CN118792273A - Rheum emodi AspAT gene, recombinant vector and construction method - Google Patents
Rheum emodi AspAT gene, recombinant vector and construction method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a large yellow vine AspAT gene, a recombinant vector and a construction method. The invention takes the Chinese rhubarb vine as a material, successfully clones the AspAT gene for the first time and expresses the AspAT gene in a prokaryotic system, which lays a foundation for the functional research of the AspAT gene in the Chinese rhubarb vine on the one hand and provides a reference for the analysis of the upstream synthetic route of isoquinoline alkaloid and the heterologous synthesis thereof on the other hand.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a large yellow vine AspAT gene, a recombinant vector and a construction method.
Background
The main active ingredient fibrauretine of caulis Fibraureae Fibraurea RECISA PIERRE has antibacterial and antiinflammatory effects, and is developed into fibrauretine tablet for treating gynecological inflammation, bacillary dysentery, enteritis, respiratory tract infection, urinary tract infection, surgical infection, conjunctivitis, etc. Fibrauretine belongs to isoquinoline alkaloid, the biological synthesis path starts from L-tyrosine, 4-hydroxy phenylacetaldehyde (4-hydroxyphenylacetaldehyde) and dopamine (dopamine) are respectively synthesized through two paths, then isoquinoline skeleton-S-norlinderamine [ (S) -norcoclaurine ] is formed by condensation under the catalysis of (S) -norlinderamine synthetase [ (S) -norcoclaurine synthase, NCS ], and a key intermediate-S-inuline is synthesized through series catalysis [ (S) -oxhide alkaloid [ (S) -reticuline ], which is an upstream synthesis path shared by most isoquinoline alkaloids. Aspartate aminotransferase (AspAT) is an enzyme involved in the process of synthesizing 4-hydroxyphenylacetaldehyde, and in the early multi-group study of the caulis et folium fibraureae recisae in the subject group, aspAT and fibrauretine are found to have obvious correlation, but the target protein is expressed by using cDNA of the caulis fibraureae recisae as a template and through prokaryotes.
Disclosure of Invention
In order to overcome the defects of the technology, the invention provides a Chinese rhubarb vine AspAT gene, a recombinant vector and a construction method, which provide references for analysis of an upstream synthesis path of isoquinoline alkaloid and heterologous synthesis thereof.
To achieve the above object, in one aspect, the present invention provides a protein which is (a) or (b);
(a) A protein consisting of the amino acids shown in SEQ ID NO. 1;
(b) And the derivative protein with the same functions is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.
In some embodiments, the invention provides a protein having an amino acid sequence that has 80% identity to the sequence shown in SEQ ID NO. 1; preferably 85% identical, more preferably 90% identical, more preferably 95% identical, and most preferably 99% identical.
In another aspect, the present invention provides a gene encoding the above protein, wherein the nucleotide sequence of the gene is (a), (b) or (c);
(a) A nucleotide sequence as shown in SEQ ID NO. 2;
(b) A nucleotide sequence which hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 2;
(c) A nucleotide sequence which has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO.2 and codes.
It is well understood by those skilled in the art that since the same amino acid may have a plurality of different codons, the nucleotide sequence encoding the protein is not limited to only one type, and may be a nucleotide sequence obtained by mutating one or more nucleotides from the nucleotide sequence shown in SEQ ID NO.1 to form a synonymous mutation, and may also encode the mutant amino acid sequence of the present invention, or may be a nucleotide sequence capable of encoding the mutant amino acid sequence of the present invention according to codon optimization.
In some specific embodiments, the invention provides a protein having a gene nucleotide sequence that has 80% identity to the sequence shown in SEQ ID NO. 2; preferably 85% identical, more preferably 90% identical, more preferably 95% identical, and most preferably 99% identical.
In another aspect, the present invention provides a method for constructing a recombinant vector, comprising the steps of:
s1, performing PCR amplification by taking cDNA of caulis et folium Rhei as a template to obtain a nucleotide sequence shown in SEQ ID NO. 2;
S2, inserting a nucleotide sequence shown in SEQ ID NO.2 into a pMD TM -T vector plasmid to obtain a cloning recombinant vector;
s3, carrying out PCR amplification by taking the cloning recombinant vector as a template to obtain a nucleotide sequence shown as SEQ ID NO. 2;
s4, inserting the amplified sequence of the S3 Transforming Trans1-T1 competent cells in Blunt E1 expression vector plasmid, screening positive bacterial liquid with the correct connection direction of target fragments, and extracting the plasmid after amplification culture to obtain the recombinant expression vector.
Further, in the S1 and the S3, the PCR amplified primer comprises a forward primer and a reverse primer, the nucleotide sequences of which are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4.
Further, the PCR amplification system in S1 is calculated as 50. Mu.L, including Premix Ex Taq Hot Start Version. Mu.L; cDNA 4. Mu.L; forward primer 2.5 μl; reverse primer 2.5 μl; ddH 2 O16. Mu.L.
Further, the PCR amplification conditions in S1: 98℃10sec,55℃30sec,72℃1min 52sec,30 cycles.
Further, the PCR amplification system in S3 was calculated as 50. Mu.L, and the plasmid DNA was 2. Mu.L; forward primer 1. Mu.L; reverse primer 1. Mu.L; 5X FastPfu Buffer. Mu.L; 2.5Mm dNTPs 4. Mu.L; PCR Stimulant. Mu.L; DNA Polymerase 1. Mu.L; ddH 2 O21. Mu.L.
Further, the PCR amplification conditions in S3: 95 ℃ for 2min;95℃20sec,53℃20sec,72℃45sec,35 cycles; and at 72℃for 5min.
On the other hand, the large yellow crostem AspAT expression vector prepared by the construction method also belongs to the protection scope of the invention.
In another aspect, the invention provides the use of the above genes, recombinant vectors, in the production of a protein of interest. Further, the recombinant vector is transformed into competent cells to obtain recombinant thalli, and the target protein is obtained through the processes of amplifying culture, induced expression, collecting thalli, breaking thalli and purifying protein in sequence.
The beneficial effects are that: the invention takes the caulis et folium fibraureae recisae as the material, successfully clones the AspAT gene for the first time, and expresses in a prokaryotic system, thereby laying a foundation for the function research of the AspAT gene on the one hand, and providing a reference for the analysis of the upstream synthesis path of isoquinoline alkaloid and the heterologous synthesis thereof on the other hand.
Drawings
FIG. 1 is an electrophoresis chart of total RNA of caulis et folium fibraureae recisae;
FIG. 2 is an electrophoresis chart of the amplification of the sequence ASPAT CDNA of caulis et folium Rhei;
FIG. 3 is an AspAT bacterial liquid PCR positive verification electrophoresis chart;
FIG. 4 is an electrophoresis chart of the AspAT protein of caulis et folium Rhei under different induction conditions.
Detailed Description
The present invention will be described in detail with reference to specific embodiments thereof, so that those skilled in the art can better understand the technical solutions of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
The inventor finds that AspAT has remarkable relevance with fibrauretine based on multiple groups of study on caulis et folium fibraureae recisae. The invention uses the rhubarb vine as the material, uses RT-PCR to clone an AspAT sequence, and connects the full-length fragment of the target gene-Blue-ray E1 constructs an expression vector, transforming competent cells to express the protein of interest.
Experimental materials: the plant of the fibraurea stem Fibraurea RECISA PIERRE comes from laboratory cultivation seedlings; the pMD TM -T cloning vector was purchased from Takara Bio-engineering (Dalian) Inc.,The Blunt E1 expression vector, strains Trans5α, trans1-T1, TRANSETTA (DE 3) were all purchased from Beijing full gold Biotechnology Co.
Experimental reagent: super Total RNA Extraction Kit available from Promega (Beijing) biotechnology Co., ltd; III 1st Strand cDNA Synthesis Kit (GDNA DIGESTER plus) was purchased from the company Highway Biotechnology (Shanghai) Inc., next; premix Ex Taq TM Hot Start Version was purchased from Takara Bio-engineering (Dalian) Inc.; PCR product recovery kit, plasmid miniprep kit, ampicillin (AMPICILLIN), PCR Supermix (+dye) was purchased from Beijing full gold Biotechnology Co., ltd; SDS-PAGE gel preparation kit, 4 Xprotein loading buffer (containing DTT) was purchased from Beijing Soy Corp technologies Co.
EXAMPLE 1AspAT Gene cloning
(1) Using prasugrelSuper Total RNA Extraction Kit extracting total RNA of caulis et folium Brassicae Capitatae (see figure 1); using next holyIII 1st Strand cDNA Synthesis Kit (GDNA DIGESTER plus) first strand cDNA was synthesized and stored at-20℃until use. PCR amplification was performed using cDNA as a template according to the Premix Ex Taq TM Hot Start Version protocol, and the PCR product was checked as a single band by 1.5% gel electrophoresis, consistent with the predicted product length 1358bp (FIG. 2), usingPCR Purification Kit purifying the PCR product, measuring the concentration, and preserving at 4 ℃;
The amplification primers are as follows,
The reaction system is as follows:
In 50 μl, including Premix Ex Taq Hot Start Version μl; cDNA 4. Mu.L; forward primer 2.5 μl; reverse primer 2.5 μl; ddH 2 O16. Mu.L.
The reaction procedure is: 98℃10sec,55℃30sec,72℃1min 52sec,30 cycles.
(2) The purified PCR product was ligated with pMD TM 19-T vector as described in pMD TM -T Vector Cloning Kit, by the following procedure:
| Reagent(s) | Usage amount |
| pMD19-T Vector | 1μL |
| Insert DNA | 0.2pmol |
| ddH2O | up to 5μL |
Reacting at 16 ℃ for 30min to finish the connection;
The full golden Trans5 alpha CHEMICALLY COMPETENT CELL product instruction book is used for the transformation of the escherichia coli, and the specific operation is as follows:
taking out the Trans5α competent cells from the temperature of-80 ℃, rapidly inserting the competent cells into ice, melting fungus blocks after 5 minutes, adding 5 mu L of the connection product, stirring the EP tube bottom by hands, gently mixing, and standing in ice for 30 minutes;
b.42 ℃ water bath heat shock for 45 seconds, rapidly putting back on ice and standing for 2 minutes (shaking can reduce conversion efficiency);
c. Adding 500 mu L LB without antibiotics, mixing uniformly, and resuscitating at 37 ℃ and 200rpm for 60 minutes;
d.1 Centrifuging at 500Xg for 1min, collecting thallus, collecting about 120 μl supernatant, gently blowing to resuspend thallus, and coating on LB plate containing AMPICILLIN;
e. The plates were placed in an incubator at 37℃overnight.
After overnight culture, the monoclonal was selected and usedPCR Supermix (+dye) was used for PCR positive verification (FIG. 3), positive bacterial solutions were amplified and subjected to first generation sequencing, plasmids were extracted from positive bacterial solutions with correct sequencing results, and the plasmids were stored at-20 ℃.
EXAMPLE 2 expression vector construction
The plasmid extracted from the clone positive bacterial liquid is used as a template according toFastPfu DNA Polymerase instructions for PCR amplification, the amplification conditions were as follows:
2. Mu.L of plasmid DNA; forward primer 1. Mu.L; reverse primer 1. Mu.L; 5X FastPfu Buffer. Mu.L; 2.5Mm dNTPs 4. Mu.L; PCR Stimulant. Mu.L; DNA Polymerase 1. Mu.L; ddH 2 O21. Mu.L.
The PCR procedure was: 95 ℃ for 2min;95℃20sec,53℃20sec,72℃45sec,35 cycles; and at 72℃for 5min.
According toInstructions for use of the Blunt E1 Expression Kit to combine the purified PCR product with-Blue E1 vector ligation, specifically as follows:
and (3) reacting at 25 ℃ for 12min to finish the connection.
The ligation product was transformed into Trans1-T1 competent cells overnight and cultured in the same manner as in example 1 usingAnd (3) carrying out PCR positive verification on the T7P primer attached to the Blunt E1 Expression Kit and the AspAT reverse primer of the patent, selecting a monoclonal, extracting plasmids from positive clones with positive results and correct sequencing, and preserving the extracted plasmids at the temperature of-20 ℃.
EXAMPLE 3 expression of the protein of interest
According to TRANSETTA (DE 3) competent cells, the expression vector obtained in example 2 was transformed into TRANSETTA (DE 3) competent cells by the same method as in example 1, and after overnight culture, 10mL of LB medium (100. Mu.g. ML -1 ampicillin) was added to the selected single clone and cultured overnight to obtain a seed bacterial liquid; the seed bacterial liquid is subjected to ice bath ultrasonic crushing after induction culture, and the conditions are as follows: 300W, crushing for 5sec at intervals of 8sec for 20min, and clarifying the crushed bacterial liquid; centrifuging the crushed bacterial liquid at 4 ℃ and 12 000Xg for 5min, pouring the supernatant into a new centrifuge tube, re-suspending the sediment with 2mL PBS, respectively spotting the supernatant and the sediment, performing SDS-PAGE, and verifying the expression condition of the target protein.
Example 4 test of influence of Induction conditions on expression of protein of interest
The recombinant expression vector is transformed into TRANSETTA (DE 3) competent cells containing rare codons lacking in escherichia coli, protein expression is induced by different inducer concentrations and different temperatures, bacterial liquid without inducer and empty vector are used as contrast, and supernatant and sediment are respectively subjected to gel running after protein disruption.
The induction culture is specifically as follows: adding a new LB culture medium into the seed bacterial liquid according to a ratio of 1:100, culturing at 37 ℃ and 200rpm until OD 600 = 0.6-0.8, adding an inducer IPTG, and rotating a shaking table at 160rpm; after the induction culture is finished, 10mL of the induced bacterial liquid is taken, 5 000Xg is centrifuged for 5min to collect submerged bacteria, 1/4 volume of PBS is used for washing twice, 5mL of PBS is added for resuspension, the protein ultrasonic disruption method in the embodiment 3 is adopted, and the supernatant and the sediment are preserved at the temperature of minus 80 ℃ for standby after ultrasonic disruption; wherein the final concentration of inducer IPTG is 0mM,0.5mM and 1mM respectively; the induction conditions are respectively 16 ℃ for 18 hours, 25 ℃ for 8 hours and 37 ℃ for 3 hours.
Mixing the supernatant and the precipitate with 4 Xloading buffer, heating at 98deg.C for 10min, taking 30 μL, and performing SDS-PAGE electrophoresis under 80V concentration gel and 120V separation gel. Staining with coomassie brilliant blue stain at room temperature at 50rpm for 1h, decolorizing with decolorizing agent for 1h, replacing decolorizing agent, decolorizing for 2h, and directly observing the band or photographing after ddH 2 O washing (FIG. 4).
The protein bands in FIG. 4 are SDS-PAGE results of proteins in the pellet after ultrasonication, A 0,A1,A2 corresponds to induction of the inducer at 0mM,0.5mM,1mM,16℃for 18h, respectively; protein band B 0,B1,B2 corresponds to induction of 8h at 25℃with 0mM,0.5mM,1mM, respectively, final inducer concentration; protein band C 0,C1 corresponds to induction of 3h at 37℃with a final concentration of 0mM,0.5mM, respectively, of the inducer. In FIG. 4, it can be seen that the inducer-added experimental group exhibited a band at the 47.5kDa position of the target protein, and that the non-inducer-added group and the empty vector control group both exhibited a lighter band at the position of the target protein, indicating that the strain itself contained a protein corresponding to the size of the position, and that the band at the position of the target protein of the experimental group was significantly thicker than that of the control group, and it was considered that the target protein was indeed expressed. From the thickness of the target protein band, the concentration of the inducer in the study has little influence on the total amount of the target protein, and the total amount of the target protein induced at 25 ℃ and 37 ℃ is obviously more than 16 ℃ as a result of induction.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A protein, characterized in that: the protein is (a) or (b);
(a) A protein consisting of the amino acids shown in SEQ ID NO. 1;
(b) And the derivative protein with the same functions is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.
2. A gene encoding the protein of claim 1, wherein the nucleotide sequence of the gene is (a), (b) or (c);
(a) A nucleotide sequence as shown in SEQ ID NO. 2;
(b) A nucleotide sequence which hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 2;
(c) A nucleotide sequence which has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO.2 and codes.
3. The construction method of the prokaryotic expression vector based on basic cloning is characterized by comprising the following steps:
s1, performing PCR amplification by taking cDNA of caulis et folium Rhei as a template to obtain a nucleotide sequence shown in SEQ ID NO. 2;
S2, inserting a nucleotide sequence shown in SEQ ID NO.2 into a pMD TM -T vector plasmid, transforming a Trans5 alpha competent cell, screening positive bacterial liquid and extracting the plasmid to obtain a recombinant cloning vector;
s3, carrying out PCR amplification by taking the recombinant cloning vector as a template to obtain a nucleotide sequence shown as SEQ ID NO. 2;
s4, inserting the amplified sequence of the S3 And (3) transforming the Trans1-T1 competent cells in the Blunt E1 expression vector plasmid, screening positive bacterial liquid with correct connection direction, and extracting the plasmid to obtain the recombinant expression vector.
4. The construction method according to claim 3, wherein the primers for PCR amplification in S1 and S3 comprise a forward primer and a reverse primer having nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.
5. The construction method according to claim 3, wherein the PCR amplification system in S1 comprises Premix Ex Taq Hot Start Version. Mu.L in terms of 50. Mu.L; cDNA 4. Mu.L; forward primer 2.5 μl; reverse primer 2.5 μl; ddH 2 O16 μL; the PCR amplification system in S3 was 50. Mu.L, and the plasmid DNA was 2. Mu.L; forward primer 1. Mu.L; reverse primer 1. Mu.L; 5X FastPfu Buffer. Mu.L; 2.5Mm dNTPs 4. Mu.L; PCR Stimulant. Mu.L; DNAPolymerase. Mu.L; ddH 2 O21. Mu.L.
6. A construction method according to claim 3, characterized in that the PCR amplification conditions in S1: 98℃10sec,55℃30sec,72℃1min 52sec,30 cycles; PCR amplification conditions in S3: 95 ℃ for 2min;95℃20sec,53℃20sec,72℃45sec,35 cycles; and at 72℃for 5min.
7. A rhubarb AspAT expression vector prepared by the construction method of any one of claims 3-6.
8. Use of the gene according to claim 2, the recombinant vector according to claim 7 for the production of a protein of interest.
9. The use according to claim 8, wherein the recombinant vector is transformed into competent cells to obtain recombinant cells, and the recombinant cells are subjected to the processes of amplification culture, induced expression, cell collection, cell disruption and protein purification in sequence to obtain the target protein.
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