CN115505582A - Chrysalid pteromalid venom kynurenine transaminase PpVKAT and application thereof - Google Patents
Chrysalid pteromalid venom kynurenine transaminase PpVKAT and application thereof Download PDFInfo
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- CN115505582A CN115505582A CN202210534010.8A CN202210534010A CN115505582A CN 115505582 A CN115505582 A CN 115505582A CN 202210534010 A CN202210534010 A CN 202210534010A CN 115505582 A CN115505582 A CN 115505582A
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- ppvkat
- kynurenine
- venom
- transaminase
- protein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a pteromalus puparum venom kynurenine transaminase PpVKAT, which has an amino acid sequence shown in SEQ ID NO:2, respectively. The invention also discloses the application of the pteromalus puparum venom kynurenine transaminase PpVKAT, which comprises the following steps: the recombinant protein pCOLD-TF-PpVKAT has a lethal effect on the pieris rapae blood cells.
Description
Technical Field
The present invention relates to the fields of molecular biology, genetic engineering, and protein engineering. In particular to kynurenine transaminase PpVKAT expressed in pteromalus puparum venom, a coded nucleic acid sequence and application thereof.
Background
People eat as the day, china is a big agricultural country, and major plant diseases and insect pests frequently occur. It is estimated that the number of main crop pests in China is more than 300, and the annual average loss caused by the damage is more than 100 hundred million yuan. The chemical pesticide industry developed in the 20 th century has the advantages of quick acting, economy, simplicity, convenience and the like, and makes great contribution to agricultural production. However, with the use of a large amount of chemical pesticides, a series of problems such as pest resurgence, pest resistance to drugs, pesticide residue in the environment and the like are also brought about.
The biological pest controlling technology is to utilize natural enemies of pests to control pests, and to control pests below the threshold of economic damage level through the parasitic or predatory effect of natural enemies. Biological pest control is an important component of modern agriculture, can reduce the use of field pesticides, ensure the quality safety of agricultural products and reduce environmental pollution, and is a necessary strategy for sustainable development of green agriculture. Although transgenic crops represented by the transgenic Bt gene play an important role in pest control and obtain remarkable economic and social benefits, the resistance problem still needs to be solved urgently.
Currently, more scientific research is focusing on the discovery of novel insecticidal, anticancer and antitumor genes from a variety of microorganisms, plants and animals. Metarhizium anisopliae is an entomopathogenic bacterium with broad-spectrum effect, and researches report that the gene of the blue mountain funnel spider is used for carrying out gene modification on the metarhizium anisopliae, so that the toxin Hybrid can be generated, mosquitoes can be killed quickly, and the gene is very important for resisting malaria. The breast cancer seriously harms the health of women, and researchers find that Melittin can effectively kill triple-negative breast cancer cells and has very low toxicity to normal cells. Glioblastoma is a fatal brain tumor, and scientists use the high targeting of chlorotoxin CLTX in the venom of the israel scorpion, so that T cells equipped with CLTX-CAR can rapidly and specifically regress the tumor without causing any adverse reactions.
Although the insecticidal gene resources of parasitic wasps are abundant, the examples of successful applications are rare. For example, tnBVANK1 gene in parasitic factor PDV of the Toxoneuron nigrceps of the branchionus nigricans is transferred into tobacco, and the larvae of the Egyptian Spodoptera littoralis show delayed development after eating the transgenic tobacco.
The function and structure of kynurenine transaminase related to the invention are explored in mammals and insects, including related diseases of central nervous system of mammals and molecular targets of novel mosquito killing drugs, but the related research on cell lethal activity is not reported.
Disclosure of Invention
The invention aims to solve the technical problem of providing a pteromalus puparum venom kynurenine aminotransferase PpVKAT gene and a protein coded by the same and application of the gene.
In order to solve the technical problems, the invention provides a pteromalus puparum venom kynurenine transaminase PpVKAT, which has the amino acid sequence shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.
Remarks explain: the amino acid sequence of SEQ ID NO:2 comprises a signal peptide (Met Gly Leu Thr he Met he Lys Val Ala Cys he Val Ala Val Leu he Cys Leu Pro Val Arg Pro Ser Val Gly) corresponding to SEQ ID NO:2 from position 1 to position 26 of the amino acid sequence.
The improvement of kynurenine transaminase PpVKAT as the pteromalus puparum venom of the invention comprises the following steps: the protein is conservative variant protein, active fragment or active derivative thereof.
The invention also provides a gene for coding the pteromalus puparum venom kynurenine transaminase PpVKAT, and the nucleotide sequence of the gene is SEQ ID NO:1, the preparation method comprises the following steps of; or to SEQ ID NO:1 has at least 70% homology with the nucleotide sequence in the sequence table; or the nucleotide sequence of the polypeptide can be matched with the nucleotide sequence shown in SEQ ID NO:1 under stringent conditions.
The amino acid sequence of SEQ ID NO: the last three bases "TAA" in 1 is a stop codon.
As an improvement of the gene of the present invention: the sequence comprises SEQ ID NO:1, 8-66 continuous nucleotides.
The invention also provides the application of the pteromalus puparum venom kynurenine transaminase PpVKAT: lethal Papilia brassicae blood cells.
As an improvement of the use of the present invention: the venom kynurenine transaminase PpVKAT secreted by the pteromalus puparum venom gland can be used for killing the cabbage butterfly blood cells. Namely, the pteromalus puparum venom kynurenine transaminase PpVKAT has cell lethal activity.
As a further improvement of the use of the invention: the recombinant protein pCOLD-TF-PpVKAT has lethal effect on the blood cells of the pieris rapae.
The pteromalus puparum venom kynurenine transaminase PpVKAT and the nucleic acid sequence coded by the same can be applied to the amino acid sequence and the coding sequence of the pteromalus puparum venom kynurenine transaminase PpVKAT, can be developed into insect-resistant crops and biological pesticides with application values, and can be applied to a plurality of fields of agricultural pest control and the like.
The invention is realized by the following technical scheme: the invention obtains a venom kynurenine transaminase PpVKAT gene complete sequence by using a pteromalus puparum genome and a transcriptome, carries out reverse transcription on extracted RNA of the pteromalus puparum to obtain cDNA, and carries out molecular cloning, prokaryotic expression and purification under a non-denaturing condition to obtain PpVKAT protein (an amino acid sequence is shown as SEQ ID NO: 2) of the prokaryotic expression. The pronucleus expressed PpVKAT can kill the blood cells of the pieris rapae, and has the function of inhibiting host cell immunity.
The DNA molecules isolated by the invention comprise: the code has a nucleotide sequence of chrysalis chrysomelidis venom kynurenine transaminase PpVKAT, and the nucleotide sequence is similar to the nucleotide sequence shown in SEQ ID NO:1 has at least 70% homology with the nucleotide sequence in the sequence table; or the nucleotide sequence can be matched with the nucleotide sequence shown in SEQ ID NO: 1. Preferably, the sequence encodes a polypeptide having the sequence of SEQ ID NO:2 in sequence shown in the specification. More preferably, the sequence has the sequence shown in SEQ ID NO: 1.
The chrysalid pteromalid venom kynurenine transaminase PpVKAT separated by the invention comprises: has the sequence shown in SEQ ID NO:2, or a conservative variant thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the protein is a polypeptide having the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.
The DNA molecule of the invention comprises 8-66 consecutive nucleotides in said DNA molecule.
The host cell transformed with the DNA molecule of the invention is a prokaryotic cell.
In the present invention, "isolated", "purified" DNA means that the DNA or fragment has been isolated from the sequences which flank it in its natural state, and that the DNA fragment has been separated from the components which accompany the nucleotides in its natural state, and from the proteins which accompany it in the cell.
In the invention, the nucleic acid sequence of the chrysalid pteromalid venom kynurenine transaminase PpVKAT refers to: the nucleotide sequence with the chrysalid pteromalid melittis venom kynurenine transaminase PpVKAT activity is coded and shown as SEQ ID NO:1 and degenerate sequences thereof. The degenerate sequence is SEQ ID NO:1 sequence having one or more codons substituted by a degenerate codon encoding the same amino acid. Due to codon degeneracy, compared to SEQ ID NO:1 can also encode a degenerate sequence having as little as about 70% homology to the nucleotide sequence of SEQ ID NO: 1.
Also included are compositions that hybridize under moderately stringent conditions, more preferably under highly stringent conditions, to the nucleotide sequence of SEQ ID NO:1, and a nucleotide sequence that hybridizes to the nucleotide sequence in 1. Also included are the sequences substantially identical to SEQ ID NO:1, preferably at least 80%, more preferably at least 90%, most preferably at least 95%. Also included are variants of the sequence of SEQ ID No.1 encoding proteins with the same function of the native pteromalus puparum venom kynurenine transaminase PpVKAT. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 90, preferably 1 to 60, more preferably 1 to 20, most preferably 1 to 10) nucleotides, and addition of several (usually less than 60, preferably less than 30, more preferably less than 10, most preferably less than 5) nucleotides at the 5 'and/or 3' end.
In the invention, the pteromalus puparum venom kynurenine transaminase PpVKAT refers to: the polypeptide has the amino acid sequence shown in SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. The term also includes SEQ ID NO:2 variant forms of the sequence. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids of similar or similar properties will generally not alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes chrysalis venom kynurenine aminotransferase PpVKAT active fragments and active derivatives.
The pteromalus puparum venom kynurenine transaminase PpVKAT conservative variant protein in the invention refers to: and SEQ ID NO:2, or a protein formed by substituting up to 10, preferably up to 8, more preferably up to 5 amino acids having similar or similar properties.
The invention also comprises a pteromalus puparum venom kynurenine transaminase PpVKAT or a protein analogue. These analogs may differ from the venom kynurenine transaminase PpVKAT by amino acid sequence differences, by modifications that do not affect the sequence, or by both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It is to be understood that the proteins of the present invention are not limited to the representative proteins listed above.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those proteins that result from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins modified to enhance their proteolytic properties or to optimize solubility properties.
In the present invention, various vectors known in the art, such as commercially available vectors including plasmids, cosmids, and the like, can be used. When the pteromalus puparum venom kynurenine transaminase PpVKAT is produced, the pteromalus puparum venom kynurenine transaminase PpVKAT coding sequence can be operably connected with an expression control sequence, so that a pteromalus puparum venom kynurenine transaminase PpVKAT expression vector is formed.
"operably linked" according to the present invention refers to the situation where certain parts of a linear DNA sequence are capable of affecting the activity of other parts of the same linear DNA sequence. For example, if the signal peptide DNA is expressed as a precursor and involved in secretion of the protein, the signal peptide (secretory leader) DNA is operably linked to the protein DNA; a promoter is operably linked to a coding sequence if it controls the transcription of that sequence; a ribosome binding site is operably linked to a coding sequence if it is placed in a position that enables translation. Generally, "operably linked" means adjacent, and for secretion leaders means adjacent in reading frame.
In the present invention the host cell is a prokaryotic cell. A commonly used prokaryotic host cell is referred to as an E.coli cell.
The expression of the gene product of the pteromalus puparum venom kynurenine aminotransferase PpVKAT can also be analyzed by Northern blotting technology or fluorescent quantitative PCR, namely, the existence and the quantity of the RNA transcript of the pteromalus puparum venom kynurenine aminotransferase PpVKAT in cells are analyzed.
In addition, the nucleic acid molecules useful as probes in the present invention typically have 8-66 contiguous amino acids, preferably 15-50 contiguous nucleotides, of the nucleotide coding sequence for pteromalus puparum venom kynurenine transaminase PpVKAT. The probe can be used for detecting whether nucleic acid molecules for coding the chrysalid pteromalid venom kynurenine aminotransferase PpVKAT exist in a sample.
The invention relates to a method for detecting whether a chrysalis venom kynurenine aminotransferase PpVKAT nucleotide sequence exists in a sample, which comprises the steps of hybridizing the probe and the sample, and detecting whether the probe is combined. Preferably, the sample is a product after PCR amplification, wherein the PCR amplification primers correspond to the nucleotide coding sequence of the pteromalus puparum venom kynurenine aminotransferase PpVKAT, and can be located at both sides or in the middle of the coding sequence. Primers are typically 15-50 nucleotides in length.
In addition, according to the nucleotide sequence and the amino acid sequence of the pteromalus puparum venom kynurenine transaminase PpVKT, homologous genes or homologous proteins of the pteromalus puparum venom kynurenine transaminase PpVKT can be screened on the basis of nucleic acid homology or expression protein homology.
The pteromalus puparum venom kynurenine transaminase PpVKAT nucleotide full-length sequence or the fragment thereof can be obtained by a PCR amplification method, a recombination method or an artificial synthesis method. For PCR amplification, the relevant sequence can be obtained by amplifying the relevant nucleotide sequence disclosed herein using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis. By utilizing the pteromalus puparum venom kynurenine transaminase PpVKAT, substances or receptors and the like which interact with the pteromalus puparum venom kynurenine transaminase PpVKAT can be screened out by various conventional screening methods.
The invention has obvious killing effect in the blood cell lethal test of important pests of brassicaceous vegetables, namely pieris rapae, and has obvious inhibition effect on the cellular immunity of pieris rapae. The harm of agricultural pests in China is very serious, the negative effect of using chemical pesticides is great, and the pteromalus puparum venom kynurenine transaminase PpVKAT has great application value on the biological control of the agricultural pests.
In addition, since parasitic factors of parasitic wasps are specific to hosts, most hosts are insects, and thus have certain safety to mammals. In conclusion, the kynurenine aminotransferase has wide application prospect, is expected to become safe and efficient anticancer and antitumor natural compounds and biological pesticides, and lays a foundation for culturing transgenic crops of the insect-resistant gene in the future.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a diagram showing the prokaryotic expression and purification of kynurenine aminotransferase PpVKAT of pteromalus puparum venom of the present invention;
note: m is standard protein, 1 is supernatant after induction of Escherichia coli BL21 containing pCOLD-TF-CAT plasmid, 2 is pCOLD-TF-CAT protein purified in Escherichia coli, 3 is supernatant after induction of Escherichia coli BL21 containing pCOLD-TF-PpVKT plasmid, and 4 is pCOLD-TF-PpVKT protein purified in Escherichia coli, namely chrysalis King wasp venom kynurenine transaminase PpVKT fusion protein.
FIG. 2 is a diagram of the lethal effect of a butterfly pupa King wasp venom kynurenine aminotransferase PpVKAT fusion protein (pCOLD-TF-PpVKAT) expressed by pronucleus on a pieris rapae blood cell, negative control is pCOLD-TF-CAT protein expressed by pronucleus, and the protein contents of pCOLD-TF-PVKAT and pCOLD-TF-CAT are both 4 mu g.
FIG. 3 shows the relative fluorescence value of the butterfly pupa and poliomyelitis kynurenine transaminase PpVKTA fusion protein (pCOLD-TF-PpVKTA) lethal pieris brassicae blood cells of the prokaryotically expressed butterfly pupa and poliomyelitis brassicae, wherein the negative control is the pCOLD-TF-CAT protein of the prokaryotically expressed, the protein contents of the pCOLD-TF-PKTA and the pCOLD-TF-CAT are both 4 mu g, the excitation wavelength Ex is 485nm, and the emission wavelength Em is 525nm.
Note: the experimental principle of FIGS. 2 and 3 is CellTox TM Asymmetric Cytosexicity Assay of Green Cytosexicity Assay (Promega, madison, USA) CylTox TM Green Dye cannot enter living cells, but can firmly bind DNA in dead cells, and the generated fluorescent signal is proportional to cytotoxicity.
Detailed Description
The invention is further illustrated below with reference to laboratory specific test data and with reference to specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1 expression and purification of recombinant pCOLD-TF-PpVKAT
1.1 extraction of RNA
In an ultra-clean workbench, female bees of pteromalus puparum are disinfected and wiped clean by 75% alcohol, and RNA is extracted by using a TRIzol method, wherein the specific method comprises the following steps:
1) 1-2 sterilized steel balls were placed in a 2ml sterile centrifuge tube.
2) 3-5 female pteromalus puparum on the third day of eclosion are prepared, collected and placed into the sterile centrifuge tube in the step 1), and 1ml of Trizol (Invitrogen, CA, USA) is added.
3) The polypide was vigorously shaken using a tissue homogenizer Tissuelyzer II (QIAGEN, hilden, germany) until a homogenate was broken, and then allowed to stand at room temperature for 5min.
4) 0.2ml of chloroform was added to the centrifuge tube, vigorously shaken for 15s, and allowed to stand at room temperature for 2-3min.
5) Centrifuge at 12000g for 15min at 4 deg.C, collect the supernatant and transfer it to a new 1.5ml sterile centrifuge tube.
6) 0.5ml of isopropanol is added into the centrifuge tube, the liquid in the tube is gently mixed evenly, and the mixture is kept stand for 10min at room temperature.
7) Centrifuge at 12000g for 10min at 4 ℃ and discard the supernatant.
8) Add 1ml 75% ethanol to the centrifuge tube and wash the pellet gently.
9) Centrifuging at 7500g for 5min at 4 deg.C, and removing supernatant. (at this time, absolute ethanol is added, and the product can be stored in a refrigerator at-80 deg.C for a long time).
10 Dry the tube in an ultra clean bench and add an appropriate amount of rnase-free water to dissolve. The OD260/OD280 value is measured using a spectrophotometer (Nano-drop Technologies, wilmington, DE), preferably at a ratio of about 2.0.
11 ) subpackaged, and stored in a refrigerator at-80 ℃ or subjected to the next experiment.
1.2 first Strand cDNA Synthesis
Use of
One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, beijing, china) kit reverse transcribes 1. Mu.g of RNA into cDNA as follows:
total RNA:1 μ g
Anchored Oligo(dT) 18 Primer(0.5μg/μl)：0.5μl
Random Primer(0.1μg/μl)：0.5μl
TransScript RT/RI Enzyme Mix：1μl
2×TS Reaction Mix：10μl
gDNA Remover：1μl
RNase-free water：to 20μl
After mixing the system, incubation was carried out for 10min at 25 ℃, 30min at 42 ℃ and 5s at 85 ℃, and finally the cDNA was stored in a refrigerator at-20 ℃ or subjected to the next experiment.
1.3 Gene cloning and PCR product recovery
Gene fragment sequences were obtained from the pteromalus puparum transcriptome and genome data, and primers (forward Primer 5'-3': ATGGGGCTAACCATAATGATT; reverse Primer 5'-3': TTATAAGGTCAGCAGCTTTTT) were designed by Primer Premier 5 (PREMIER Biosoft, CA, USA). Using cDNA as a template, taKaRa LA was used
The enzyme (TaKaRa, beijing, china) is used for PCR amplification, and the reaction solution system is as follows:
TaKaRa LA Taq(5U/μl)：0.5μl
10×LA Taq Buffer II(Mg 2+ Plus)：5μl
dNTP Mixture(2.5mM each)：8μl
PCR Forward primer(10μM)：2μl
PCR Reverse primer(10μM)：2μl
cDNA：500ng
Nuclease-free water：to 50μl
after the reagents are mixed evenly, the mixture is slightly sucked, beaten and centrifuged at low speed and put into a PCR amplification instrument, and the reaction conditions are as follows: 3min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 90s at 72 ℃ and 35 cycles; 10min at 72 ℃.
After the PCR amplification product is subjected to 1% agarose Gel electrophoresis to check the size of the fragment, gel Extraction and Clean-up Kit (EASY-DO, ZHejiang, china) is used for Gel cutting and recovery, and the specific method is as follows:
1) The cut agarose gel was placed in a 1.5ml sterile centrifuge tube, 400. Mu.l of Buffer A1 was added and heated at 65 ℃ until it was completely melted.
2) The solution was pipetted onto a HiPure DNA adsorption column set in a 2ml collection tube, centrifuged at 12000rpm for 30 seconds at room temperature, and the waste solution was discarded. If the volume of the solution is more than 700. Mu.l, the solution needs to be loaded on the column in several times.
3) The adsorption column was returned to the collection tube, 500. Mu.l of Buffer A2 (100 ml of absolute ethanol was added to Buffer A2 according to the instructions before use), centrifuged at 12000rpm for 30s at room temperature, and the waste liquid was discarded.
4) Repeating the step 3).
5) The adsorption column was returned to the collection tube and centrifuged at 12000rpm for 1min at room temperature.
6) Transferring the adsorption column to a new sterile 1.5ml centrifuge tube, adding 30-50 μ l of Elution2.0 (heating to 65 deg.C is more beneficial to improve elution efficiency) to the center of the adsorption column membrane, and standing at room temperature for 1-2min.
7) Centrifuging at 12000rpm for 1min at room temperature, and eluting to obtain the target DNA. The OD260/OD280 was measured by a spectrophotometer, and the ratio was preferably around 1.8.
1.4 sequencing verification of PCR products
Connecting the recovered product to
Easy Vector Systems (Promega, madison, USA), the linkage system is as follows:
2×Rapid ligation Buffer，T4 DNA Ligase：5μl
pGEM-T Easy Vector(50ng)：1μl
PCR product：65ng
T4 DNA ligase：1μl
Nuclease-free water：to 10μl
the prepared system is sucked and uniformly mixed by using a pipette gun, and is placed at room temperature for 1h or is kept overnight at 4 ℃.
The ligation products were transformed into Competent cells Transgen, beijing, china Cell, transns 1-T1 Phage resist chemical Complex Cell, by the following specific steps:
1) Taking out the frozen competent cells from a refrigerator at the temperature of-80 ℃, quickly placing on ice, and waiting for the cells to melt for about 5-7min.
2) The ligation product was added to 50. Mu.l of competent cells, mixed gently and incubated in ice for 20-30min.
3) The water bath was heat shocked at 42 ℃ for 45-50s, and then the tube was quickly transferred to an ice bath for 2min without shaking the centrifuge tube.
4) Adding 900 μ l LB liquid culture medium without antibiotics into the centrifuge tube, mixing well, culturing at 37 deg.C and 200rpm for 1h to recover bacteria.
5) Pipette 8. Mu.l of IPTG (50 mM) and 40. Mu.l of X-gal (20 mg/mL), mix well and spread on LB plates with ampicillin resistance until they are absorbed.
6) Centrifuging 1000g of bacteria recovered in the step 4) for 10min, discarding the supernatant, resuspending the pellet with 200. Mu.l of non-resistant LB liquid medium, and smearing 100. Mu.l of the pellet on the plate prepared in the step 5).
7) The plate is placed in a constant temperature incubator at 37 ℃ for overnight culture for about 16-24 h, and is placed in a refrigerator at 4 ℃ after the clonal bacteria grow out, which is beneficial to screening blue white spots.
8) 1ml of LB liquid medium having ampicillin resistance was added to a 1.5ml sterile centrifuge tube, and white monoclonals selected by the blue-white selection were transferred to the tube using a pipette gun.
9) Shaking at 37 deg.C and 200rpm to turbidity, and sequencing by Zhejiang Shanghai Biotechnology Ltd.
The nucleotide sequence of the obtained PCR product is SEQ ID NO:1, the preparation method is as follows. The pteromalus puparum venom canine urinary alanine transaminase PpVKAT coded by the gene has an amino acid sequence shown as SEQ ID NO:2, wherein the signal peptide is predicted online by software SignalP 5.0 (http:// www.cbs.dtu.dk/services/SignalP /).
1.5 construction of recombinant plasmid
The PpVKAT full length obtained by pteromalus puparum transcriptome and genome data is used for designing and constructing primers of a prokaryotic expression vector (the enzyme cutting sites of PpVKAT recombinant plasmid are KpnI and XbaI) by using a full length ORF (signal peptide is removed, and His tag is added at the C end), prokaryotic expression is carried out by an escherichia coli expression system, and a prokaryotic primer (forward primer 5'-3': agcataggagcatgggcgtgcttaccATACCTTCGGAAAATCTCTCTCATCA; reverse primer 5'-3': agcagaggagatacccatcttactagTGATGATGATGATGAAGTGATGAAGTC) is designed by CE Design (Vazyme, nanjing, china). Using the cDNA synthesized in step 1.2 as a template, KOD One was used TM PCR was performed using PCR Master Mix (TOYOBO, shanghai, china) in the following amplification scheme:
KOD One TM PCR Master Mix：25μl
PCR Forward primer(10μM)：1.5μl
PCR Reverse primer(10μM)：1.5μl
cDNA：750ng
Nuclease-free water：to 50μl
after the reagents are mixed evenly, the mixture is slightly sucked, beaten and centrifuged at low speed and put into a PCR amplification instrument, and the reaction conditions are as follows: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 5s, and elongation at 68 ℃ for 10s, repeated for 35 cycles.
After the PCR amplification product was checked for fragment size by 1% agarose Gel electrophoresis, it was recovered by Gel Extraction & Clean-up Kit (EASY-DO, ZHEjiang, china) by Gel cutting as described in 1.3.
Subsequently, the pCOLD-TF no-load plasmid was double digested with FastDigest KpnI and FastDigest XbaI (Thermo Fisher, calif., USA) restriction enzymes as follows:
1) Preparing a reaction solution:
10×Fast Digest Buffer：2μl
FastDigest KpnI：1μl
FastDigest XbaI：1μl
pCOLD-TF plasmid：1μg
Nuclease-free water：to 20μl
2) Gently sucking and beating the mixture, and incubating the mixture for 15 to 30min at 37 ℃ to obtain the linearized pCOLD-TF plasmid.
After the size of the fragment was checked by 1% agarose Gel electrophoresis, the digested product was recovered by Gel Extraction & Clean-up Kit (EASY-DO, ZHEjiang, china) Gel cutting as described in 1.3.
The target fragment obtained by PCR amplification was subjected to homologous recombination with linearized pCOLD-TF plasmids (restriction sites: kpnI and XbaI) using the Clonexpress Ultra One Step Cloning Kit (Vazyme, nanjing, china), the homologous recombination system being:
2×ClonExpress Mix：5μl
linearized pCOLD-TF plasmid: 100ng
PCR fragment amplification product: 50ng
Nuclease-free water：to 10μl
Gently sucking and beating the mixture by using a pipettor, mixing the mixture evenly, centrifuging the mixture for a short time to collect reaction liquid to the bottom of the tube, and reacting under the conditions that: 50 ℃ for 5min.
After the reaction is finished, immediately placing on ice to cool for 15min, and directly converting or storing at-20 ℃ for later use.
The ligation products were transformed into Transgen 1-T1 Phage resist chemical Complex Cell (Transgen, beijing, china) and the ampicillin-screened monoclonal strains were shaken to turbid (without blue-white screening) as described in 1.3 and sent to sub biotechnologies of Zhejiang province for sequencing. After verification, the pCOLD-TF-PpVKAT recombinant plasmid was transformed into BL21 chemical component Cell (Transgen, beijing, china) by the following specific steps:
1) 50 μ l of the competent cells thawed on ice bath were added with the objective DNA, mixed gently and placed in ice bath for 30min.
2) The tube was placed in a 42 ℃ water bath for 45s and then quickly transferred to an ice bath for 2min without shaking the tube.
3) Add 500. Mu.l sterile LB liquid medium (without antibiotics) to the centrifuge tube, mix well and incubate at 37 ℃ for 1h at 200rpm to resuscitate the bacteria.
4) The resuscitated bacteria were centrifuged at 1000g for 10min, the supernatant was discarded, the pellet was resuspended in 200. Mu.l of sterile LB medium (without antibiotics), and 50. Mu.l of the pellet was smeared onto LB plates with ampicillin resistance.
5) The plate was incubated overnight at 37 ℃ for about 16h to 24h.
6) 1ml of LB liquid medium having ampicillin resistance was added to a 1.5ml sterile centrifuge tube, and the ampicillin-resistant-selected monoclonal bacteria were transferred to the tube using a pipette gun.
7) Shaking at 37 ℃ and 200rpm until the solution is turbid, and sending the turbid solution to Zhejiang Shanghai SubBiotechnology Limited for sequencing.
Thus, escherichia coli containing the pCOLD-TF-PpVKAT recombinant plasmid is obtained, and prokaryotic expression can be carried out.
The same procedure (i.e., changing the PCR primers in step 1.5 above to forward primers 5'-3': agcatatgagctcgggtaccATGGAGAAAAAAATCACTGGATATACC; reverse primers 5'-3': agcagaggatattactcata TTAATGATGATGATGATGATGCCCCCGCCCCTGTCCGACCATCCA, changing the PCR template to a negative control plasmid containing the Catalase (CAT) gene stored in the laboratory, the remainder being identical to step 1.5 above) gave Escherichia coli containing the pCOLD-TF-CAT recombinant plasmid as a negative control for prokaryotic expression.
1.6 prokaryotic expression, purification and desalting of proteins
1) The Escherichia coli liquid with correct sequencing is sucked into LB liquid culture medium with ampicillin resistance, and is placed into a shaking table to be cultured overnight at 37 ℃ and 200 rpm.
2) Taking 2ml of activated bacteria liquid, adding into 200ml of fresh LB liquid culture medium, culturing for 1-2h until OD 600nm is about 0.5.
3)15℃，230rpm，30min。
4) 200. Mu.l of 1M IPTG was added and expression was induced at 15 ℃ for 16h-24h on a 120rpm shaker.
5) The cells were collected by centrifugation at 4 ℃ at 3000g for 15min, and the supernatant was discarded.
6) Adding into
Master Mix (Novagen, MA, USA), add 5ml of this lysis solution per gram of pellet, pipette until the pellet is completely dissolved, and incubate at 4 ℃ for 30min on a shaker.
7) Centrifuging at 12000g for 20min at 4 deg.C, collecting supernatant, and placing in ice bath for use.
8) Mu.l of cOmplete His-Tag Purification Resin (Roche, mannheim, germany) was pipetted into a 6ml affinity column (Sangon Biotech, shanghai, china), and the mixture was allowed to stand for layer separation and ethanol was eluted.
9) 4ml binding buffer (50 mM NaH) was used 2 PO 4 300mM NaCl, pH =8.0, 10mM imidazole) was gently mixed with the resin, and the mixture was allowed to settle naturally and then flowed out.
10 ) repeat step 9) twice.
11 Add the supernatant prepared in step 7) and combine overnight in a rotary mixer at 4 ℃.
12 Discard the column liquid every day, rinse buffer (50 mM NaH) with 4ml 2 PO 4 300mM NaCl, pH =8.0, 30mM imidazole) was gently mixed with the resin and the mixture was allowed to settle naturally and then the liquid was discharged.
13 ) repeat step 12) once.
14 With 4ml of rinsing buffer (50 mM NaH) 2 PO 4 300mM NaCl, pH =8.0, 50mM imidazole) was gently mixed with the resin and the mixture was allowed to settle naturally and then drained.
15 ) repeat step 14) once.
16 Add 500. Mu.l of elution buffer (50 mM NaH) 2 PO 4 300mM nacl, ph =8.0, 250mM imidazole), allowed to bind well to the resin in an ice bath for 30min, and the supernatant was collected by centrifugation.
Using Zeba TM Spin Desalting Columns,7K mwco,2ml (Thermo Scientific, waltham, USA) desalt the eluate from step 16) as follows:
17 The cap of the desalting column was unscrewed and the tail was broken off, and the column was placed in a 12ml centrifuge tube.
18 4 ℃ C., 1000g centrifugation for 2min removed the stock solution, a mark was made on the tube wall at the top of the tapered resin, after which all centrifugation steps were performed with the column mark facing outward into the centrifuge.
19 Add 1ml of ice-precooled PBS to the column.
20 ) the PBS was removed by centrifugation at 1000g for 2min at 4 ℃.
21 Repeat steps 19) and 20) two to three times and discard the PBS from the centrifuge tube.
22 The column was placed in a fresh sterile centrifuge tube and the eluent containing the protein of interest was slowly added to the center of the resin.
23 Sample was collected by centrifugation at 1000g for 2min at 4 ℃ and this was desalted supernatant (containing the protein of interest).
The protein concentration of the desalted supernatant was determined by Quick Start Bradford Dye Reagent (Bio-Rad, calif., USA), and the protein was separated by 8% -16% SDS-PAGE (GenScript, nanjing, china), and if the molecular weight was correct, the collected protein was stored in an ice bath for further experiments.
Thus, cell lysis supernatants of pCOLD-TF-CAT and pCOLD-TF-PpVKAT were obtained according to step 7) of 1.6, and recombinant proteins of pCOLD-TF-PpVKAT and pCOLD-TF-CAT were obtained according to step 23) of 1.6.
The results are shown in FIG. 1, and it can be seen from lanes 2 and 4 in FIG. 1 that the bands of the purified pCOLD-TF-CAT protein and the target protein pCOLD-TF-PpVKT are single and the sizes of the fragments are correct, and they can be used for the subsequent functional studies.
Example 2 lethal assay of recombinant protein pCOLD-TF-PpVKAT on Pieris rapae blood cells
The recombinant expression proteins pCOLD-TF-PpVKAT and pCOLD-TF-CAT are incubated with host blood cells, and CellTox is adopted TM Green cytotoxin Assay (Promega, madison, USA) for detecting cell mortality (asymmetric cyanine fluorescent dye CellTox) TM Green Dye can not enter into alive fineCell, but can firmly combine with DNA in dead cells, and the generated fluorescence signal is proportional to cytotoxicity), and the pteromalus puparum venom kynurenine transaminase PpVKAT is determined to have cell lethal activity.
1) In a clean bench, the pupated cabbage caterpillars (cabbage butterfly pupation stage) and the surfaces of the insect needles are cleaned by using 75% alcohol, the cabbage caterpillars pupae are naturally dried in the clean bench, and the insect needles are sterilized by using an alcohol lamp outer flame.
2) Preparing a culture medium Mix for in vitro culture of blood cells in a clean bench: 800. Mu.l Grace's Instrument, supplemented (Life Technologies Corporation, NY, USA) + 50. Mu.l saturated phenylthiourea solution (Sigma-Aldrich, MO, USA) + 50. Mu.l Penicillin-Streptomycin (Life Technologies Corporation, NY, USA) + 0.9. Mu.l CellTox TM Green Dye (Promega, madison, USA), in which the Insect cell culture fluid Grace's Instrument, supplemented contains 10% Fetal Bovine Serum (Total Bovine Serum) (WISENT, nanjing, china), and the concentration of penicillin-streptomycin is 10000U/ml.
3) Sucking and stirring Mix obtained in the step 2), and subpackaging into 1.5ml sterile centrifuge tubes with 90 mu l of each tube.
4) Piercing the winged bud of pupa with sterilized insect needle until hemolymph naturally flows out.
5) Sucking hemolymph with a 10. Mu.l sterile pipette tip into a 1.5ml sterile centrifuge tube in step 3), ratio: 10 μ l haemolymph +90 μ l Mix.
6) Gently sucking, mixing, subpackaging into 384-well plates with each well being 40 μ l, and culturing in a cell culture box at 27 ℃ overnight for 12 h.
7) The following day 4. Mu.g of sample protein was added to each well and after incubation for 24h at 27 ℃ in a cell incubator, observed and photographed under an inverted fluorescence microscope Leica DMi8 (Leica, wetzlar, germany), as shown in FIG. 2. After the photographs were taken, the supernatant culture solution in each well was discarded and immediately placed in a microplate reader Thermo Scientific Varioskan Flash (Thermo Scientific, vantaa, finland) to measure the fluorescence of adherent cells at the bottom of each well plate, as shown in fig. 3.
Wherein, the sample proteins are respectively: negative control pCOLD-TF-CAT recombinant protein; experiment of the inventionAnd (3) treating the pCOLD-TF-PpVKAT recombinant protein. The Leica DMi8 of the inverted fluorescence microscope respectively observes blood cells by using a bright field and green fluorescence and takes pictures, the excitation wavelength Ex of the microplate reader is 485nm, the emission wavelength Em is 525nm, and each treatment is 3 biological repetitions. Comparison of mean values between two samples was done by unpaired Student's t test, data was analyzed for variance using DPS data processing software (both chequer and von brighten, 2007), fig. 3<0.01, the data are statistically very different. Meanwhile, graphPad Prism 5.0 (GraphPad Prism, CA, USA) was used for the drawing, and Image J (Image J, NIH, USA) was used for the Image processing, and the specific results are shown in fig. 2 and 3. As can be observed from fig. 2: in a 24h light field, the blood cells after incubation of pCOLD-TF-PpVKAT recombinant protein become round in shape, have reduced volume and cluster, and the blood cells after incubation of negative control pCOLD-TF-CAT recombinant protein extend normally without cluster phenomenon; in 24h of green fluorescence, the green fluorescence of blood cells incubated by the pCOLD-TF-PpVKAT recombinant protein is more than that of a negative control group (pCOLD-TF-CAT recombinant protein). Meanwhile, as can be seen from the data of fig. 3: the relative fluorescence value of blood cells after incubation of pCOLD-TF-PpVKAT recombinant protein is higher than that of a negative control group (pCOLD-TF-CAT recombinant protein), has very significant difference in statistics, and is combined with CellTox TM The experimental principle of the Green Dye (the Dye can not enter living cells, but can firmly bind to DNA in dead cells, and the generated fluorescent signal is proportional to cytotoxicity) indicates that the cytotoxicity and the number of dead cells generated by blood cells incubated by the pCOLD-TF-PpVKAT recombinant protein are larger than those of a negative control group.
The above test results thus demonstrate that: the PpVKAT protein has lethal effect on the pieris rapae blood cells.
Finally, it should also be noted that the above list is only a specific implementation example of the present invention. It is obvious that the invention is not limited to the above embodiment examples, but that many variations are possible. All modifications which can be derived or suggested by the person skilled in the art from the present disclosure are to be considered within the scope of the present invention.
Sequence listing
<110> Zhejiang university
<120> pteromalus puparum venom kynurenine transaminase PpVKAT and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1311
<212> DNA
<213> Pteromalus puparum (Pteromalus puparum)
<400> 1
atggggctaa ccataatgat taaggtggcg tgtatagttg ccgtattaat ttgcctgcca 60
gtaaggccaa gtgtcggaat acctttgtcg gaaaatctct caaagagact cgcgaggagt 120
gtaacaaatg aggagcccat cgttgacctc cagattgaca agactgacgg tttcgcaccg 180
ccacatttgg taaaggctct gctccaggct gtcgcttcca acgacacttc actcaaccag 240
tatgcagcag gcattggaca cccaagactt cgcaaagcta tagcagcttt ttacggtaag 300
gtgatagaac gcgaacttga ttggcaaaag aacgtgatcg tgacgatcgg tgcgaccgag 360
gcggtatggg acagcttcca tgccctcacc caacccggcg acgagtggat cgtcgttgag 420
ccatttttct ccaagtatgc cccaaccatc aaactcgctg gtggcatacc tcgatttact 480
tctatgaaat tgacgaaaac gagcggtgaa ataactggtg ccgactgggt gctggataag 540
gaagagctaa gaagcctctt taacaacaaa accagaggta taataattaa caatccaaac 600
aaccctgcgg gcaaggttct cacgatggac gatcttcagt tcattgccga tttgattaag 660
ctgcataatg cttacgtcat cgccgatgac gctcacgaat gggtgctatt cgacccgatc 720
aaaactccct tcattagaat ggcacaatta cctggaatgt gggagcgcac cattaccatc 780
ggctctgcca gcaagtcctt cacggtctca ggatggagag tcggttgggc ctatgctccg 840
gcagatctca tcactcgtct cctcaagatt cacaccaacg ccgtacagag tgttcccact 900
ccacaacagg aagccgtagc cttcggcttt gaagaagagc ttcgtcttta cggtaaaccc 960
gaagcctact tcgcctccaa ttcacgcgcc gtccgcgaga agcacaactt catataccag 1020
gctttcgtcg acgtcggtat gatccctatc cgtcctgacg gcggatactg catgcttgca 1080
aagtggccca gcttcaatga tcacaaagag ctcttcgaag gccaggagaa ggccgttgcc 1140
ttcgagaact tcatgagcca aaggatcaag atacttggta ccacaatggc cgactactac 1200
agcgacgagc atcgctatat gggcgaggac tacatcaggt tctgtctgca aaagagaaac 1260
gagactttgg ttagaactgc tgaaaacttg aaaaagctgc tgaccttata a 1311
<210> 2
<211> 436
<212> PRT
<213> Pteromalus puparum
<400> 2
Met Gly Leu Thr Ile Met Ile Lys Val Ala Cys Ile Val Ala Val Leu
1 5 10 15
Ile Cys Leu Pro Val Arg Pro Ser Val Gly Ile Pro Leu Ser Glu Asn
20 25 30
Leu Ser Lys Arg Leu Ala Arg Ser Val Thr Asn Glu Glu Pro Ile Val
35 40 45
Asp Leu Gln Ile Asp Lys Thr Asp Gly Phe Ala Pro Pro His Leu Val
50 55 60
Lys Ala Leu Leu Gln Ala Val Ala Ser Asn Asp Thr Ser Leu Asn Gln
65 70 75 80
Tyr Ala Ala Gly Ile Gly His Pro Arg Leu Arg Lys Ala Ile Ala Ala
85 90 95
Phe Tyr Gly Lys Val Ile Glu Arg Glu Leu Asp Trp Gln Lys Asn Val
100 105 110
Ile Val Thr Ile Gly Ala Thr Glu Ala Val Trp Asp Ser Phe His Ala
115 120 125
Leu Thr Gln Pro Gly Asp Glu Trp Ile Val Val Glu Pro Phe Phe Ser
130 135 140
Lys Tyr Ala Pro Thr Ile Lys Leu Ala Gly Gly Ile Pro Arg Phe Thr
145 150 155 160
Ser Met Lys Leu Thr Lys Thr Ser Gly Glu Ile Thr Gly Ala Asp Trp
165 170 175
Val Leu Asp Lys Glu Glu Leu Arg Ser Leu Phe Asn Asn Lys Thr Arg
180 185 190
Gly Ile Ile Ile Asn Asn Pro Asn Asn Pro Ala Gly Lys Val Leu Thr
195 200 205
Met Asp Asp Leu Gln Phe Ile Ala Asp Leu Ile Lys Leu His Asn Ala
210 215 220
Tyr Val Ile Ala Asp Asp Ala His Glu Trp Val Leu Phe Asp Pro Ile
225 230 235 240
Lys Thr Pro Phe Ile Arg Met Ala Gln Leu Pro Gly Met Trp Glu Arg
245 250 255
Thr Ile Thr Ile Gly Ser Ala Ser Lys Ser Phe Thr Val Ser Gly Trp
260 265 270
Arg Val Gly Trp Ala Tyr Ala Pro Ala Asp Leu Ile Thr Arg Leu Leu
275 280 285
Lys Ile His Thr Asn Ala Val Gln Ser Val Pro Thr Pro Gln Gln Glu
290 295 300
Ala Val Ala Phe Gly Phe Glu Glu Glu Leu Arg Leu Tyr Gly Lys Pro
305 310 315 320
Glu Ala Tyr Phe Ala Ser Asn Ser Arg Ala Val Arg Glu Lys His Asn
325 330 335
Phe Ile Tyr Gln Ala Phe Val Asp Val Gly Met Ile Pro Ile Arg Pro
340 345 350
Asp Gly Gly Tyr Cys Met Leu Ala Lys Trp Pro Ser Phe Asn Asp His
355 360 365
Lys Glu Leu Phe Glu Gly Gln Glu Lys Ala Val Ala Phe Glu Asn Phe
370 375 380
Met Ser Gln Arg Ile Lys Ile Leu Gly Thr Thr Met Ala Asp Tyr Tyr
385 390 395 400
Ser Asp Glu His Arg Tyr Met Gly Glu Asp Tyr Ile Arg Phe Cys Leu
405 410 415
Gln Lys Arg Asn Glu Thr Leu Val Arg Thr Ala Glu Asn Leu Lys Lys
420 425 430
Leu Leu Thr Leu
435
Claims (7)
1. The pteromalus puparum venom kynurenine transaminase PpVKAT is characterized in that: the amino acid sequence is shown as SEQ ID NO:2, respectively.
2. The pteromalus puparum kynurenine transaminase PpVKAT of claim 1, which is characterized in that: the protein is a protein, a conservative variant protein thereof, an active fragment thereof or an active derivative thereof.
3. The gene encoding chrysalid pteromalid venom kynurenine aminotransferase PpVKAT of claim 1 or 2, which is characterized in that: the nucleotide sequence is shown as SEQ ID NO:1 is shown in the specification; or to SEQ ID NO:1 has at least 70% homology with the nucleotide sequence in the sequence table; or the nucleotide sequence of the polypeptide can be matched with the nucleotide sequence shown in SEQ ID NO: 1.
4. The gene according to claim 3, which is characterized in that: the nucleotide sequence comprises 8-66 continuous nucleotides.
5. The use of chrysalid pteromalid venom kynurenine aminotransferase PpVKAT according to claim 1 or 2, which is characterized by: lethal pieris rapae blood cells.
6. Use according to claim 5, characterized in that: the venom kynurenine transaminase PpVKAT secreted by the pteromalus puparum venom gland can be used for killing the cabbage butterfly blood cells.
7. Use according to claim 5 or 6, characterized in that: the recombinant protein pCOLD-TF-PpVKAT has lethal effect on the blood cells of the pieris rapae.
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| CN102387701A (en) * | 2008-08-29 | 2012-03-21 | 洛斯阿拉莫斯国家安全有限公司 | Plant glutamine phenylpyruvate transaminase gene and transgenic plants carrying same |
| CN105440131A (en) * | 2015-12-16 | 2016-03-30 | 浙江大学 | Pteromalus puparumvenom serine protease inhibitor PpSerpin protein and application thereof |
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| CN108892721A (en) * | 2018-06-28 | 2018-11-27 | 浙江大学 | Pteromalus Puparum L venom Kazal-type serpin PpSPI20 albumen and application |
| CN110590925A (en) * | 2019-09-10 | 2019-12-20 | 浙江大学 | Pteromalus puparum venom protein PpVPG and application thereof |
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| CN102387701A (en) * | 2008-08-29 | 2012-03-21 | 洛斯阿拉莫斯国家安全有限公司 | Plant glutamine phenylpyruvate transaminase gene and transgenic plants carrying same |
| WO2016154047A2 (en) * | 2015-03-20 | 2016-09-29 | Memorial Sloan-Kettering Cancer Center | Monoclonal antigen-binding proteins to intracellular oncogene products |
| CN105440131A (en) * | 2015-12-16 | 2016-03-30 | 浙江大学 | Pteromalus puparumvenom serine protease inhibitor PpSerpin protein and application thereof |
| CN108892721A (en) * | 2018-06-28 | 2018-11-27 | 浙江大学 | Pteromalus Puparum L venom Kazal-type serpin PpSPI20 albumen and application |
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