CN111394359A - Cloning, prokaryotic expression and protein purification method of carminespider mite calmodulin gene - Google Patents
Cloning, prokaryotic expression and protein purification method of carminespider mite calmodulin gene Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a cloning, prokaryotic expression and protein purification method of tetranychus cinnabarinus calmodulin genes, which comprises the steps of extracting total RNA of tetranychus cinnabarinus, transcribing to obtain cDNA of the cDNA, amplifying specific gene tetranychus cinnabarinus calmodulin genes from tetranychus cinnabarinus by an RT-PCR method, cloning and sequencing, using the full length of the obtained tetranychus cinnabarinus calmodulin genes as a template, amplifying by using upstream and downstream primers of CaM to obtain a target fragment, digesting pGEX-6P-1(+) vectors and the target fragment by using Bam HI and Sma I respectively, extracting the target fragment to be connected to the vectors by a homologous recombination method, obtaining TcCaM-pGEX-6P-1(+) plasmids, transferring the TcCaM-pGEX-6P-1(+) plasmids into B L21 competent cells, carrying out IPTG induced protein expression, identifying by SDS-PAGE, and obtaining the tetranychus cinnabarinus calmodulin protein with the molecular weight of 19.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a cloning, prokaryotic expression and protein purification method of tetranychus cinnabarinus calmodulin genes.
Background
Tetranychus cinnabarinus (Tetranychus cinnabarinus) which is called spider mites, Tetranychus cinnabarinus and Tetranychus urticae is one of the most important phytophagous harmful mites, and the Tetranychus cinnabarinus mainly harms more than 100 kinds of ornamental plants and economic crops such as cowpea, cotton, vegetables, willow, tea, Chinese rose, jujube and the like. In addition, tetranychus cinnabarinus has the characteristics that individuals are very small and are difficult to find by naked eyes; the compound feed has extremely strong reproductive capacity and a large egg laying amount under a proper growth environment; the biological pesticide also has the characteristics of easy generation of pesticide resistance, multiple generations, high mutation rate, strong adaptability and the like, and is one of the pest communities which are recognized in the world and are the most difficult to control. Tetranychus cinnabarinus mainly gathers on the back of the leaves and two sides of veins of plants, sucks plant juice through a mouth needle, and is knotted into a silk screen, so that the damaged leaves gradually fade green, countless white spots are formed, and serious damage is caused to plant tissues.
Ca2+Ions are central signaling molecules that regulate in various biofunctional neurons, including neural delivery release, gene expression, and muscle contraction. Many cytoplasmic Ca2+Binding proteins mediate intracellular Ca2+A signal. In these Ca2+Among the binding proteins, calmodulin (CaM) plays the most important role. Calmodulin (CaM) is mainly expressed in the brain and plays an important role in various physiological functions such as gene transcription, muscle contraction and relaxation, and signal transduction. Although calmodulin (CaM) itself does not exhibit any catalytic activity, it modulates the activity of numerous calmodulin-dependent enzymes, including phosphodiesterases, protein kinases, phosphatases and nitric oxide synthases. Calmodulin (CaM) consists of roughly symmetrical N-and C-terminal helices, each of which can bind two Ca in the micromolar range together via two EF-hand motifs2+。Ca2+Several hydrophobically exposed residues promote C after binding to either terminusInteraction of aM with the target sequence of the regulated enzyme. Notably, studies have found that CaM can be considered as a potential target for pest control; for example, CaM mediates the insecticidal mechanism of lycopene against cotton bollworm, while certain antagonists of CaM mediated by Trifluoperazine (TFP), a specific inhibitor of CaM, inhibit the production of natural pheromones in silkworms. Up to now, there have not been any reports on in vitro expression of the calmodulin (CaM) from spider mites.
Disclosure of Invention
In view of the above, the invention aims to provide a method for cloning, prokaryotic expression and protein purification of tetranychus cinnabarinus calmodulin gene, construct a recombinant expression vector of tetranychus cinnabarinus calmodulin gene, express recombinant protein of calmodulin through a prokaryotic system, and lay a foundation for in vitro research of tetranychus cinnabarinus calmodulin gene in future.
The invention solves the technical problems by the following technical means:
on one hand, the invention provides a tetranychus cinnabarinus calmodulin gene, and the nucleotide sequence of the gene is shown in SEQ ID No. 3.
Furthermore, the amino acid sequence of the gene is shown in SEQ ID NO. 4.
The invention provides a method for cloning and recombining the calmodulin gene of tetranychus cinnabarinus, which comprises the following steps:
s1, extracting carminespider mite total RNA, carrying out reverse transcription on the total RNA into cDNA, carrying out polymerase chain reaction by using TcCaM upstream and downstream primer amplification, and carrying out cutting, recovery, DNA fragment connection, connection product conversion, bacterium shaking, spot coating, spot picking and bacterium liquid detection on an obtained PCR amplification product to obtain a full-length gene of carminespider mite calmodulin;
s2, using the full length of the tetranychus cinnabarinus calmodulin gene as a template, using an upstream primer and a downstream primer of CaM to amplify to obtain a target fragment, using Bam HI and Sma I to enzyme-cut pGEX-6P-1(+) vector and the target fragment respectively, using a homologous recombination method to connect the target fragment to the vector, and extracting to obtain a TcCaM-pGEX-6P-1(+) plasmid.
Furthermore, the nucleotide sequences of the upstream and downstream primers of TcCaM are shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the nucleotide sequences of the upstream and downstream primers of the CaM are shown as SEQ ID NO.5 and SEQ ID NO. 6.
Further, the amplification conditions in the steps S1 and S2 are both: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s, for 30 cycles.
Further, the steps of the ligation product conversion are as follows:
① DH5 α of 50. mu. L was added slowly to the ligation product, mixed well,
② is ice-cooled for 30min, heat-shocked at 42 ℃ for 40s,
③ adding L B liquid culture medium 500 μ L, transferring to ice to quench for 2min,
④ shaking at 37 deg.C and 200rpm for 40min,
⑤ and finally, coating the obtained bacterial liquid on a plate, and culturing for 14-16 hours in an incubator at the temperature of 37 ℃ to obtain the blue white spots.
In another aspect, the invention provides a protein purification method of tetranychus cinnabarinus calmodulin, which comprises the following steps:
the TcCaM-pGEX-6P-1(+) plasmid was transferred to B L21 competent cells, plated on L B plates, and cultured to OD600When the molecular weight reaches 0.6-0.8, IPTG is added to induce protein expression, and tetranychus cinnabarinus calmodulin with the molecular weight of 19kDa is obtained.
The method comprises the steps of extracting the total RNA of the tetranychus cinnabarinus, transcribing to obtain cDNA of the cDNA, amplifying a specific gene tetranychus cinnabarinus calmodulin gene from the tetranychus cinnabarinus by adopting an RT-PCR method, cloning and sequencing, using the obtained total length of the tetranychus cinnabarinus calmodulin gene as a template, amplifying by using CaM upstream and downstream primers to obtain a target fragment, digesting pGEX-6P-1(+) vector and the target fragment by using BamHI and Sma I respectively, connecting the target fragment to the vector by using a homologous recombination method, extracting to obtain TcCaM-pGEX-6P-1(+) plasmid, transferring the plasmid into B L21 competent cells, carrying out IPTG induced protein expression, and identifying by adopting SDS-PAGE to obtain the tetranychus cinnabarinus calmodulin protein with the molecular weight of 19 kDa.
Drawings
FIG. 1 is a total RNA agarose gel electrophoresis of Tetranychus cinnabarinus;
FIG. 2 is an SDS-PAGE picture of recombinant tetranychus cinnabarinus calmodulin expressed in Escherichia coli.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures used in the following examples are conventional ones without specific mention.
The test consumables used in the examples described below were commercially available without specific reference.
Example 1
Cloning and sequence analysis of the calmodulin gene of tetranychus cinnabarinus:
(1) extraction of Total RNA
The total RNA of tetranychus cinnabarinus is extracted by a Trizol method, and the extraction electrophoresis result is shown in figure 1. Extraction reference of total RNA of tetranychus cinnabarinus
plus Micro Kit instructions, performed as follows:
① preparing experimental reagent, wherein Buffer R L T plus solution is prepared according to the proportion of R L T, β -ME, 1m L and 10 mul, 70% ethanol solution is prepared according to the proportion of absolute ethyl alcohol, RNase-Free ddH2O, 7 and 3, and 80% ethanol solution is prepared according to the proportion of absolute ethyl alcohol, RNase-Free ddH2O, 8 and 2;
② pouring liquid nitrogen into the holes of the foam board, covering the hard paper sheet on the foam board to prevent the liquid nitrogen from losing, quickly inserting the collected 1.5m L centrifugal tube filled with mites into the holes to keep the mites at the bottom of the centrifugal tube at low temperature, grinding with a grinding rod cooled by liquid nitrogen, and preventing the mites from leaking out;
③ adding Buffer R L T plus solution into the centrifuge tube, continuing grinding until no granular substances exist, and then carrying out refrigerated centrifugation at 13500rpm for 3 min;
④ adding the supernatant in the centrifuge tube into gDNA Elimator spin column, centrifuging at 10000rpm for 1min, retaining the solution, and removing the column;
⑤ transferring 70% ethanol solution 350 μ L into ④, mixing with pipette tip, transferring into RNeasy spin column, centrifuging at 10000rpm for 1min, discarding the waste liquid, and leaving the adsorption column;
⑥ adding 700 μ l Buffer W1 into the adsorption column, centrifuging at 10000rpm for 1min, discarding the waste liquid, and leaving the adsorption column;
⑦ adding 500m L Buffer RPE into the adsorption column, centrifuging at 10000rpm for 1min, discarding the waste liquid, and leaving the adsorption column;
⑧ placing the adsorption column into a collecting tube, sucking 500 microliters of 80% ethanol solution into the collecting tube, setting the rotating speed at 10000rpm, centrifuging for 2 minutes, pouring the waste liquid and the collecting tube, and leaving the adsorption column;
⑨ placing the column into a new 2m L collecting tube, setting 13000rpm, centrifuging for 5 minutes, keeping the adsorption column, pouring the waste liquid and discarding the collecting tube;
⑩ the column was placed in a new 1.5m L collection tube, 30 μ L RNase-Free ddH2O was added to the center of the column, and centrifuged at 13500rpm for 1 minute to obtain RNA samples, which were stored in an ultra-low temperature freezer at-80 ℃.
(2) First Strand cDNA Synthesis
The first Strand cDNA synthesis was performed according to the instructions of the Primescript II Strand cDNAsSynthesis Kit (D621OA) of TAKARA. The method comprises the following steps:
the reagents were added to the PCR tube in the order of the amounts shown in Table 1 and mixed well.
TABLE 1
Then, the temperature is kept for 5min at 65 ℃ in a PCR instrument, and the mixture is rapidly cooled for 2 min. The following reagents of table 2 were added and mixed well:
TABLE 2
And finally, placing the mixture into a PCR instrument, storing the mixture for 60min at 42 ℃, then storing the mixture for 5min at 95 ℃, and storing the mixture for later use at-20 ℃ after the reaction is finished.
(3) full-Length PCR amplification of Gene
Designing a specific Primer sequence by using Primer 3 software, wherein an upstream Primer is marked as SEQ ID NO.1, an upstream Primer is marked as SEQ ID NO.2:
TcCaM-Forward:ATGTTAAACGACTCCATGCATC
TcCaM-Reverse:TCATTTAGGTGCCATCAAAACA
the reagent composition of the polymerase chain reaction is shown in table 3:
TABLE 3
The conditions for the polymerase chain reaction are shown in table 4:
TABLE 4
(4) PCR product recovery
And adding all the obtained PCR amplification products into agarose gel for electrophoresis, photographing in a gel imager, and cutting and recovering the obtained amplified target band. The recovery process was performed according to the instructions in the DNA purification recovery kit of TAKARA, and a DNA solution was finally obtained.
(5) Ligation of DNA fragments
DNA fragmentAnd (3) connecting with a carrier: pMD according to TAKARATMThe 19-T Vector ligation kit instructions were used to perform the experiments to obtain ligation products.
(6) Conversion of ligation products
The conversion steps of the ligation products were as follows:
① DH5 α of 50. mu. L was added slowly to the ligation product, mixed well,
② is ice-cooled for 30min, heat-shocked at 42 ℃ for 40s,
③ adding L B liquid culture medium 500 μ L, transferring to ice to quench for 2min,
④ shaking at 37 deg.C and 200rpm for 40min,
⑤ and finally, coating the obtained bacterial liquid on a plate, and culturing for 14-16 hours in an incubator at the temperature of 37 ℃ to obtain the blue white spots.
(7) Screening and identification of blue and white spots
Taking out the culture dish, placing the culture dish on a clean bench, picking white single bacterial plaque, gently picking the bacterial plaque by using a pipette, adding the bacterial plaque into L B liquid culture medium of 800 mu L, uniformly mixing, then placing the mixture into a constant temperature oscillator to culture for 4-6 hours at constant temperature of 220rpm and 37 ℃, finally taking 2 mu L bacterial solution to perform PCR amplification and electrophoresis detection, obtaining a single clear strip, wherein the size of the obtained strip is consistent with that of a target fragment, and then roughly judging that the bacterial solution contains the target fragment.
(8) Sequencing of cloned products
And (4) numbering the sample seal sample, and then sending the sample to Shenzhen Shenhua Dagenecompany for sequencing.
The complete length of the tetranychus cinnabarinus calmodulin gene cDNA obtained by sequencing is shown as SEQ ID NO. 3.
The amino acid sequence of the tetranychus cinnabarinus calmodulin gene obtained by sequencing is shown as SEQ ID No. 4.
(9) Analysis of target Gene sequence
B1ast comparison and evolutionary tree construction analysis are carried out at NCBI, the obtained target gene and different substances are analyzed, the gene is found to have a characteristic structural domain EF-hand of a calmodulin gene, and finally the obtained target gene calmodulin gene is determined to be a calmodulin gene in Tetranychus cinnabarinus.
Example 2
Procaryon expression and protein purification of the calmodulin gene of tetranychus cinnabarinus:
(1) expression plasmid construction
TcCaM-pMDTM19-T Vector and pGEX-6P-1(+) Plasmid are subjected to double enzyme digestion by respectively using Bam HI and Sma I (the enzyme digestion method refers to the enzyme digestion instruction of TaKaRa company), the double enzyme digestion is carried out, then T4 DNA ligase is used for connection (the connection method is according to the instruction of T4 DNA ligase of TaKaRa company), then the connection product is transformed into escherichia coli (DH5 α), the recombinant Plasmid is verified by a PCR method, wherein the PCR amplification condition is 94 ℃ for pre-denaturation 3min, 94 ℃ for denaturation 30s, 50 ℃ for annealing 30s, 72 ℃ for extension 30s, 30 cycles, the amplification is carried out, then the amplified product is sent to Shenhua Dagen scientific and technology company for sequencing, after the constructed Plasmid is determined to be correct, TcCaM-pGEX-6P-1(+) Plasmid is extracted according to the instruction of QIAGEN Plasmid Pus Midi by Qiagen company, the Primer 3 software is used for designing the specific expression Vector, the following primers are marked as SEQ ID NO.5, and the downstream Primer is marked as SEQ ID No. 6P-1(+) Plasmid:
CaM-F:CGCGGATCCGCGATGTTAAACGACTCCATGCATC
CaM-R:TCCCCCGGGGGATCATTTAGGTGCCATCAAAACA
(2) recombinant plasmid transformed Escherichia coli
The positive recombinant plasmid TcCaM-pGEX-6P-1(+) was transformed into competent B L21 (DE3) and applied to L B plates (Amp)+) Then, the cells were cultured overnight at 37 ℃ in an inverted manner.
(3) IPTG induced protein expression
Monoclonal to L B (Amp)+) In (c), the cells were cultured overnight at 37 ℃ and 220rpm, and inoculated overnight at a ratio of 1:50 to 20m L L B (Amp)+) In the middle, the cultivation is continued for about 4h, OD600When reaching 0.6-0.8, IPTG is added to l mM to induce protein expression, and l m L bacterial liquids are collected after 0h, 2h, 4h, 6h, 8h, 10h, 12h and 14h respectively.
(4) SDS-PAGE identification
L m L bacterial liquid is taken to be centrifuged at 12000rpm for l min to collect thalli, 10 mu L of 10 ×L-attaching Buffer solution is added to the thalli, the thalli are boiled for 10min, the thalli are centrifuged at 12000rpm for l 0min after being cooled, 10 mu L samples are taken to be subjected to 10% SDS-PAGE, the electrophoresis condition is 180mA, the gel is taken down to be shaken and dyed l h in Coomassie brilliant blue staining solution after 1h, the gel is moved into eluent to be shaken and eluted overnight, the change of protein bands before and after induction is observed, and the result shows that the protein expression reaches the peak after 12h of induction.
(5) Protein purification of calmodulin
The protein purification of tetranychus cinnabarinus calmodulin is completely carried out according to Beyogold produced by BiyunshiTMThe instructions for the GST-tagPurification Resin kit were performed. The final harvested protein was analyzed by SDS-PAGE and showed a purified protein molecular weight of about 19kDa, as shown in FIG. 2, in which channel 1: pGEX-6p-1 supernatant (without insert); and (3) a channel 2: recombinant CaM without IPTG induction; and (3) passage: IPTG-induced recombinant CaM; and (4) passage: and (5) purifying the recombinant CaM.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.
Sequence listing
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Claims (8)
1. The tetranychus cinnabarinus calmodulin gene is characterized in that the nucleotide sequence of the gene is shown in SEQ ID No. 3.
2. The tetranychus cinnabarinus calmodulin gene of claim 1, wherein the amino acid sequence of the gene is shown in SEQ ID No. 4.
3. The tetranychus cinnabarinus calmodulin gene cloning and recombinant expression method according to claim 1, comprising the steps of:
s1, extracting carminespider mite total RNA, carrying out reverse transcription on the total RNA into cDNA, carrying out polymerase chain reaction by using TcCaM upstream and downstream primer amplification, and carrying out cutting, recovery, DNA fragment connection, connection product conversion, bacterium shaking, spot coating, spot picking and bacterium liquid detection on an obtained PCR amplification product to obtain a full-length gene of carminespider mite calmodulin;
s2, using the full length of the tetranychus cinnabarinus calmodulin gene as a template, using an upstream primer and a downstream primer of CaM to amplify to obtain a target fragment, using Bam HI and Sma I to enzyme-cut pGEX-6P-1(+) vector and the target fragment respectively, using a homologous recombination method to connect the target fragment to the vector, and extracting to obtain a TcCaM-pGEX-6P-1(+) plasmid.
4. The tetranychus cinnabarinus calmodulin gene cloning and recombinant expression method according to claim 3, wherein the nucleotide sequences of the upstream and downstream primers of TcCaM are shown as SEQ ID No.1 and SEQ ID No. 2.
5. The tetranychus cinnabarinus calmodulin gene cloning and recombinant expression method according to claim 3, wherein the nucleotide sequences of the CaM upstream and downstream primers are shown as SEQ ID No.5 and SEQ ID No. 6.
6. The tetranychus cinnabarinus calmodulin gene cloning and recombinant expression method according to claim 3, wherein the amplification conditions in the steps S1 and S2 are both: pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s, for 30 cycles.
7. The method for cloning and recombinant expression of the tetranychus cinnabarinus calmodulin gene according to any one of claims 3 to 6, wherein the step of transformation of the ligation product is as follows:
① DH5 α of 50. mu. L was added slowly to the ligation product, mixed well,
② is ice-cooled for 30min, heat-shocked at 42 ℃ for 40s,
③ adding L B liquid culture medium 500 μ L, transferring to ice to quench for 2min,
④ shaking at 37 deg.C and 200rpm for 40min,
⑤ and finally, coating the obtained bacterial liquid on a plate, and culturing for 14-16 hours in an incubator at the temperature of 37 ℃ to obtain the blue white spots.
8. The protein purification method of tetranychus cinnabarinus calmodulin is characterized by comprising the following steps of:
transferring the TcCaM-pGEX-6P-1(+) plasmid of claim 3 into B L21 competent cells, plating on L B plates, and culturing to OD600When the molecular weight reaches 0.6-0.8, IPTG is added to induce protein expression, and tetranychus cinnabarinus calmodulin with the molecular weight of 19kDa is obtained.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN113234133A (en) * | 2021-04-19 | 2021-08-10 | 扬州大学 | Method for prokaryotic expression of liriomyza trifoliata heat shock transcription factor protein |
| CN113234133B (en) * | 2021-04-19 | 2022-04-26 | 扬州大学 | Method for prokaryotic expression of liriomyza trifoliata heat shock transcription factor protein |
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