CN118027168A - Preparation method and application of MSL recombinant plant protein based on eukaryotic expression - Google Patents
Preparation method and application of MSL recombinant plant protein based on eukaryotic expression Download PDFInfo
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- CN118027168A CN118027168A CN202410315393.9A CN202410315393A CN118027168A CN 118027168 A CN118027168 A CN 118027168A CN 202410315393 A CN202410315393 A CN 202410315393A CN 118027168 A CN118027168 A CN 118027168A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method and application of mannose-specific lectin recombinant plant protein based on eukaryotic expression. The method comprises the steps of culturing cells comprising a nucleic acid molecule encoding the recombinant plant protein under conditions suitable for expression of the mannose-specific lectin recombinant plant protein, obtaining a culture, and isolating the recombinant plant protein from the culture. The mannose-specific lectin recombinant plant protein obtained by eukaryotic expression can promote the level of NO/cGMP in the tissue of the cavernous body, restore the activity of oxidation injury of the endothelial cells of the cavernous body, improve the male erection function and further improve the male reproductive function.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a preparation method and application of MSL recombinant plant protein based on eukaryotic expression.
Background
Erectile dysfunction (erectile dysfunction, ED) is the most common male sexual dysfunction, meaning that the penis continues to fail to achieve or maintain a sufficient erection to complete satisfactory sexual life, over 3 months of disease. ED is mainly divided into psychogenic ED and organic ED. ED occurrence is closely related to a variety of risk factors, including aging with incidence rate of more than 10% of the population, diabetes, obesity, smoking, hypertension, cardiovascular diseases, depression, spinal cord injury, etc.
The medicines for treating erectile dysfunction commonly used in the medical field at present comprise a 5-type phosphodiesterase inhibitor and a 5-hydroxytryptamine reuptake inhibitor, and the 5-type phosphodiesterase inhibitor can cause dizziness, headache, diarrhea, priapism and the like along with some side effects during treatment, and the 5-hydroxytryptamine reuptake inhibitor belongs to psychosis medicines with anxiolytic effect, and can cause dizziness, headache, palpitation, nausea, somnolence, anejaculation, hypotension and the like.
ED has become a common chronic disease, seriously harms male reproductive health, reduces life quality, and affects family harmony. Thus, there is a need for a medicament for preventing, ameliorating or treating erectile dysfunction.
Disclosure of Invention
In order to solve at least part of the problems in the prior art, the invention provides a preparation method and application of mannose-specific lectin (Mannose SPECIFIC LECTIN, MSL) recombinant plant protein based on eukaryotic expression. Specifically, the present invention includes the following.
In a first aspect of the present invention there is provided a method of preparing a mannose-specific lectin recombinant plant protein, or an active fragment thereof, comprising the steps of culturing a cell comprising a nucleic acid molecule encoding said recombinant plant protein under conditions suitable for expression of the mannose-specific lectin recombinant plant protein, resulting in a culture, and isolating the recombinant plant protein from said culture, wherein said cell is a eukaryotic cell.
In certain embodiments, the method of making a mannose-specific lectin recombinant plant protein, or active fragment thereof, according to the invention, comprises the steps of:
(1) Connecting a base sequence containing recombinant plant proteins encoding mannose-specific lectin with a vector to construct a recombinant protein expression vector;
(2) Transforming the expression vector into eukaryotic cells and inducing expression of the recombinant protein under suitable culture conditions;
(3) Isolating the mannose-specific lectin recombinant plant protein.
In certain embodiments, the method of making a mannose-specific lectin recombinant plant protein, or active fragment thereof, according to the invention, wherein the nucleic acid molecule encoding the recombinant plant protein has a nucleotide sequence depicted as (I) or (II):
(I) A nucleotide sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and having the same function;
(II) the nucleotide sequence shown in SEQ ID No.1 is obtained by replacing, deleting or adding one or more nucleotides and has the same function.
In certain embodiments, the method of preparing a mannose-specific lectin recombinant plant protein, or an active fragment thereof, according to the invention, wherein the nucleic acid molecule encoding the mannose-specific lectin recombinant protein comprises a coding sequence resulting from codon optimization.
In certain embodiments, the method of preparing a mannose-specific lectin recombinant plant protein, or active fragment thereof, according to the invention, wherein the nucleic acid molecule encoding the recombinant plant protein has the nucleotide sequence depicted in SEQ ID NO. 1.
In a second aspect, the invention provides the use of a recombinant plant protein obtained according to the preparation method of the invention in the preparation of a medicament for promoting or improving reproductive function.
In a third aspect of the invention, there is provided the use of a recombinant plant protein obtained according to the preparation method of the invention in the manufacture of a medicament for the prevention or treatment of erectile dysfunction.
In a fourth aspect of the invention, there is provided the use of a recombinant plant protein obtainable by a method according to the invention for the preparation of a medicament for increasing the level of NO in cavernous tissue.
In a fifth aspect of the invention there is provided the use of a recombinant plant protein obtainable by a method according to the invention for the manufacture of a medicament for increasing cGMP levels in spongy tissue.
In a sixth aspect, the invention provides the use of the recombinant plant protein obtained by the preparation method of the invention in the preparation of a medicament for restoring the viability of oxidative damage cavernous endothelial cells.
The MSL recombinant protein or the active fragment thereof prepared by the invention can promote the level of NO/cGMP in the cavernous tissue, recover the activity of oxidation injury cavernous endothelial cells, improve the male erection function and further improve the male reproductive function.
Drawings
FIG. 1 shows PCDNA 3.1.1-MSL-His vector maps.
FIG. 2 shows an electrophoretogram of the recombinant plasmid after PCR amplification, wherein the leftmost band is MSL and the right two bands are markers.
FIG. 3 shows SDS-PAGE electrophoresis of recombinant proteins at different elution volumes, wherein M: DNA MARKER, lane 1:20mM imidazole elution, lane 2:300mM imidazole elution-1, lane3:300mM imidazole elution-2.
FIG. 4PCDNA3.1-MSL-His recombinant protein electrophoretogram, wherein M: DNA MARKER, lane 1: recombinant proteins.
FIG. 5 immunofluorescence analysis MCECs purity (magnification: 100X).
FIG. 6MSL recombinant protein increases H 2O2 -induced MCECs oxidative damage model cell viability, wherein # # # P <0.0001vs. control; * P <0.05, < P <0.01, < P <0.0001vs.h 2O2.
Fig. 7 effect of MSL recombinant protein on NO levels in spongy tissue of kidney-yang deficient rats (n=10), wherein # # # P <0.0001 compared to control group; p <0.05, < P <0.001 compared to model group.
Fig. 8 effect of MSL recombinant protein on cGMP levels in kidney-yang deficient rat cavernous tissue (n=10), wherein # # # P <0.0001 compared to control group; p <0.05, < P <0.01 compared to model group.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
Preparation method
In one aspect of the invention, methods are provided for the preparation of mannose-specific lectin (sometimes referred to herein simply as "MSL") recombinant plant proteins, or active fragments thereof. The terms "protein," "peptide," and "polypeptide" are used interchangeably herein to refer to a plurality of covalently linked amino acid residues joined by amide linkages. "protein" also includes post-translational modifications introduced by chemical or enzymatic reactions, it being understood that the term may encompass variants or fragments of a protein.
Any suitable MSL protein may be used in the present invention, and the MSL protein may include a full-length MSL protein or a biologically active fragment thereof. As used herein, a biologically active fragment of MSL protein is meant to be a polypeptide that retains all or part of the function of MSL protein. Typically, the biologically active fragment retains at least 50% of the activity of the MSL protein. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the MSL protein.
In a preferred embodiment, the MSL proteins of the invention are isolated or purified from plants. In another preferred embodiment, the MSL protein may also be artificially prepared, such as a recombinant MSL protein produced according to conventional genetic engineering recombinant techniques.
The proteins or polypeptides of the invention may have covalent modifications at the C-terminus and/or N-terminus. They may also exist in various forms (e.g., native, fusion, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimerised, particulate, etc.).
MSL proteins derived from other species are encompassed by the present invention, particularly those proteins having homology to MSL proteins. As used herein, the term "homology" refers to a polymeric molecule, e.g., an overall correlation between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In general, the term "homology" means the evolutionary relationship between two molecules. Thus, two homologous molecules will have a common evolutionary ancestor. In the context of the present invention, the term "homology" includes identity and similarity.
"Homology" can be determined using methods known in the art (such as sequence comparison algorithms) when compared and aligned with maximum identity over a comparison window, two or more sequences have a specified percentage of nucleotides that are identical over a specified region. The percentage of "homology" between two sequences can be determined using the BLASTP algorithm version 2.2.2 (Altschul, stephen f., thomas l.madden, alejandro a. Jinghui Zhang, zheng Zhang, webb Miller, and David J.Lipman(1997),"Gapped BLAST and PSI-BLAST:a new generation of protein database search programs",Nucleic Acids Res.) using default parameters.
In some embodiments, polymer molecules are considered "homologous" to each other if at least 25%,30%,35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%,96%,97%,98%,99% or 100% of the monomers in the molecule are identical (identical monomers) or similar (conservative substitutions). The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
In the present invention, the nucleic acid molecule encoding a recombinant MSL protein, a variant or a fragment of an MSL protein has a nucleotide sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID No.1, for example a nucleic acid sequence having at least 95%, preferably at least 96%, still preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.9% homology with the nucleotide sequence according to SEQ ID No.1, and the nucleic acid molecule having the above-mentioned homology sequence has the same function as the nucleic acid molecule shown in SEQ ID No. 1.
In certain embodiments, the nucleic acid molecule encoding the MSL recombinant protein has a nucleotide sequence obtained by substitution, deletion or addition of one or several nucleotides to the nucleotide sequence shown in SEQ ID No.1, and the nucleic acid molecule having the above sequence has the same function as the nucleic acid molecule shown in SEQ ID No. 1.
Herein, the term "substitution" refers to the replacement of one or more nucleotides by a different nucleotide. "deletion" refers to the deletion of one or more nucleotides in a nucleotide sequence. "insertion" or "addition" refers to an alteration in the nucleotide sequence that results in an increase in one or more nucleotides as compared to the naturally occurring molecule.
The nucleic acid molecules of the invention include coding sequences obtained by codon optimization. Codons refer to a group of every adjacent three nucleotides in a messenger RNA molecule that represent a certain amino acid sequence during protein synthesis. "codon optimization" is intended to include the modification of the codon composition of a recombinant nucleic acid without changing the amino acid sequence.
In a preferred embodiment, the nucleic acid molecule encoding the MSL recombinant protein has the nucleotide sequence shown in SEQ ID NO. 1.
The term "nucleic acid" as used herein is intended to include polymeric forms of nucleotides of any length, containing deoxyribonucleotides, ribonucleotides and/or analogs thereof, including DNA, RNA and DNA/RNA hybrids, and also DNA or RNA analogs, such as those containing a modified backbone (e.g., peptide Nucleic Acid (PNA) or phosphorothioate) or modified bases. Thus, the nucleic acids of the present invention include DNA, cDNA, mRNA, recombinant nucleic acids, and the like.
Once the coding sequence of the protein of the present invention has been isolated, recombinant techniques can be used to obtain the protein in large quantities. An exemplary method is to clone the coding gene into a vector, transfer the vector into a cell, and isolate the vector from the proliferated host cell by a conventional method.
In a preferred embodiment, the method comprises:
(1) Connecting a base sequence for encoding the MSL recombinant protein with a vector to construct a recombinant protein expression vector;
(2) Transforming the recombinant vector into eukaryotic cells and inducing expression of MSL recombinant proteins;
(3) Purifying MSL recombinant protein.
The vectors of the invention may also comprise expression control sequences operably linked to the sequences of the nucleic acid molecules to facilitate expression of the protein. By "operably linked" is meant a condition in which certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, if a promoter controls transcription of a sequence, it is operably linked to a coding sequence. The vector may be, for example, an expression vector designed to express a nucleotide sequence in a host cell, or a viral vector designed to produce a recombinant virus or virus-like particle.
The host cells of the present invention are preferably eukaryotic cells, and examples of eukaryotic host cells useful in the present invention include, but are not limited to, yeast cells, insect cells, and mammalian cells, such as Saccharomyces cerevisiae cells, spodoptera frugiperda insect cells, fibroblasts (e.g., CHO cells, COS-7 cells, etc.), HEK293 cells, and the like.
In a preferred embodiment, the recombinant expression vector is obtained by ligating the plant MSL coding sequence with the eukaryotic expression vector pCDNA3.1 (+) via cleavage sites NheI and HindIII.
The culture conditions suitable for the present invention are not particularly limited, and those skilled in the art can easily determine conditions such as temperature, rotation speed, and culture medium during the culture process based on the eukaryotic cells selected.
Use of the same
The invention also provides application of the MSL recombinant protein or the active fragment thereof in preparing medicines for promoting or improving reproductive function. It should be noted that the MSL recombinant plant protein after eukaryotic expression of the present invention has bioactivity and corresponding biological function.
In particular embodiments, promoting or enhancing reproductive function includes, but is not limited to, preventing or treating erectile dysfunction, increasing NO levels in cavernous tissue, increasing cGMP levels in cavernous tissue, restoring oxidative damage to cavernous endothelial cell viability.
The term "preventing or treating" as used herein refers to ameliorating a disease or disorder before or after such a condition has occurred. The extent of such remission or prophylaxis is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% as measured by any standard technique, as compared to an untreated reference group under equivalent conditions. In the present invention, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (reduce) an undesired physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, results that are either detectable or undetectable, including alleviation of symptoms, diminishment of extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and palliation (whether partial or total). The need for treatment includes those already suffering from a condition or disorder, as well as those susceptible to a condition or disorder, or those in need of prophylaxis of such a condition or disorder.
Examples
1. Experimental method
1.1 Experimental cells and animals
HEK293 cells: the culture conditions are 293 serum-free culture medium, which is purchased from Shanghai cell bank of China academy of sciences, placed in a cell incubator with 5% CO 2 at 37 ℃ and replaced every other day, and passaged when the cells are in logarithmic growth phase (confluence 70-90%).
C57BL/6 mice: purchased from Yiss laboratory animal technologies Co., ltd, grade SPF, 22-24g, eligibility number: SCSK 2018-0007.
SD rats: purchased from Yiss laboratory animal technologies Co., ltd, grade SPF, 180-200g, eligibility number: SCSK 2018-0007.
1.2 Recombinant expression amino acid sequences and vector maps
Mannose SPECIFIC LECTIN (MSL) recombinant protein expression sequence is shown in SEQ ID NO. 1:
ATGGATGCCATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTGTTTGTCTCTCCCAGCATGGCTGATTTCATCCTGTACAGCGGCGAGAGCCTGAGAAGCGGACAGGCTCTGTACAGAGGCTCCTACACATTCATTATGCAGAATGACTGCAACCTGGTGCTGTACGATAATGGCAAGGCCATCTGGGCCAGCGGCACAAATGGCAGAGGCTCTGGATGTTACTGTGCCATGCAGAGCGATGGCAATCTGGTGGTGTACACAAGCAATAATAACGCCGTGTGGGCCAGCAACACCAACGTGGGACAGGGACACTACGTGTGTATCCTGCAGAAGGACAGGAACGTGGTCATCTATGGCGGCGCCAGGTGGGCTACAAATACCAACACAGTGGGCGTGTCCGGCGGCATGTTTATCGAGTCCAAGGCCACAATCTTCGGCAGCCTGCCCGCTAACGAGACAACAGCTGAGGCCAAGGCCGCCAGAATCAGCATGGTGGTGAACAAGCACCACCACCACCATCACTGA.
According to the synthesized sequence, codon optimization is carried out according to an escherichia coli protein expression system, his tag is added at the C end, the structure is synthesized and constructed on a pCDNA3.1 (+) vector (cleavage site is NheI/HindIII), and the construction strategy is shown in figure 1.
1.3 Recombinant plasmid amplification
The forward primer and the reverse primer are designed according to the primer design principle, the target sequence is amplified by PCR, the amplification system is shown in table 1, the amplification program is shown in table 2, and the electrophoresis result after PCR amplification is shown in fig. 2.
TABLE 1PCR amplification System
TABLE 2PCR amplification procedure
1.4 Vector plasmid cleavage
PCDNA 3.1.1 vector was digested with NheI/HindIII and the digestion system is shown in Table 3.
Table 3 double cleavage System
And (3) uniformly mixing the enzyme cutting systems, then placing the mixture into a water bath kettle at 37 ℃ for reaction for 15min, and then carrying out gel cutting recovery.
1.5 Construction of recombinant plasmids
And (3) carrying out recombinase connection on the vector recovered by cutting the gel and the target fragment, and reacting for 15-20min at 50 ℃, wherein the reaction system is shown in Table 4.
TABLE 4 recombinase ligation reaction System
1.6 Conversion
The ligation system was transformed into DH 5. Alpha. Competent cells as follows:
(1) 1 tube of competent cells was taken from a-80℃refrigerator and immediately placed on ice.
(2) Adding the connecting system (volume not more than 10 μl), mixing gently, and standing on ice for 30min.
(3) Accurately heat-striking in a water bath at 42 ℃ for 90sec, and rapidly cooling on ice for 3-5min after heat-striking.
(4) 1ML of LB liquid medium (without antibiotics) is added into the tube, and after uniform mixing, shaking culture is carried out for 1h at 37 ℃ and 200rpm, so that the bacteria are recovered to a normal growth state.
(5) Centrifuging the bacterial liquid, removing 800 mu L of supernatant, sucking and beating the rest culture medium, uniformly mixing, and coating on a screening plate containing corresponding antibiotics.
(6) The dishes were inverted and incubated at 37℃for 16-24h.
(7) And (5) picking bacteria on the transformed flat plate, collecting clone bacterial liquid, sending the clone bacterial liquid to a sequencing company for sequencing, and verifying the accuracy of the recombinant plasmid.
1.7MSL histone expression
HEK293 cells were subcultured with 293 serum-free medium, and the plasmids to be expressed were mixed with the transfection reagent TF2 and added to the cells for plasmid co-transfection, followed by culture for 7 days. Shake flask culture conditions: 5% CO 2, temperature 37℃and shaking table rotation 175rpm.
1.8 Purification of MSL recombinant proteins
Taking the cell supernatant after culture, and centrifuging in a low-temperature centrifuge. The supernatant obtained after the centrifugation was filtered with a 0.45 μm filter. 5mL of His filler was added to the suction-filtered sample, and the mixture was incubated at 4℃for 2 hours, followed by protein purification by chromatography. The method comprises the following steps:
(1) The column was equilibrated with Buffer A (PBS, pH 7.4);
(2) Flowing the sample through the chromatographic column at a speed of 2 mL/min;
(3) Elution with 20mM and 300mM imidazole;
(4) And respectively performing SDS-PAGE electrophoresis on the eluted samples to analyze whether target proteins exist. Proteins eluted with 300mM imidazole-2 were collected, dialyzed to Buffer A, at 4℃overnight, and the purity of the target protein was detected by SDS-PAGE gel.
1.9 Isolated culture of mouse cavernous endothelial cells MCECs based on Matrigel 3D culture System
The C57BL/6 mice at the age of 8 weeks are taken out of the neck and killed, and the lower abdomen of the mice is disinfected by alcohol cotton balls. The lower abdomen incision is made by forceps and surgical scissors, and the abdominal fascia and prepuce glands are peeled off to expose the cavernous tissue. The cavernous body is separated by surgical scissors and placed in HBSS containing 10% of diabody, urethra and neurovascular bundles are removed under a lens, clean cavernous body tissues are obtained, and the cavernous body tissues are washed 3 times in PSB containing 10% of diabody. Cutting into 1-2mm 3 pieces by fine surgical scissors, placing at the bottom of a precooled 24-hole plate, and cutting into 2 pieces per hole. mu.L of Matrigel coating containing 50ng/ml VEGF was added to each well, cut, and mouse cavernous endothelial cells (mouse cavernous endothelial cells, MCCs) were cultured and induced to proliferate. Culturing in a 5% CO 2 incubator at 37 ℃ for 14 days until the bottom of the hole is 80%. The medium was aspirated and digested with 200. Mu.L of Dispase enzyme per well in the incubator for 1h. Adding 10mM EDTA with equal volume to stop digestion, and precipitating after centrifugation to obtain the cell. The MCECs obtained was cultured in a culture dish previously coated with 0.2% gelatin, supplemented with complete medium. And carrying out subsequent experiments for 2-3 generations.
Purity identification of 1.10MCECs
And carrying out identification on MCECs purity by using an immunofluorescence method. MCECs with a density of 5×10 5 cells/mL was used for cell slide treatment in 6-well plates. After 24h of adherence, the medium was aspirated and washed 3 times with pre-chilled PBS. 500 μl of 4% paraformaldehyde was added to each well and the mixture was fixed at room temperature for 15min and washed 3 times with pre-chilled PBS. mu.L of 0.5% Triton X-100 permeate containing 5% goat serum was added to each well and left at room temperature for 0min. The blocking solution was aspirated, primary antibody (PECAM-1: endothelial cell marker, 1:20; desmin: smooth muscle cell marker, 1:200) was added and incubated overnight at 4 ℃. The primary antibody was recovered and washed 3 times with pre-chilled PBS. Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 594-labeled goat anti-rat IgG were added and incubated for 1h at room temperature, and pre-chilled PBS was washed 3 times. DAPI reagent is dripped to dye the cell nucleus, the cell nucleus is protected from light for 5min at room temperature, and the cell nucleus is washed 3 times by precooled PBS. And (5) dripping a sealing liquid of the fluorescence quenching agent, and sealing the sheet. Observed under a fluorescence microscope and photographed, image Proplus Image software analyzed MCECs purity.
1.11 Cell viability assay
MCECs cells were plated at a density of 2X 10 4/mL in 96 plates, and after adherence old medium was aspirated and starved for 24h with M199 basal medium. MSL recombinant proteins (320, 80, 20, 5. Mu.g/mL) were added at different concentrations, respectively, and culture was continued for 24h. Adding 0.5mmol/L H 2O2 to make oxidation damage model, and culturing for 2 hr. Cell viability was measured by CCK-8: after the cell culture was completed, 10. Mu.L of CCK-8 solution was added to each well, and incubated at 37℃for 1 hour, and absorbance was measured at A450 using an enzyme-labeled instrument.
1.12 Construction and administration of Kidney yang deficiency model
50 Male SD rats weighing 180-200g are randomly divided into 5 groups, namely a control group, a model group, an MSL low-dose group (0.6 mg/kg MSL), an MSL high-dose group (0.9 mg/kg MSL) and a positive control group (0.9 mg/kg yam protein DP 1), and the male SD rats are obtained through laboratory separation and purification. After the rats are adapted to the environment for one week, the rats are continuously perfused with 25mg/kg Hydrocortisone (HCT) for 10 days, and a kidney-yang deficiency model is constructed. After the model was established, distilled water was administered to the control group and the model group, and the other three groups were administered with different drugs for 10 consecutive days (the administration concentration was confirmed by the pre-experiment results).
1.13 Detection of erectile function
After the last administration, the rats were placed in a transparent observation cage and allowed to acclimate for 10min. 100 μg/kg apomorphine (apomorphine, APO) solution (prepared at a concentration of 40 μg/mL in physiological saline containing 0.2mg/mL vitamin C as a solvent) was subcutaneously injected, the penis of the rat was erected by exciting the central dopamine receptor, the erection latency of the rat was observed for 30min and the erection rate was calculated (erection rate% = number of erections per group of rats/total number of rats per group x 100). Complete exposure of the distal end of the penis is noted as a single erection.
1.14 Biochemical index detection
Nitric oxide (nitric oxide, NO) and guanosine cyclophosphate (cyclic guanosine monophosphate, cGMP) levels were measured in rat cavernous body using ELISA. After the rats were anesthetized, the cavernous body was taken and stored in liquid nitrogen for use. The level of NO or cGMP in the rat corpus cavernosum was measured according to ELISA kit instructions and absorbance was measured at 450nm using a microplate reader.
1.15 Statistical analysis
The experimental results are expressed as mean.+ -. Standard deviation (means.+ -. SD). Group differences were analyzed by one-way ANOVA and Tukey-Kramer using GRAPHPAD PRISM V6.0.0 software. * P <0.05 indicates that the difference is statistically significant.
2. Experimental results
2.1 Amplification of the Gene fragment of interest
The result of electrophoresis after PCR amplification is shown in FIG. 2. The target fragment of about 537bp can be seen by agarose gel electrophoresis analysis, and is consistent with the theoretical value.
2.2 Recombinant protein expression and purification
The results of protein electrophoresis of recombinant proteins eluted according to different procedures are shown in FIG. 3. As a result, it was found that the recombinant protein was successfully expressed and purified under the elution condition of300 mM imidazole.
The 300mM imidazole elution-2 protein was dialyzed and purity was checked by SDS-PAGE electrophoresis, and the results are shown in FIG. 4. The purity of the obtained purified protein is more than 85%, and the concentration is 0.64mg/mL.
2.3 Immunofluorescence analysis MCECs purity
To identify the true nature of primary cells, it is verified whether isolation methods are viable, immunofluorescence techniques being the analytical method most commonly used today to detect primary cell type and purity. In addition to endothelial cells, smooth muscle cells were most abundant in the corpora cavernosa, so this experiment labeled positive cells MCECs with platelet and endothelial cell adhesion molecule (PECAM-1), mouse cavernosa smooth muscle cells (mouse cavernous smooth muscle cells, MCSMCs) with desmin (Desmin) expressed by muscle tumor as negative control, and the type and MCECs purity of the primary cells obtained were analyzed. As a result, it was found that MCECs positive rate was 90% or more (FIG. 5).
2.4MSL recombinant protein improving H 2O2 -induced MCECs oxidative damage model cell viability
The CCK-8 method is adopted to examine the change of the cell activity of the MSL recombinant protein after the MSL recombinant protein intervenes in oxidative damage MCECs cells. As a result, as shown in FIG. 6, the cell viability was significantly reduced after 2 hours of H 2O2 action compared to the Control group. 320 μg/ml MSL had the most pronounced recovery effect on oxidative damage MCECs cells (P < 0.0001) following drug administration dry prognosis. Subsequent experiments were performed with 320. Mu.g/ml as the optimal concentration of MSL.
2.5 Effect of MSL recombinant protein on rat kidney-yang deficiency syndrome
As is clear from Table 5, rats developed aversion to cold, hair-holding, hair-drying, and back-bows after 10 days of HCT molding, and accompanied by a decrease in body mass (P < 0.0001), indicating the occurrence of kidney-yang deficiency in rats. Compared with a model group, the phenomena of aversion to cold, cluster, hair dryness, bow back and the like of rats in the low-MSL and high-dose group are obviously relieved after the administration for 10 days; volume mass was significantly increased (P <0.001 or P < 0.0001); wherein the MSL high dose group has similar effect with the positive control group. The results show that MSL has an improving effect on the kidney-yang deficiency syndrome of rats caused by HCT induction.
TABLE 5 influence of MSL recombinant protein on the body mass of rats with kidney-yang deficiency (n=10)
Note that: # # P <0.0001 compared to the control group; p <0.001 and P <0.0001 compared to model group.
2.6 Effect of MSL recombinant proteins on rat erectile function
As can be seen from Table 6, the model rats had significantly prolonged erection latency (P < 0.0001) and significantly reduced erection rate (P < 0.001) compared to the control group. Compared with the model group, the MSL group rats have significantly reduced erection latency (P <0.05, P < 0.01), significantly increased erection rate (P <0.05, P < 0.01) and are dose dependent. Wherein the MSL high dose group has similar effect with the positive control group. The results show that MSL can improve the erection function of rats with kidney-yang deficiency.
TABLE 6 influence of MSL recombinant proteins on erectile function in rats with kidney-yang deficiency (n=10)
Note that: compared to the control group, # # P <0.001, # # P <0.0001; p <0.05, P <0.01, P <0.001 compared to model group.
2.7 Effect of MSL recombinant proteins on NO and cGMP levels in rat cavernous tissue
The study of erectile physiology indicates that the non-adrenergic non-cholinergic nerve endings and the corpora cavernosa endothelial cells release NO under the catalysis of nitric oxide synthase, rapidly diffuse into smooth muscle cells from cell membranes, activate guanylate cyclase, increase cGMP synthesis and induce penile erection through a series of cascade reactions. Thus, the NO/cGMP signaling pathway is a key signaling pathway for measurement of the corpora cavernosa erection function. The effect on the levels of NO and cGMP was examined by ELISA. As a result, as shown in FIGS. 7 and 8, the NO and cGMP levels in the corpora cavernosa tissues of rats in the model group were significantly reduced (P < 0.0001) as compared with those in the control group. Compared with the model group, the MSL group rat has significantly increased NO and cGMP levels (P < 0.05, P < 0.01 or P < 0.001) in the corpora cavernosa tissues of the penis, and is dose dependent. Wherein the MSL high dose group has similar effect with the positive control group. The above results indicate that MSL can enhance NO/cGMP signaling in the corpora cavernosa of rats with kidney-yang deficiency.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that: the technical scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents. Such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for the preparation of a mannose-specific lectin recombinant plant protein, or an active fragment thereof, comprising the steps of culturing a cell comprising a nucleic acid molecule encoding said recombinant plant protein under conditions suitable for expression of the mannose-specific lectin recombinant plant protein, obtaining a culture, and isolating the recombinant plant protein from said culture, wherein said cell is a eukaryotic cell.
2. The method for preparing mannose-specific lectin recombinant plant proteins, or active fragments thereof, according to claim 1, characterized in that it comprises the following steps:
(1) Connecting a base sequence containing recombinant plant proteins encoding mannose-specific lectin with a vector to construct a recombinant protein expression vector;
(2) Transforming the expression vector into eukaryotic cells and inducing expression of the recombinant protein under suitable culture conditions;
(3) Isolating the mannose-specific lectin recombinant plant protein.
3. The method for producing a mannose-specific lectin recombinant plant protein or active fragment thereof according to claim 2, wherein the nucleic acid molecule encoding the recombinant plant protein has a nucleotide sequence shown in (I) or (II):
(I) A nucleotide sequence having at least 90% homology with the nucleotide sequence shown in SEQ ID NO.1 and having the same function;
(II) the nucleotide sequence shown in SEQ ID No.1 is obtained by replacing, deleting or adding one or more nucleotides and has the same function.
4. A method for the preparation of a mannose-specific lectin recombinant plant protein, or an active fragment thereof, according to claim 3, characterised in that the nucleic acid molecule encoding the mannose-specific lectin recombinant protein comprises a coding sequence optimised by codons.
5. The method for preparing mannose-specific lectin recombinant plant proteins, or active fragments thereof, according to claim 4, wherein the nucleic acid molecule encoding the recombinant plant protein has the nucleotide sequence shown in SEQ ID NO. 1.
6. Use of a recombinant plant protein obtainable by a method according to any one of claims 1 to 5 for the preparation of a medicament for promoting or enhancing reproductive function.
7. Use of a recombinant plant protein obtainable by a process according to any one of claims 1 to 5 for the preparation of a medicament for the prevention or treatment of erectile dysfunction.
8. Use of a recombinant plant protein obtained by the method according to any one of claims 1-5 for the preparation of a medicament for increasing NO levels in spongy tissue.
9. Use of a recombinant plant protein obtained by the method according to any one of claims 1-5 for the preparation of a medicament for increasing cGMP levels in spongy tissue.
10. Use of a recombinant plant protein obtained according to the method of any one of claims 1-5 for the preparation of a medicament for restoring the viability of oxidatively damaging cavernous endothelial cells.
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