Therefore, the development of a more inexpensive and efficient eukaryotic expression system is urgently required.
Disclosure of Invention
The invention provides a phycocyanin/chimeric calcitonin fusion gene, a fusion protein, a recombinant plasmid, transgenic saccharomyces cerevisiae and application thereof, aiming at solving a series of problems that the expression level of calcitonin in an expression system is low, purification is required, activity is low, cost is high, oral administration cannot be carried out and the like.
In a first aspect, the present invention provides a phycocyanin/chimeric calcitonin fusion protein, which is achieved by the following technical scheme.
A phycocyanin/chimeric calcitonin fusion protein has an amino acid sequence shown in SEQ ID No. 2.
In a second aspect, the present invention provides a nucleic acid sequence encoding phycocyanin/chimeric calcitonin fusion protein, which is achieved by the following technical scheme.
A nucleic acid sequence for coding the phycocyanin/chimeric calcitonin fusion protein, wherein the nucleic acid sequence is shown as SEQ ID NO. 1.
Furthermore, the upstream amplification primer of the nucleic acid sequence is shown as SEQ ID NO.7, and the downstream amplification primer is shown as SEQ ID NO. 8.
In a third aspect, the present invention provides a recombinant plasmid, which is realized by the following technical scheme.
A recombinant plasmid, which comprises the nucleic acid sequence for coding the phycocyanin/chimeric calcitonin fusion protein.
Further, the recombinant plasmid comprises a pgk gene promoter; the upstream amplification primer of the pgk gene promoter is shown as SEQ ID NO.9, and the downstream amplification primer is shown as SEQ ID NO. 10.
Further, the recombinant plasmid comprises leu2 genetic selection marker gene; the upstream amplification primer of the leu2 genetic selection marker gene is shown as SEQ ID NO.11, and the downstream amplification primer is shown as SEQ ID NO. 12.
Further, the recombinant plasmid comprises two fragments of rDNA1 and rDNA 2; the upstream amplification primer of rDNA1 is shown in SEQ ID NO.13, and the downstream amplification primer is shown in SEQ ID NO. 14; the upstream amplification primer of rDNA2 is shown in SEQ ID NO.15, and the downstream amplification primer is shown in SEQ ID NO. 16.
In a fourth aspect, the invention provides saccharomyces cerevisiae, which is realized by adopting the following technical scheme.
A Saccharomyces cerevisiae, the preservation name is: saccharomyces cerevisiae (BY4742-LPB5) with a collection unit of: china general microbiological culture Collection center (CGMCC), abbreviated as: CGMCC with preservation date as follows: 12/25/2020, with a deposit number: CGMCC 21552.
In a fifth aspect, the invention provides a preparation method of saccharomyces cerevisiae, which is realized by adopting the following technical scheme.
A preparation method of Saccharomyces cerevisiae BY4742-LPB5 comprises the following steps:
(1) cloning of phycocyanin/chimeric calcitonin fusion gene:
performing PCR (polymerase chain reaction) amplification on the phycocyanin/chimeric calcitonin fusion gene (cpcB/5hscT), connecting a cloning vector pMD19-T, and constructing a recombinant plasmid pMD19-cpcB/5 hscT;
(2) cloning of the promoter sequence of the pgk Gene:
taking Saccharomyces cerevisiae BY4742 genome DNA as a template, carrying out PCR reaction amplification to obtain a pgkpromoter sequence, connecting a cloning vector pMD19-T, and constructing a recombinant plasmid pMD 19-pgkpromoter;
(3) cloning of the selectable marker gene leu 2:
carrying out PCR amplification by taking the genomic DNA of Saccharomyces cerevisiae YS (Saccharomyces cerevisiae, China center for culture Collection of Industrial microorganisms, the strain number: CICC 1964) as a template to obtain leu2 gene, and connecting with a cloning vector pMD19-T to construct a recombinant plasmid pMD19-leu 2;
(4) cloning of rDNA fragment:
taking Saccharomyces cerevisiae BY4742 genome DNA as a template, respectively carrying out PCR reaction amplification to obtain two fragments of rDNA1 and rDNA2, respectively connecting into a cloning vector pMD19-T, and constructing recombinant plasmids pMD19-rDNA1 and pMD19-rDNA 2;
(5) preparation of a fusion gene expression plasmid containing cpCB/5 hscT:
firstly, inserting rDNA1 fragment cut by BamHI and EcoRI into cloning vector pUC19 digested by the same endonuclease, secondly, inserting cpCB/5hsCT cut by XbaI and SacI into the upstream of rDNA1 fragment, then inserting pgkpromoter cut by SalI and XbaI into the upstream of cpCB/5hsCT, then inserting leu2 cut by XhoI and SalI into the upstream of pgkpromoter, and finally inserting rDNA2 fragment cut by SphI and XhoI into the upstream of leu2 to construct pUC19-rDNA2-leu2-PpgkcpcB/5hscT-rDNA1 (pUC 19-RLPB5R for short);
(6) the conversion of the phycocyanin/chimeric calcitonin fusion gene into saccharomyces cerevisiae:
after the plasmid pUC19-RLPB5R is subjected to enzyme digestion BY Sph I and Not I, a linear DNA fragment containing a phycocyanin/chimeric calcitonin fusion gene is obtained, the linear fragment is transformed into leucine-deficient saccharomyces cerevisiae BY4742 competent cells BY a LiAc mediated method, and a transformant is screened for recombinants on an SD/-Leu plate;
(7) the culture steps of the phycocyanin/chimeric calcitonin fusion gene yeast are as follows:
transgenic Saccharomyces cerevisiae BY4742-LPB5 was grown in SD/-Leu medium to logarithmic phase at 30 deg.C, 1/10 of bacterial liquid was aspirated into YPD medium to continue culturing cells to 5 × 107cells/ml; the shaker is set to 30 ℃ and 200 rpm; detecting the phycocyanin/chimeric calcitonin fusion gene yeast, and freeze-drying for storage.
Further, in the step (1), the PCR reaction amplification is carried out by taking the cpCB/5hsCT-F and the cpCB/5hsCT-R as primers:
primer cpb5 hsCT-F: 5'-GTCTAGAATGTTTGATGCCTTCACCA-3' (SEQ ID NO: 7);
primer cpb5 hsCT-R: 5'-CGAGCTCTCATTACGGGGTGCCGGAG-3' (SEQ ID NO: 8);
the phycocyanin/chimeric calcitonin fusion gene cpcB/5hsCT is shown as SEQ ID NO: 1.
Further, in the step (2), a primer P is usedpgk-F and Ppgk-R performing PCR reaction amplification:
primer Ppgk-F：5′-GTCGACTATTTTAGATTCCTGACTTCAAC-3′(SEQ ID NO:9)；
Primer Ppgk-R：5′-TCTAGAATATTTGTTGTAAAAAGTAGATAATTACT-3′ (SEQ ID NO:10)；
The sequence of the pgkpromoter gene is shown in SEQ ID NO. 3.
Further, in the step (3), the PCR reaction amplification is carried out by using primers leu2-F and leu 2-R:
primer leu 2-F: 5' -GCATGCCTCGAGTAACGAGAACACACAGGG-3′(SEQ ID NO:11)；
Primer leu 2-R: 5' -GTCGACAGATTGCGTATATAGTTTCGTC-3′(SEQ ID NO:12)；
The leu2 gene sequence is shown in SEQ ID NO. 4.
Further, in the step (4), PCR amplification is carried out by using primers rDNA1-F and rDNA1-R, rDNA2-F and rDNA2-R respectively:
primer rDNA 1-F: 5' -GAGCTCACCTACCGACCAACTTTCA-3′(SEQ ID NO:13)；
Primer rDNA 1-R: 5' -GAATTCGCGGCCGCAGGACATGCCTTTGATAT-3′ (SEQ ID NO:14)；
Primer rDNA 2-F: 5' -GCATGCTATAGGAAATGAGCTTTTCT-3′(SEQ ID NO:15)；
Primer rDNA 2-R: 5' -CTCGAGGCGAAACCACAGCCAAGG-3′(SEQ ID NO:16)；
The rDNA fragment is respectively positioned in the linear DNA fragment rDNA2-leu2-PpgkThe both sides of the cpcB/5hsCT-rDNA1, the rDNA1 fragment sequence is shown as SEQ ID NO. 5, and the rDNA2 fragment sequence is shown as SEQ ID NO. 6.
Further, in the step (6), the formula of the SD/-Leu solid medium is as follows, and is measured in w/v: 0.67% yeast nitrogen source, 2% glucose, 0.004% adenine sulfate, 0.002% arginine hydrochloride, 0.01% aspartic acid, 0.01% glutamic acid, 0.002% histidine, 0.003% lysine, 0.002% methionine, 0.005% phenylalanine, 0.0375% serine, 0.02% threonine, 0.004% tryptophan, 0.003% leucine.
In a sixth aspect, the invention provides a medicament or health product for treating osteoporosis, which is realized by adopting the following technical scheme.
A medicine or health product for treating osteoporosis comprises freeze-dried Saccharomyces cerevisiae.
Further, the minimum dosage of the saccharomyces cerevisiae is 0.02 g/kg.
The present application has the following advantageous effects.
The invention connects a single-copy phycocyanin gene (deleting a downstream stop codon sequence) with a 5-copy human-salmon chimeric calcitonin gene (deleting an upstream transcription initiation codon sequence) to synthesize a novel phycocyanin/chimeric calcitonin fusion gene (cpcB/5hscT), improves the stability of molecules, adopts a constitutive strong promoter (pgkpemoter) of saccharomyces cerevisiae to efficiently start the expression of phycocyanin/chimeric calcitonin, then mediates the high copy number stable integration of the phycocyanin/chimeric calcitonin fusion gene in a saccharomyces cerevisiae genome by taking rDNA as a homologous recombination site, realizes the efficient stable expression of exogenous genes in saccharomyces cerevisiae cells, greatly improves the expression amount of calcitonin, meanwhile, the leu2 gene is used as a genetic selection marker gene of the saccharomyces cerevisiae, so that the problem of adverse effect or insecurity brought by selection markers such as antibiotics and the like to later-stage industrial production is solved. The transgenic saccharomyces cerevisiae BY4742-LPB5 gastrically normal adult female SD rat has the continuous capability of reducing blood calcium and blood phosphorus. The invention provides a new way for oral administration of calcitonin, increases the stability of calcitonin in the purification and clinical application processes, prolongs the biological half-life of calcitonin, widens the application of calcitonin to clinical treatment of osteoporosis and the like, and provides a basis for wider application of calcitonin in the medical industry.
Drawings
FIG. 1 is a plasmid map of recombinant plasmid pUC19-rDNA2-leu2-Ppgk-cpcB/5hscT-rDNA1 of the present invention;
FIG. 2 is the restriction enzyme digestion verification result of the recombinant plasmid pUC19-rDNA2-leu2-Ppgk-cpcB/5hscT-rDNA1 of the invention;
FIG. 3 is a graph showing the results of screening Saccharomyces cerevisiae transformants using SD/-Leu plates according to the present invention;
FIG. 4 is a diagram showing the result of agarose gel electrophoresis of a Saccharomyces cerevisiae transformant screened by the PCR method according to the present invention;
FIG. 5 is a diagram showing the detection result of Western Blot of transgenic Saccharomyces cerevisiae of the present invention;
FIG. 6 is a graph showing the results of the blood calcium-lowering activity of the transgenic Saccharomyces cerevisiae of the present invention in SD rats;
FIG. 7 is a graph showing the results of the serum phosphorus-reducing activity of the transgenic Saccharomyces cerevisiae of the present invention in SD rats.
Detailed Description
The invention is further described below with reference to the figures and examples.
The invention preserves strains of the Saccharomyces cerevisiae (BY4742 for short) and the recombinant Saccharomyces cerevisiae (BY4742-LPB5 for short) after the transgenosis.
Saccharomyces cerevisiae (BY 4742): name of the depository: china Center for Type Culture Collection (CCTCC); address: wuhan university depository in Wuchang district, Wuhan city, Hubei province, No. 299 one-path; the preservation date is as follows: 2007.12.03, respectively; the preservation number is: CCREM SDMCC 011184 are provided.
Transgenic recombinant Saccharomyces cerevisiae (BY4742-LPB 5): name of the depository: china general microbiological culture Collection center (CGMCC); address: western road No.1, north west city of township, beijing, institute of microbiology, china academy of sciences; the preservation date is as follows: 12 month and 25 days 2020; the preservation number is: CGMCC 21552.
Chimeric Calcitonin gene (hsCT) takes human and salmon Calcitonin as a primer, so as to reduce the antigenicity and increase the biological activity of the Calcitonin gene, and the Calcitonin gene is modified and designed and synthesized by computer-aided software. The gene has a single copy total length of 96bp, encodes 32 amino acids, wherein the first 16 amino acids comprise 14 human amino acids, the last 16 amino acids are all salmon amino acids, the activity of the gene is higher than that of hCT, and the antigenicity of the gene is lower than that of sCT. Because the molecular weight of the single-copy hsCT is only 3.5KD, the single-copy hsCT is extremely unstable in a solution environment, and in order to improve the expression quantity and stability of molecules of the single-copy hsCT, a section of flexible peptide sequence (DNA sequence is GGCTCGTCGAGTGGTGTG, and amino acid sequence is Gly-Ser-Ser-Gly-Val) is designed as a connecting peptide, 5 single-copy hscts are connected in series end to end through the flexible peptide, and then the carboxyl end of the cpcB is connected with the amino end of the 5hsCT, so that the fusion protein cpcB/5hsCT is synthesized.
The phycocyanin/chimeric calcitonin is expressed in a saccharomyces cerevisiae eukaryotic system, the phycocyanin/chimeric calcitonin is based on the advantages that saccharomyces cerevisiae is easy to produce, a fermentation technology is quite mature, the expression level is high, saccharomyces cerevisiae is a food-safety-level model eukaryotic microorganism and can be directly eaten, the yeast after freeze drying can relieve the degradation capability under a digestive juice environment, the degradation of calcitonin is reduced, and calcitonin can directly pass through epidermal cells of animal gastrointestinal tracts to enter blood in a polypeptide form. The obtained saccharomyces cerevisiae of the phycocyanin/chimeric calcitonin fusion gene can be directly orally taken without complicated processes such as protein purification, cutting processing, purification and the like, and has good safety, thereby greatly reducing the production cost and providing an effective and cheap administration way for the clinical application of calcitonin.
Moreover, an efficient homologous recombination repair double-stranded DNA break (DSB) mechanism exists in the saccharomyces cerevisiae, and systematic researches show that a homologous region with the length of 15bp can possibly obtain recombinant clone, and when the homologous region is 30bp long, the frequency of effective homologous recombination can be close to 80%, so that the saccharomyces cerevisiae is more suitable for carrying out the recombinant cloning of an exogenous gene by applying a homologous recombination technology than other organisms. The yeast genome has 100-200 repeated rDNA units, and the invention uses the repeated rDNA units as integration sites to improve the integration copy number of the exogenous genes.
The existence of the screening marker can facilitate screening of the recombinants, improve the screening efficiency and greatly reduce the screening workload. For Saccharomyces cerevisiae, the selection markers are mainly classified into 2 types: auxotrophic markers and antibiotic markers. If the transformed engineering strain is to be applied to industrial and agricultural production, especially food industrial production, the antibiotic screening marker cannot meet the industrial requirements. Because the existence of the antibiotic screening marker can have adverse effects on the fermentation of the engineering bacteria in the aspects of food safety, environmental pollution and the like. Nowadays, people have higher and higher requirements on food safety, and the trend of finding a safe and reliable screening method to replace antibiotic markers is inevitable. The breeding method using the auxotrophic marker gene transformation can solve the above problems well. Many studies have shown that transformants obtained by auxotrophic markers do not have antibiotic markers and do not suffer from antibiotic problems in metabolite production. According to the invention, the leucine-deficient saccharomyces cerevisiae strain is used as a receptor strain, the wild leucine gene (leu2) is introduced into a saccharomyces cerevisiae genome by taking rDNA repetitive units as homologous recombination sites, and transformants are screened through expression of leu2 gene, so that industrial production of phycocyanin/chimeric calcitonin fusion protein is facilitated.
The specific experimental process is as follows:
preparation of phycocyanin-transferring/chimeric calcitonin fusion gene saccharomyces cerevisiae
1. Design of fusion genes and primer Synthesis
A pair of primers, namely, cpcB-F, cpcB-R, 5hscT-F and 5hscT-R, are respectively designed by taking published gene sequences of cpcB (GenBank: KF052038.1) and 5hscT (GenBank: KR065696.1) as templates. In order to ensure that the spatial structures of the proteins expressed by the two genes are relatively independent, a connecting peptide gene is designed at the gene fusion position; introducing an EcoRI site at the 5 'end of the cpCB-F primer, introducing a BamHI site at the 5' end of the cpCB-R primer, and simultaneously carrying out reverse coding on 2 amino acids (GS) of the joint, and then carrying out reverse coding on 5 amino acids (GGGSG) at the N end of the joint and 8 amino acids at the C end of a cpCB subunit; to prevent hairpin formation at the 3' end, which affects amplification, the cpcB-R was designed as a degenerate primer. A BamHI site and Gly (G) are introduced into a primer 5hsCT-F, a Hand III site and a stop codon are introduced into a primer 5hsCT-R, and a Not I site is introduced into the primer 5hsCT-R so as to facilitate entering an expression vector after enzyme digestion. The primer synthesis was performed by Biotech (Shanghai) and the amplification was performed using Pyrobest DNA Polymerase, and the primers were as follows:
CpcB-F:5’-GGAATTCTTTGATGCCTTCAC-3’(SEQ ID NO.17)；
CpcB-R:5’-CGGGATCCACCAGATCCACCGCCGGAAACYGCYGCAGCAG CACGGTC-3’(SEQ ID NO.18)；
5hsCT-F:5’-CGGGATCCGGTCTGTGCGGTAACCTGTC-3’(SEQ ID NO.19)；
5hsCT-R:5’CCCAAGCTTGCGGCCGCTCATTACGGAGTACCAGAACCG-3’ (SEQ ID NO.20)。
2. construction of fusion Gene
Performing PCR amplification by using spirulina genome DNA and recombinant plasmid pMD18-5hsCT (both presented by the teaching of the Zao Xiao nan of the academy of Marine Life of China) as templates; the obtained cpCB gene is cut by EcoRI and BamHI and is connected into a cloning vector pUC19 to obtain a recombinant plasmid pUC-cpCB; the obtained 5hscT gene (gene sequence GenBank: KR065696.1) was digested with BamHI and Hand III, ligated into recombinant plasmid pUC19-cpcB, to obtain a recombinant plasmid containing the fusion gene cpcB/5hscT, named pUC19-cpcB/5 hscT.
3. Cloning of phycocyanin/chimeric calcitonin fusion gene
The DNA sequence of the phycocyanin/chimeric calcitonin fusion gene cpcPCB/5 hsCT is as follows (SEQ ID NO. 1):
primers are designed according to the DNA sequence of phycocyanin/chimeric calcitonin fusion gene cpcB/5hscT, and XbaI and SacI sites are respectively introduced into the primers cpcB/5hscT-F and cpcB/5 hscT-R. The primer sequences are respectively as follows:
cpcB/5 hscT-F: 5'-GTCTAGAATGTTTGATGCCTTCACCA-3' (SEQ ID NO: 7); cpcB/5 hscT-R: 5'-CGAGCTCTCATTACGGGGTGCCGGAG-3' (SEQ ID NO: 8). Carrying out PCR reaction by using the recombinant plasmid pUC19-cpcB/5hscT as a template, wherein the PCR reaction program is that the PCR reaction program is pre-denatured at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 80s, 20 cycles; keeping the temperature at 72 ℃ for 5 min. The PCR product is detected by 2% agarose gel electrophoresis and recovered about 1200bp, and then cloned to pMD18-T carrier, transformed Escherichia coli DH5 alpha competent cell, cultured and selected single colony, screened positive clone strain by PCR and enzyme digestion identification, and analyzed by sequencing, the phycocyanin/chimeric calcitonin fusion gene (shown as sequence SEQ ID NO:1 in sequence table) can code 367 amino acid residues, the 1 st position is methionine of initiation codon, connecting peptide (Gly Gly Gly Ser Gly Gly Ser Gly Leu) is arranged between cpCB and 5hsCT, two amino acids behind the 365 st position are continuous stop codons, the molecular weight is 37.4KDa, and the coding generated amino acid sequence is shown as follows (SEQ ID NO: 2):
4. cloning of the promoter of the pgk Gene
The phycocyanin/chimeric calcitonin fusion gene is preceded by pgkpromoter (abbreviated as P)pgkPhosphoglycerate kinase gene promoter) which is one of constitutive promoters with the highest starting efficiency in a saccharomyces cerevisiae system, is not inhibited by any carbon source, and can realize continuous expression of the cpcB/5hsCT protein in saccharomyces cerevisiae cells under the condition of not changing culture conditions.
According to a pgk gene sequence published by GenBank (GenBank accession No. X59720.2), an upstream primer and a downstream primer are respectively designed at the upstream of-778 bp and-7 bp of an ATG start codon, the primers are named as Ppgk-F and Ppgk-R, a Sal I site is introduced at the 5 'end of the upstream primer, and an Xba I site is introduced at the 3' end of the downstream primer.
The DNA sequence of the pgk gene is as follows (SEQ ID NO: 3):
the primer sequences are respectively as follows:
Ppgk-F：5′-GTCGACTATTTTAGATTCCTGACTTCAAC-3′(SEQ ID NO:9)；
Ppgk-R：5′-TCTAGAATATTTGTTGTAAAAAGTAGATAATTACT-3′(SEQ ID NO:10)。
taking Saccharomyces cerevisiae BY4742 genomic DNA as template, PCR reaction program: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 46 ℃ for 30s, and extension at 72 ℃ for 60s, for 20 cycles; keeping the temperature at 72 ℃ for 5 min. The PCR product is detected by 1.5% agarose gel electrophoresis, recovered to 1490bp, cloned to pMD18-T carrier, transformed into Escherichia coli DH5 alpha competent cell, cultured, selected single colony, screened out positive clone bacterial strain by PCR and enzyme digestion identification, and analyzed by sequencing to be consistent with the expected sequence (shown as sequence SEQ ID NO:3 in sequence table).
5. Cloning of the leu2 genetic selection marker Gene
According to the sequence of the leu2 gene published by GenBank (GenBank Accession No. NC-001137.3), upstream and downstream primers are designed at the upstream-333 bp and downstream +122bp of an ATG initiation codon, named as leu2-F and leu2-R respectively, and a SphI site and a XhoI site are introduced at the 5 'end of the upstream primer and a SalI site is introduced at the 3' end of the downstream primer.
The DNA sequence of leu2 gene is as follows (SEQ ID NO: 4):
the primer sequences are respectively as follows:
leu2-F：5′-GCATGCCTCGAGTAACGAGAACACACAGGG-3′(SEQ ID NO:11)；
leu2-R：5′-GTCGACAGATTGCGTATATAGTTTCGTC-3′(SEQ ID NO:12)。
taking Saccharomyces cerevisiae genome DNA as a template, and carrying out PCR reaction program of pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 80s, 20 cycles; keeping the temperature at 72 ℃ for 5 min. And (3) detecting the PCR product by 1.5% agarose gel electrophoresis, recovering about 1600bp, cloning the PCR product to a pMD18-T vector, transforming escherichia coli DH5 alpha competent cells, culturing, selecting a single colony, and screening out positive clones by PCR and enzyme digestion identification. After sequencing, the leu2 gene is positioned in front of pgkpromoter and is used as a selection marker gene for saccharomyces cerevisiae genetic transformation, the full length of the leu2 gene sequence is 1550bp, sequence comparison analysis is carried out to obtain a promoter sequence of leu2 gene in 48-334 bp, a gene coding region in 334-1428 bp, and a polyA tail sequence after 1428 bp.
6. Cloning of rDNA fragment
Lopes and the like are consulted to find that a DNA sequence between 5S and 25S in rDNA can mediate the stable integration of the exogenous gene, two rDNA sequences at two sides of HpaI locus in the rDNA sequence (GenBank accession No. NC-001144.5) are selected to mediate the homologous recombination of the exogenous gene and a saccharomyces cerevisiae genome, and primers are designed and named as rDNA1-F, rDNA1-R, rDNA2-F and rDNA2-R respectively. When designing the primer, SacI site is introduced into 5 'end of rDNA1-F, and Not I and EcoRI sites are introduced into 3' end of rDNA 1-R. The 5 'end of primer rDNA2-F introduced SphI site, and the 3' end of rDNA2-R introduced XhoI site. The primer sequences are respectively as follows:
rDNA1-F：5′-GAGCTCACCTACCGACCAACTTTCA-3′(SEQ ID NO:13)；
rDNA1-R：5′-GAATTCGTTAACAGGACATGCCTTTGATAT-3′(SEQ ID NO:14)；
rDNA2-F：5′-GCATGCTATAGGAAATGAGCTTTTCT-3′(SEQ ID NO:15)；
rDNA2-R：5′-CTCGAGGCGAAACCACAGCCAAGG-3′(SEQ ID NO:16)；
carrying out PCR reaction by using Saccharomyces cerevisiae genome DNA as a template, wherein the PCR reaction program is pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 80s, 20 cycles; keeping the temperature at 72 ℃ for 5 min. Detecting and recovering about 1100bp and 1460bp of PCR product by 1.5% agarose gel electrophoresis, cloning to pMD18-T vector, transforming Escherichia coli DH5 alpha competent cell, culturing, selecting single colony, screening out positive clone strain by PCR and enzyme digestion identification, sequencing and analyzing, wherein the rDNA fragment is located in linear DNA fragment rDNA2-leu2PpgkFlanking the cpcB/5hscT-rDNA1, linear DNA fragment rDNA2-leu2-Ppgkthe-cpCB/5 hsCT-rDNA1 carries out homologous recombination with saccharomyces cerevisiae through rDNA locus, the lengths of the rDNA1 fragment and the rDNA2 fragment are 1102bp and 1474bp respectively, the oligonucleotide long-chain sequence rDNA1 is shown as SEQ ID NO:5, the oligonucleotide long-chain sequence rDNA2 is shown as SEQ ID NO:6, and the sequence is consistent with the expected sequence.
The DNA sequence of the long oligonucleotide sequence rDNA1 is as follows (SEQ ID NO: 5):
the DNA sequence of the long oligonucleotide sequence rDNA2 is as follows (SEQ ID NO: 6):
7. construction of recombinant clone plasmid pUC19-rDNA2-leu2-Ppgk-cpcB/5hscT-rDNA1
According to the sequence analysis and experimental design requirements of each gene, a recombinant clone plasmid pUC18-rDNA2-leu2-Ppgk-cpCB/5hsCT-rDNA1 is constructed on the basis of a clone vector pUC 19. Inserting rDNA1 fragment obtained by cutting pMD18-rDNA1 with EcoR I and Sac I into pUC19 vector digested with the same endonuclease, inserting cpB/5 hsCT obtained by cutting pMD 18-cpB/5 hsCT with Xba I and Sac I into the upstream of rDNA1 fragment, inserting Ppgk obtained by cutting pMD18-Ppgk with Sal I and Xba I into the upstream of cpB/5 hsCT, inserting Ppu 2 obtained by cutting pMD18-leu2 with Xho1 and Sal I into the upstream of Ppgk, and inserting rDNA2 fragment obtained by cutting pMD18-rDNA2 with Sph I and leXho I into the upstream of leu2 to construct pUC 638-rDNA 2-rDNA 2-rDNA 1-prb-5-rDNA 1 (as shown in the figure).
The recombinant plasmid is digested by five groups of restriction endonucleases (the digestion verification result of the recombinant plasmid is shown in figure 2): no.1 obtains a product rDNA1 gene by double enzyme digestion of Not I and Sac I, and the fragment is about 1100bp and is the same as the expected product according to the display of a picture 2, thereby proving that rDNA1 is successfully connected between the sites of the Not I and Sac I of the vector; the product of the target gene of the cpcB/5hsCT is obtained by double digestion of XbaI and SacI No.2, and the fragment is about 1200bp which is shown according to the figure 2 and is the same as the expected product, thereby proving that the cpcB/5hsCT is successfully connected between the sites of the vector SacI and XbaI; the product pgk promoter sequence is obtained by double digestion of SalI and XbaI No. 3, and the fragment is about 750bp and is the same as the expected product according to the display of a graph in FIG. 2, so that the Ppgk is proved to be successfully connected between the sites of the XbaI and SalI of the vector; the product Leu2 screening marker gene is obtained by double enzyme digestion of No. 4 SphI and SalI, and the fragment is about 1600bp and is the same as the expected product according to the display of a picture 2, so that Leu2 is proved to be successfully connected between the SalI site and the XhoI site of the vector; SphI No. 5 and XhoI double enzyme digestion to obtain product rDNA2 gene, according to the picture 2, the fragment is about 1500bp, the same size with the expected product, prove rDNA2 successfully connected between vector XhoI and SphI site; the genes were successfully ligated into the vector in the expected order to construct a fusion gene cloning vector pUC19-rDNA1-cpcB/5hscT-Ppgk-leu2-rDNA 2.
8. Conversion of phycocyanin/chimeric calcitonin fusion gene into Saccharomyces cerevisiae
After the recombinant plasmid pUC19-rDNA2-leu 2-Ppgk-cpB/5 hsCT-rDNA1 is subjected to Not I and Sph I enzyme digestion, a rDNA2-leu 2-Ppgk-cpB/5 hsCT-rDNA1 linear fragment is obtained, and the linear fragment is transformed into a leucine-deficient saccharomyces cerevisiae BY4742 competent cell BY a LiAc mediated method. Transformants the recombinant BY4742-LPB5 was screened on SD/-Leu (leucine deficient selection medium) plates. Positive clones were identified by PCR, and the results are shown in FIG. 3 (A: screening of recombinants on SD/-Leu plates initially; B: detection of genetic stability of recombinants on SD/-Leu plates; C: growth of recombinants on YPD plates and SD/-Leu plates, respectively).
The formulation of SD/-Leu medium is as follows (w/v): 0.67% yeast nitrogen source, 2% glucose, 0.004% adenine sulfate, 0.002% arginine hydrochloride, 0.01% aspartic acid, 0.01% glutamic acid, 0.002% histidine, 0.003% lysine, 0.002% methionine, 0.005% phenylalanine, 0.0375% serine, 0.02% threonine, 0.004% tryptophan, 0.003% leucine.
9. Culture of transgenic and non-transgenic Saccharomyces cerevisiae
Transgenic Saccharomyces cerevisiae BY4742-LPB5 and non-transgenic Saccharomyces cerevisiae BY4742 were pre-cultured in YPD medium at 30 ℃ to log phase, respectively. Sucking 1/10 bacterial liquid into fresh YPD culture medium to continue culturing cells to 108cells/mL. The shaker was set at 30 ℃ and 200 rpm.
The YPD medium was formulated as follows (w/v): 2% glucose; 2% peptone; 1% yeast extract.
10. Expression of recombinant phycocyanin/chimeric calcitonin fusion protein in saccharomyces cerevisiae cells
The promoter of phosphoglycerate kinase gene (Ppgk) is a highly efficient constitutive promoter in Saccharomyces cerevisiae system, and is not controlled by carbon source, and as yeast cells grow continuously, exogenous genes are expressed at any time and accumulated in the yeast cells. Therefore, continuous expression of the foreign protein in the transgenic yeast cell is achieved without changing the culture conditions.
(1) A transgenic yeast BY4742-LPB5 was picked up and monocloned into 10mLYPD liquid medium, while a non-transgenic yeast BY4742 was picked up and monocloned into 10mLYPD liquid medium as a negative control and cultured overnight in a 30 ℃ constant temperature shaker.
(2) Reading the absorbance, OD, of the culture at a wavelength of 600nm600Should be between 2 and 5. When OD is reached600When the concentration is less than 2 or more than 5, the culture is continued to the concentration range or the dilution is carried out to the concentration.
(3) Centrifugation was carried out at 3000-.
(4) OD modulation in fresh YPD Medium of resuspended cells600The value reaches between 0.5 and 0.6.
(5) Culturing the yeast cells on a shaker at 30 ℃ and aspirating an amount of 2OD in 48-72 hours600Is thin and thinCell cultures were placed on ice.
(6) The cell culture is analyzed.
11. Freeze drying of phycocyanin-transferring/chimeric calcitonin fusion gene yeast
Saccharomyces cerevisiae cells were harvested in a refrigerated centrifuge at 4 deg.C, 5000rpm, 5min, and then resuspended in physiological saline. The resuspension was frozen to-40 ℃ at a rate of 1 ℃/min. The samples were freeze dried on a freeze dryer at a pressure of 6Pa and during the last two hours of freeze drying the samples were heated to 23 ℃ to reduce residual water vapour.
Second, gene detection (PCR technology) of phycocyanin/chimeric calcitonin fusion gene Saccharomyces cerevisiae
1. The cultivation steps before the genetic testing of the transgenic and non-transgenic Saccharomyces cerevisiae are as described above.
2. Extraction of Saccharomyces cerevisiae genomic DNA
(1) Taking the mixture containing 1-2 x 108The overnight culture of cells yeast cells was placed in a 1.5mL centrifuge tube and centrifuged at 12000rpm for 1 min.
(2) The supernatant was aspirated as much as possible using a micropipette. To the pellet was added 500. mu.L of GenTLE Yeast Solution A, gently shaken and the pellet was well suspended, incubated at 37 ℃ for 1h, during which the centrifuge tubes were gently shaken several times.
(3) Adding 100. mu.L GenTLE Yeast Solution B, shaking slightly and mixing well, and water bathing at 70 deg.C for 10 min.
(4) Add 200. mu.L of GenTLE Yeast Solution C, mix well with GenTLE shaking, cool for 5min on ice.
(5) Centrifuge at 12000rpm at 4 ℃ for 5 min. Carefully transfer the supernatant to a new 1.5mL centrifuge tube (Do not aspirate the pellet). Add 1/2 volumes of isopropanol (ca. 400. mu.L) to the supernatant and invert the tube upside down to mix well.
(6) Centrifuge at 12000rpm at 4 ℃ for 5min and discard the supernatant. To the pellet was added 500. mu.L of pre-cooled 70% ethanol and the pellet was washed gently upside down.
(7) Centrifuge at 12000rpm at 4 ℃ for 5 min. The supernatant was aspirated as much as possible using a micropipette. The DNA precipitate was naturally dried at room temperature until free from ethanol smell.
(8) The appropriate amount of TE Buffer was added to dissolve the genomic DNA.
PCR reaction and agarose gel electrophoresis detection
(1) Carrying out PCR reaction BY taking the genomic DNA of the transgenic saccharomyces cerevisiae BY4742-LPB5 and the non-transgenic saccharomyces cerevisiae BY4742 as templates and taking primers cpcB/5hscT-F and cpcB/5hscT-R as primers, wherein the PCR reaction program is that the PCR reaction is carried out for 5min at 94 ℃; denaturation at 94 ℃ for 30s, annealing at 45 ℃ for 30s, extension at 72 ℃ for 80s, 20 cycles; keeping the temperature at 72 ℃ for 5 min.
(2) The PCR product was detected by 2% agarose gel electrophoresis, and the results showed that the negative control group (N) had no DNA band, and the experimental groups (recombinants No. 11-14) all had single DNA band of about 1200bp, which preliminarily indicated that the cpcB/5hsCT gene was successfully transferred into the Saccharomyces cerevisiae genome and stably integrated (the agarose gel electrophoresis detection results are shown in FIG. 4, wherein P is the positive control group, the template is recombinant plasmid pUC19-cpcB/5hsCT, N is the negative control group, the template is non-transgenic Saccharomyces cerevisiae genome DNA, and B is the blank control group, no template).
4. Analysis of Gene sequences
After recovering the DNA fragment of about 1200bp, the sequencing was performed directly. The obtained sequence is compared with the sequence of SEQ ID NO:1 through the Nucleotide BLAST module function of the NCBI online website, and the transgenic saccharomyces cerevisiae BY4742-LPB5 with the contrast ratio of 100% is screened out.
Third, Western Blot qualitative analysis of phycocyanin/chimeric calcitonin fusion protein expressed in Saccharomyces cerevisiae cells
1. The culturing steps before Western Blot were carried out on the transgenic and non-transgenic yeasts were as described above.
2. Processing of proteins in Yeast cells
(1) The collected yeast culture was dissolved in 160. mu.L of triple distilled water, and 40. mu.L of 5 XSDS-PAGE Loading Buffer was added and mixed well.
(2) Placing in boiling water bath for 10-15min, shaking for several times, cooling at room temperature, and allowing yeast cell lysate to precipitate completely.
SDS-PAGE detection
(1) SDS-polyacrylamide gels were prepared according to the following table
And (3) installing a device, injecting the separation gel into the interlayer of the glass plate by using a micropipette, and sealing by using a small amount of distilled water after the liquid level reaches a certain height so as to keep the gel surface flat and prevent oxygen from entering the gel to prevent the gel from solidifying. After 30min separation gel polymerization, preparing concentrated gel, absorbing upper layer water with absorbent paper, then pouring concentrated gel with a micropipettor, and inserting a sample comb.
(2) Adding Tris-Gly electrophoresis buffer solution into an electrophoresis tank, carefully pulling out a gel-making comb, washing a sample application hole by using a micropipette, and sequentially loading 10-20 mu L of processed sample supernatant.
(3) Firstly, using low voltage (70-80V) for about 30min, increasing the voltage (100-120V) after the sample just enters the separation gel or is concentrated into a straight line in the concentration gel, and stopping electrophoresis when the bromophenol blue indicator reaches about 1cm away from the bottom edge.
(4) Taking out the gel, marking, and fixing in 10% trichloroacetic acid solution for 30 min.
(5) The gel was transferred to Coomassie brilliant blue staining solution and stained for 6-8h with shaking.
(6) Transferring the gel into a decoloring solution, and shaking for decoloring for a plurality of hours until the gel background is clean and the strips are clear.
(7) The gel was stored in distilled water at 4 ℃.
4. Electrotransfer of yeast proteins
(1) The gel was equilibrated by immersion in transfer buffer for 10 min. (Note: if a small protein of less than 20kDa is detected, this step can be omitted, since the small protein readily diffuses out of the gel.)
(2) And (3) shearing 6 pieces of PVDF membrane and filter paper according to the size of the glue, soaking and activating the PVDF membrane in pure methanol for 1-5s, and then respectively putting the PVDF membrane and the filter paper into a transfer buffer solution for balancing for 10 min.
(3) A 'wet transfer' device is adopted, an electric transfer device (three layers of filter paper-PVDF membrane-polyacrylamide gel-three layers of filter paper in sequence from bottom to top) is arranged according to a 'sandwich method', the gel is close to a cathode, the membrane is close to an anode, and the electric transfer is carried out for about 60min under the conditions of 180-200 mA.
(4) After the electrotransfer is finished, the PVDF membrane is washed by distilled water at room temperature, a small strip is cut off according to the molecular weight of the target protein and the internal reference protein and is placed in ponceau to be dyed for 1min, and the dyeing solution is gently shaken during the process. After the protein bands appeared, the gel was rinsed several times with distilled water at room temperature until ponceau was washed off. And after confirming that the electrotransfer is successful, carrying out Western Blot detection.
Western Blot detection
(1) Blocking with appropriate amount of blocking solution (0.02 mol/L PBS solution of 5% skimmed milk powder, based on immersed membrane), at 37 deg.C for 1h or 4 deg.C overnight.
(2) PVDF membrane was washed 3 times (10 min/time) with an appropriate amount of 0.02mol/L PBS.
(3) Reacting with a primary antibody: the target protein adopts rabbit anti-human calcitonin antibody (titer 1: 100-; the internal control protein adopts a mouse anti-beta-tubulin antibody (the titer is 1:100-1000), and is diluted by primary anti-diluent according to the proportion of 1: 500. Incubate at 37 ℃ for 2h or 4 ℃ overnight, respectively.
(4) PVDF membrane was washed 3 times (10 min/time) with an appropriate amount of 0.02mol/L PBS.
(5) Reaction with a secondary antibody: horse Radish Peroxidase (HRP) labeled goat anti-rabbit lgG (titer 1: 1000-.
(6) PVDF membrane was washed 4 times (10 min/time) with an appropriate amount of 0.02mol/L PBS.
(7) And (3) placing the PVDF membrane on an internal plate of a chemiluminescence imaging system, dripping a proper amount of chemiluminescence liquid at a position with the same molecular weight as the expected protein, exposing by adopting the chemiluminescence imaging system, taking a picture and storing after an obvious protein band appears after exposure for 10-20 s.
(8) The Western Blot detection result shows that the negative control (N) and the human calcitonin antibody have no antigen antibody immunoreaction band, and the positive control (P) and the experimental groups (12h, 24h and 36h) and the human calcitonin antibody have obvious antigen antibody immunoreaction bands, which show that the cpcB/5hsCT fusion protein is successfully and efficiently expressed in Saccharomyces cerevisiae cells, shows stronger immunoreaction with the human calcitonin antibody and has no obvious time effect on the protein expression (the Western Blot detection result is shown in figure 5, M is a protein marker, P is a positive control, N is a negative control, a third lane is a 12h protein sample, a fourth lane is a 24h protein sample, and a fifth lane is a 36h protein sample).
Enzyme-linked immunosorbent assay (Elisa) assay of expression of cpcB/5hsCT fusion protein in saccharomyces cerevisiae cells
1. The cultivation steps before Elisa of the transgenic and non-transgenic s.cerevisiae were as described above.
2. Extracting total protein of transgenic yeast and non-transgenic yeast by ultrasonic disruption method
(1) 500mg of freeze-dried yeast was weighed into a 10mL centrifuge tube, 4mL of 0.02mol/L PBS solution was added to thoroughly suspend the precipitate, and the mixture was placed on ice.
(2) After ultrasonication for 20min, the mixture was centrifuged at 4500rpm at 4 ℃ for 5min, and the precipitate was discarded.
(3) The supernatant was transferred to multiple 1.5ml centrifuge tubes, centrifuged at 12000rpm at 4 ℃ for 5min, and the pellet was discarded.
(4) The total protein concentration of each sample was determined and the data recorded, and the samples were stored at-20 ℃.
3. Enzyme-linked immunosorbent assay (Elisa) procedure is as follows
(1) Diluting human calcitonin standard substance, negative control total protein and total protein of sample to be tested with coating solution (CB for short, pH 9.6) at a certain ratio (1: 2)2，1:23，1:24，1:25，1:26， 1:27,1:28,1:29) Enzyme plates (150. mu.L per well) were coated and triplicated for each sample at 4 ℃ overnight. Simultaneously setting 3 blank holes, 3 holes with antigen and 1 antibody without adding 1 antibody, and 3 holes with antigen and 1 antibody without adding 2 antibody;
(2) the coating solution was aspirated, and a blocking solution (0.01 mol/L PBS buffer containing 5% skimmed milk powder) was added thereto at 200. mu.L per well and blocked at 37 ℃ for 1 hour.
(3) After blocking was complete, the blocking solution was discarded and 200. mu.L of 0.02mol/L PBST was added to each well and the plates were washed 3 times.
(4) mu.L of an antibody solution containing a rabbit anti-human calcitonin antibody (titer: 1:500-1000) diluted in a ratio of 1:1000 was added to each well, and incubated at 37 ℃ for 2 hours.
(5) After the reaction was complete, the solution was discarded, 200. mu.L of 0.02mol/L PBST was added to each well, and the plate was washed 3 times.
(6) mu.L of an antibody solution containing HRP-labeled goat anti-rabbit IgG (titer: 1:3000-25000) diluted at a ratio of 1:5000 was added to each well, and incubated at 37 ℃ for 1 hour.
(7) After the reaction was complete, the solution was discarded and 200. mu.L of 0.02mol/L PBST wash plates were added 3 times per well.
(8) 100 μ L of TMB developing solution was added to each well, and the reaction was carried out for 10min at room temperature in the dark. When the positive control shows obvious color change or the negative control shows slight color change, 100 mu.L of 2mol/L H is added into each hole2SO4And (4) dissolving to terminate the reaction.
(9) Immediately after the reaction, the optical density of each well at 450nm was measured using a microplate reader.
(10) And drawing a standard curve of the human calcitonin standard substance, and calculating the expression level of the cpcB/5hsCT fusion protein in the saccharomyces cerevisiae cells to be about 3.16%.
Fifthly, the biological activity of the transgenic yeast BY4742-LPB5 in rats is measured
In vivo activity experiments in animals were performed according to methods of the british pharmacopoeia. The animals were normal SD rats (all adult female rats). In the animal in vivo activity test for detecting the transgenic yeast BY4742-LPB5, the animal test is divided into a negative control group, a positive drug group and three high, medium and low dose test groups. The negative control group was inoculated with 0.50g/kg dose of stomach-filling lyophilized non-transgenic yeast BY4742 based on the rat body weight, the positive drug group was injected subcutaneously with 0.83IU/kg dense calcium source (salmon calcitonin injection for medical use), and the high, medium and low dose groups were inoculated with 0.50g/kg, 0.10g/kg and 0.02g/kg doses of stomach-filling lyophilized transgenic yeast BY4742-LPB5 based on the rat body weight, respectively. Experimental animals whole blood was drawn from the tail vein blood vessels at different time periods (1, 2, 4, 8, 12 hours) and serum was prepared and subjected to determination of blood calcium value and blood phosphorus value.
As can be seen from the graph in FIG. 6, the positive drug and the three doses of the transgenic yeast BY4742-LPB5 with high, medium and low gavage all show obvious blood calcium reducing activity. Compared with a negative control group, the blood calcium values of other four groups basically show a trend of rapidly decreasing and then slowly rising within 12 hours; the positive control group and the low-dose experimental group both achieve the maximum effect of reducing the blood calcium at the 4 th hour, the blood calcium values are reduced by 0.36mM and 0.30mM respectively, the blood calcium values slowly rise after 4 hours, and the blood calcium values at the 12 th hour are reduced by 0.13mM and 0.18mM respectively; the blood calcium reducing effect of the medium-dose experimental group reaches the maximum at the 2 nd hour, the blood calcium value is reduced by 0.58mM, the blood calcium value slowly rises after 2 hours, and the blood calcium value at the 12 th hour is reduced by 0.39 mM; the high dose group continued to decrease over 12h, reaching essentially maximal effect at 12h, with a decrease in the serum calcium value of 0.58 mM. Compared with the positive control group, the blood calcium reducing effect of the low-dose experimental group is basically consistent with that of the positive control group, and the blood calcium reducing effect of the medium-dose and high-dose experimental groups is better than that of the high-dose and medium-dose experimental groups. Compared with the three dose experimental groups, the blood calcium reducing effect is stronger with the increase of the dose, and a certain dose effect is shown. The experimental results show that the freeze-dried transgenic yeast BY4742-LPB5 with the gavage dose of 0.02-0.10 g/kg can obviously reduce the blood calcium value (p) of rats<0.01), the maximum calcium reduction effect range is 0.30-0.58 mM, and the maximum drug effect time TmaxThe maximum blood calcium reducing capacity is slightly higher than that of a positive medicament (dense calcium information) of 2IU/kg within 2-4 hours, and the duration of the medicament effect is obviously longer than that of the dense calcium information.
As can be seen from fig. 7, the blood phosphorus values of the groups showed a gradual decrease in 12 hours. Compared with a negative control group, the blood phosphorus values of the positive drug group and the high, medium and low dose groups are obviously reduced. The blood phosphorus value of the positive control group is rapidly reduced within 4 hours, the blood phosphorus reducing effect reaches the maximum at 4 hours and is reduced by 1.06mM, and the blood phosphorus value slowly rises after 4 hours; the blood phosphorus value of the low-dose group is rapidly reduced within 2 hours, the blood phosphorus reducing effect reaches the maximum at the 2 nd hour, the blood phosphorus reducing effect is reduced by 0.57mM, the blood phosphorus value slowly rises after 2 hours, and the blood phosphorus value is basically consistent with that of a negative control group at the 12 th hour; the blood phosphorus value of the medium-dose group reaches the maximum effect of reducing blood calcium at 1 hour, the blood phosphorus value is reduced by 0.49mM, the blood phosphorus value slowly rises after 1 hour, and the blood phosphorus value is still reduced by 0.20mM at 12 hours; the blood phosphorus value of the high-dose group is continuously reduced within 4 hours, the blood phosphorus reducing effect reaches the maximum at the 4 th hour, the blood phosphorus reducing effect is reduced by 0.77mM, the blood phosphorus value slowly rises after 4 hours, and the blood phosphorus value is still reduced by 0.57mM at the 12 th hour. Compared with a positive control group, the blood phosphorus reducing effect of the low, medium and high dose test groups does not reach the corresponding level. However, compared with the three dose experimental groups, the blood phosphorus reducing effect is stronger with the increase of the dose, and a certain dose effect is shown. The experimental results show that the transgenic yeast BY4742-LPB5 with the stomach-filling dose of 0.10-0.50 g/kg has obvious blood phosphorus reduction activity, the maximum blood phosphorus reduction effect range is 0.49-0.77 mM, the maximum drug effect time Tmax is 2-4 hours, the maximum blood phosphorus reduction capability is lower than that of a positive drug (dense calcium information) of 2IU/kg, and the drug effect duration is obviously superior to that of the dense calcium information.
In conclusion, for human clinical application, according to the calculation of the drug effect of 12 hours, the transgenic yeast BY4742-LPB5 with the weight of about 3-6 g is predicted to be taken BY a patient with the weight of 60kg every day, so that the blood calcium and blood phosphorus reducing effects of the salmon calcitonin injection clinically applied at present can be achieved, and the oral administration of calcitonin can be basically realized.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (10)
1. A phycocyanin/chimeric calcitonin fusion protein, comprising: the amino acid sequence of the fusion protein is shown as SEQ ID NO. 2.
2. A nucleic acid sequence encoding the phycocyanin/chimeric calcitonin fusion protein of claim 1, wherein said nucleic acid sequence comprises seq id no: the nucleic acid sequence is shown as SEQ ID NO. 1.
3. The nucleic acid sequence encoding phycocyanin/chimeric calcitonin fusion protein according to claim 2, wherein the upstream amplification primer of said nucleic acid sequence is shown as SEQ ID No.7, and the downstream amplification primer is shown as SEQ ID No. 8.
4. A recombinant plasmid, characterized in that: comprising the nucleic acid sequence encoding a phycocyanin/chimeric calcitonin fusion protein as claimed in claim 2.
5. A recombinant plasmid according to claim 3, wherein: the recombinant plasmid comprises a pgk gene promoter; the upstream amplification primer of the pgk gene promoter is shown as SEQ ID NO.9, and the downstream amplification primer is shown as SEQ ID NO. 10.
6. A recombinant plasmid according to claim 3, wherein: the recombinant plasmid comprises leu2 genetic selection marker gene; the upstream amplification primer of the leu2 genetic selection marker gene is shown as SEQ ID NO.11, and the downstream amplification primer is shown as SEQ ID NO. 12.
7. A recombinant plasmid according to claim 3, wherein: the recombinant plasmid comprises two fragments of rDNA1 and rDNA 2; the upstream amplification primer of rDNA1 is shown in SEQ ID NO.13, and the downstream amplification primer is shown in SEQ ID NO. 14; the upstream amplification primer of rDNA2 is shown in SEQ ID NO.15, and the downstream amplification primer is shown in SEQ ID NO. 16.
8. A Saccharomyces cerevisiae is characterized in that: the preservation name is: saccharomyces cerevisiae (BY4742-LPB5) with a collection unit of: china general microbiological culture Collection center (CGMCC), abbreviated as: CGMCC with preservation date as follows: 12/25/2020, with a deposit number: CGMCC 21552.
9. A pharmaceutical or nutraceutical product for the treatment of osteoporosis, comprising freeze-dried saccharomyces cerevisiae according to claim 8.
10. The medicine or health product for treating osteoporosis of claim 9, wherein the minimum dosage of Saccharomyces cerevisiae is 0.02 g/kg.
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| CN1721446A (en) * | 2004-12-30 | 2006-01-18 | 中国海洋大学 | Fusion protein and method for preparing same |
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| WO2018224477A1 (en) * | 2017-06-06 | 2018-12-13 | Institut National De La Recherche Agronomique (Inra) | Phycocyanin composition for use in inhibiting bone resorption |
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| CN1721446A (en) * | 2004-12-30 | 2006-01-18 | 中国海洋大学 | Fusion protein and method for preparing same |
| CN104560753A (en) * | 2015-01-06 | 2015-04-29 | 中国海洋大学 | Human-salmon chimeric calcitonin tandem gene yeast YS-5hsCT and preparation method and application thereof |
| WO2018224477A1 (en) * | 2017-06-06 | 2018-12-13 | Institut National De La Recherche Agronomique (Inra) | Phycocyanin composition for use in inhibiting bone resorption |
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