CN110592113B - Sumo modification system gene TpUBC9 of taenia pisiformis and application thereof - Google Patents
Sumo modification system gene TpUBC9 of taenia pisiformis and application thereof Download PDFInfo
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Abstract
The invention provides a taenia pisiformis SUMO modification system gene TpUBC9 and application thereof, and relates to the technical field of genetic engineering. The nucleotide sequence of the gene TpUBC9 is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene TpUBC9 is shown in SEQ ID NO. 2. The invention screens the interacting protein gene TpUBC9 of soybean tapeworm leucine aminopeptidase TpLAP for the first time by a yeast two-hybrid technology, clones the full-length cDNA of TpUBC9 gene, verifies the interaction of TpUBC9-TpLAP and verifies that a fine SUMO modification system also exists in the body of tapeworm; provides a new clue for the subsequent related research of SUMO modification of tapeworm. The TpUBC9 antibody immunomagnetic beads prepared from the antigen have the characteristics of good specificity, high affinity, convenient operation, reliable result and the like.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a taenia pisiformis SUMO modification system gene TpUBC9 and application thereof.
Background
Taenia pisiformis (taeniasiformis) is a common canine intestinal parasite, belonging to the Taenia (taenidae) genus taeniae (taenidia). The life history of the rabbit needs to be completed by converting 2 hosts, adults usually parasitize in small intestines of dogs, and cysticercus pisiformis parasitizes in livers, gastric omentum, mesenterium and other parts of rabbits. In China, the average infection rate of rabbits reaches 40%, the death rate is 4.0% -23.69%, the rabbit product quality is seriously influenced, and the development of rabbit breeding in China is threatened. At present, China still faces a great problem in the prevention and treatment of tapeworm/oncosis, and a need for screening and excavating specific drug targets and vaccine candidate molecules is urgently needed, so that a new means is provided for the effective prevention, control and radical treatment of the parasitic diseases.
SUMO modification is essential for growth and development of mammals and parasites, wherein UBC9 is the only E2 binding enzyme in the SUMO modification pathway, and SUMO of substrate proteins must be dependent on expression of UBC 9. Research shows that knockout of caenorhabditis elegans UBC9 gene can lead to embryonic development retardation, severe development deformity of pharyngeal muscle, gonad and tail of larva, and finally, abnormal spawning and genital pore rupture. The deletion of the drosophila UBC9 can block the nuclear entry of a bicoid protein which is an important regulatory transcription factor for the development of the front somite, thereby influencing the normal expression of a target gene and finally causing the abnormal development of the front somite. Defects in mouse UBC9 significantly impair the ability of stem cells to differentiate, leading to apoptosis, premature embryonic death or cardiac failure.
At present, the research on the SUMO modification approach of tapeworm is not reported at home and abroad.
Disclosure of Invention
In view of this, the invention aims to provide a taenia pisiformis SUMO modification system gene TpUBC9, which lays a foundation for the protein purification and protein interaction research of TpUBC9 in the taenia pisiformis SUMO modification system.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a taenia pisiformis SUMO modification system gene TpUBC9, wherein the nucleotide sequence of the gene TpUBC9 is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the gene TpUBC9 is shown in SEQ ID NO. 2.
The invention also provides a group of primer pairs for cloning the gene TpUBC9, wherein the primer pairs comprise a forward primer TpUBC9F321 and a reverse primer TpUBC9R487, and the nucleotide sequence of the forward primer TpUBC9F321 is shown as SEQ ID NO. 3; the nucleotide sequence of the reverse primer TpUBC9R487 is shown as SEQ ID NO. 4.
The invention also provides a method for amplifying the gene TpUBC9, which comprises the following steps: (1) carrying out reverse transcription on the total RNA of the taenia pisiformis serving as a template to obtain a first cDNA chain;
(2) taking the first cDNA chain as a template and the primer pair as a primer to carry out rapid amplification of the cDNA end; the procedure for the amplification was: 30s at 94 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 70 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 68 ℃, 3min at 72 ℃ and 25 cycles;
(3) recovering the PCR band, connecting to pMD19-T vector and transforming E.coli DH5a competent cell; clones which are identified as positive by the PCR of the bacterial liquid and are sequenced correctly are spliced with 3 'and 5' amplification sequences to obtain a full-length cDNA sequence of the TpUBC9 gene.
Preferably, Clontech is used in step (1)
RACE cDNA Amplification kit was used for the reverse transcription.
The invention also provides a recombinant expression vector pET28a-TpUBC9 containing the gene TpUBC9, and the gene TpUBC9 is cloned between enzyme cutting sites Nde I and Xho I of the expression vector by taking pET-28a (+) as an expression vector.
The invention also provides a recombinant bacterium containing the gene TpUBC9 or the recombinant expression vector pET28a-TpUBC 9.
The invention also provides a preparation method of the TpUBC9 immunomagnetic bead, which comprises the following steps: (a) transforming the recombinant expression vector pET28a-TpUBC9 into escherichia coli, inducing with IPTG, and purifying with Ni-affinity column to obtain His-TpUBC9 fusion protein;
(b) immunizing rabbits with the His-TpUBC9 fusion protein to obtain polyclonal antibody;
(c) and coating Dynabeads M-280Tosylactivated magnetic beads with the polyclonal antibody to obtain the TpUBC9 immunomagnetic beads.
The invention provides a taenia pisiformis SUMO modification system gene TpUBC9, wherein the nucleotide sequence of the gene TpUBC9 is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the gene TpUBC9 is shown in SEQ ID NO. 2. In the invention, the interaction protein gene TpUBC9 of taenia pisiformis TpLAP is screened for the first time by a yeast two-hybrid technology; obtaining a core component molecule TpUBC9 of the taenia pisiformis SUMO modification system for the first time, cloning the full-length cDNA of a TpUBC9 gene, verifying the interaction of TpUBC9-TpLAP, and verifying that a fine SUMO modification system also exists in the taenia pisiformis; provides a new clue for the subsequent related research of SUMO modification of tapeworm.
The invention further prepares TpUBC9 antibody immunomagnetic beads, the method can effectively enrich TpUBC9 protein and screen and identify substrate protein stably interacted with TpUBC9, has the characteristics of good specificity, high affinity, convenient operation, reliable result and the like, can directly enrich TpUBC9 protein from total protein of a worm body, prokaryotic and eukaryotic expression systems, can effectively identify the substrate protein stably interacted with TpUBC9, and thus determines the technical feasibility of the application of the method in SUMO modified protein interaction; provides a simple and practical means for screening and verifying SUMO modified substrate molecules.
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FIG. 1 shows the results of SDS-PAGE and Western-blot analysis of His-TpUBC9 protein; wherein M: protein molecular weight standards; 1: induced His-TpUBC9 protein; 2: induction control of empty vector bacteria; 3-4: His-TpUBC9 purified protein (200mM and 300mM imidazole elution); 5: His-TpUBC9 reacted with anti-TpUBC 9 polyclonal antibody (1. mu.g/mL); 6: His-TpUBC9 did not react with rabbit negative serum;
FIG. 2 shows the results of Westernblot analysis of the immunomagnetic beads purified His-TpUBC9 protein; wherein 1: His-TpUBC9 purified by Ni column; 2: the immune magnetic bead is incubated with His-TpUBC9 protein at 4 ℃ overnight; 3: incubating the His-TpUBC9 protein for 1h at 37 ℃ by using immunomagnetic beads;
FIG. 3 is a one-to-one validation of the interaction of TpUBC9 with TpLAP by yeast; wherein 1: negative controls (pGBKT7-Lam + pGADT7-T) were grown on DDO plates and not on QDO/X/A plates; 2: positive controls (pGBKT7-p53+ pGADT7-T) were grown on DDO plates and blue yeast colonies on QDO/X/A plates; 3: the BD-TpLAP + AD-TpUBC9 experimental group was grown on DDO plates and blue yeast colonies were grown on QDO/X/A plates;
fig. 4 shows immunomagnetic beads demonstrating TpUBC9 interaction with TpLAP; wherein 1: cell whole protein lysate (Input) of co-transfected Myc-TpLAP and Flag-TpUBC9 plasmids can respectively react with Myc antibody and Flag antibody; 2: the co-transfected cell sample separated by the immunomagnetic beads reacts with both the Myc antibody and the Flag antibody; 3: cell whole protein lysates of untransfected plasmids were non-reactive with Myc antibody and Flag antibody.
Detailed Description
The invention provides a taenia pisiformis SUMO modification system gene TpUBC9, wherein the nucleotide sequence of the gene TpUBC9 is shown as SEQ ID No. 1; the amino acid sequence of the protein coded by the gene TpUBC9 is shown in SEQ ID NO. 2.
The gene TpUBC9 is preferably used for obtaining the interacting molecule TpUBC9 by screening a taenia pisiformis adult three-box yeast cDNA library by a yeast two-hybrid technology by taking TpLAP (taenia pisiformis leucine aminopeptidase) as a bait protein. The screening method of the present invention preferably comprises the following steps:
(1) construction of three-box yeast cDNA library: extracting taenia sojae imago total RNA by Trizol method, and purifying mRNA by Oligo d (T)25Magnetic Beads; constructing a yeast cDNA library after BP and LR recombination by adopting a CloneMiner II cDNA library construction kit;
(2) construction and identification of bait plasmid: PCR amplification is carried out to obtain a complete CDS sequence of TpLAP, the complete CDS sequence is connected with pGBKT7 after a target fragment is recovered by Nde I and Pst I double enzyme digestion, escherichia coli DH5a is transformed, positive clone is screened, and plasmid sequencing is extracted to verify and obtain a bait vector (BD-TpLAP); converting the BD-TpLAP plasmid into yeast Y2HGold for self-activation and toxicity detection;
(3) yeast two-hybrid: and (3) screening interaction protein of TpLAP from a three-box yeast cDNA library of the taenia pisiformis imagoes by taking BD-TpLAP as a bait. Extracting yeast plasmid from the positive bacterial plaque and transforming escherichia coli DH5 alpha; selecting positive bacterial plasmids for sequencing, and performing Blast comparison analysis to obtain a positive interaction gene sequence; then the positive plasmid and the bait plasmid are transformed into Y2HGold yeast in pairs for yeast recovery experiment.
The total length of the TpUBC9 gene cDNA is 608bp, the 3' UTR is 90bp long, and contains 28bp polyA tail; the ORF size is 516bp, codes 171 amino acids, and has a predicted molecular weight of about 19.59kDa and an isoelectric point of 8.25. BLAST alignment shows that TpUBC9 is a homologous sequence of UBCc gene superfamily, and the conserved structural domain is located between 43-480 nucleotides.
The invention also provides a group of primer pairs for cloning the gene TpUBC9, wherein the primer pairs comprise a forward primer TpUBC9F321 and a reverse primer TpUBC9R487, and the nucleotide sequence of the forward primer TpUBC9F321 is shown as SEQ ID NO. 3; the nucleotide sequence of the reverse primer TpUBC9R487 is shown as SEQ ID NO. 4. The primer pair of the invention is preferably based on the gene TpUBC9 obtained by screening by the method, and utilizes Oligo6.0 software to design 3 'and 5' specific primers in a gene conservation region.
The invention also provides a method for amplifying the gene TpUBC9, which comprises the following steps: (1) carrying out reverse transcription on the total RNA of the taenia pisiformis serving as a template to obtain a first cDNA chain;
(2) taking the first cDNA chain as a template and the primer pair as a primer to carry out rapid amplification of the cDNA end; the procedure for the amplification was: 30s at 94 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 70 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 68 ℃, 3min at 72 ℃ and 25 cycles;
(3) recovering the PCR band, connecting to pMD19-T vector and transforming E.coli DH5a competent cell; clones which are identified as positive by the PCR of the bacterial liquid and are sequenced correctly are spliced with 3 'and 5' amplification sequences to obtain a full-length cDNA sequence of the TpUBC9 gene.
The amplification of the invention is preferably performed by utilizing RACE-PCR technology for amplification splicing, firstly, the total RNA of taenia pisiformis is taken as a template, and the first chain of cDNA is obtained after reverse transcription. The invention does not specially limit the extraction of the total RNA of the taenia pisiformis, and the conventional RNA extraction method in the field can be used. The present invention preferably utilizes Clontech
The RACE cDNA Amplification kit performs the reverse transcription to obtain a first strand cDNA with a 3 'linker and a 5' linker.
Obtaining the first strand of cDNAThen, the first strand of cDNA is taken as a template, and the primer pair is taken as a primer to carry out rapid amplification of the cDNA end; the procedure for the amplification was: 30s at 94 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 70 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 68 ℃, 3min at 72 ℃ and 25 cycles. The present invention preferably utilizes Clontech
The Amplification was performed using the RACE cDNA Amplification kit, and the system of Amplification was 50. mu.L, and included: 2 XSeqAmp PCR buffer 25. mu.L, 10 XUPM 5. mu.L, 3'GSP/5' GSP (25. mu.M) 1. mu.L, template 2.5. mu.L, SeqAmp DNA Polymerase 1. mu.L, sterilized water 15.5. mu.L.
After obtaining a PCR target strip, recovering the PCR target strip by using the gel of the invention, connecting the PCR target strip to a pMD19-T vector and transforming E.coli DH5a competent cells; clones which are identified as positive by the PCR of the bacterial liquid and are sequenced correctly are spliced with 3 'and 5' amplification sequences to obtain a full-length cDNA sequence of the TpUBC9 gene. The method for recovering, connecting and converting the glue of the present invention is not particularly limited, and may be any method conventionally used in the art. The present invention preferably further comprises verifying the accuracy of the Open Reading Frame (ORF) sequence of the TpUBC9 gene after the amplification is completed. The invention preferably utilizes the primer pair shown in SEQ ID NO. 5-6 to carry out LD-PCR verification, and the specific method comprises the following steps: amplifying a target fragment by using primers shown in SEQ ID NO. 5-6, connecting a pMD19-T vector, transforming DH5a competent cells, carrying out positive clone sequencing, comparing the obtained sequence with a TpUBC9 full-length cDNA sequence, and determining a correct TpUBC9 sequence.
TABLE 1 TpUBC9 Gene amplification primers
The LD PCR system of the present invention is preferably a system comprising, in an amount of 50. mu.L: 5 XQ 5 Reaction buffer 10. mu.L, 2.5mM dNTP 4. mu.L, upstream and downstream primers 1. mu.L, 5' cDNA template 2. mu.L, Q5 DNA polymerase 1. mu.L, 5 XQ 5 high GC Enhancer 10. mu.L, and sterile water 21. mu.L. The reaction procedure of the LD PCR of the invention is preferably as follows: 30s at 98 ℃; 10s at 98 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; further extension was carried out at 72 ℃ for 2 min.
The invention also provides a recombinant expression vector pET28a-TpUBC9 containing the gene TpUBC9, and the gene TpUBC9 is cloned between enzyme cutting sites Nde I and Xho I of the expression vector by taking pET-28a (+) as an expression vector. The construction method of the recombinant expression vector pET28a-TpUBC9 is not particularly limited, and preferably, prokaryotic vector expression primers shown in SEQ ID NO. 7-8 are used for amplification (the restriction enzyme sites are underlined):
TpUBC9F：5ˊ-GGAATTCCATATGATGGGGGGAGTAATGGGTGATT-3ˊ(Nde I，SEQ ID NO.7)
TpUBC9R：5ˊ-CCGCTCGAGAGATAGATTTGGGTTACGAAAAAG-3ˊ(Xho I，SEQ ID NO.8)。
the invention also provides a recombinant bacterium containing the gene TpUBC9 or the recombinant expression vector pET28a-TpUBC 9. The construction method of the recombinant strain is not particularly limited.
The invention also provides a preparation method of the TpUBC9 immunomagnetic bead, which comprises the following steps: (a) transforming the recombinant expression vector pET28a-TpUBC9 into escherichia coli, inducing with IPTG, and purifying with Ni-affinity column to obtain His-TpUBC9 fusion protein;
(b) immunizing rabbits with the His-TpUBC9 fusion protein to obtain polyclonal antibody;
(c) and coating Dynabeads M-280Tosylactivated magnetic beads with the polyclonal antibody to obtain the TpUBC9 immunomagnetic beads.
The method for coating the magnetic beads in step (c) is not particularly limited in the present invention, and any conventional method in the art may be used.
The SUMO modification system gene TpUBC9 of Taenia pisifera and its use are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Construction of three-box yeast cDNA library of taenia pisiformis adults
1.1 extraction of Taenia pisiformis adult total RNA and purification of mRNA
Taking the taenia sojae adult segments of about 500mg, placing the taenia sojae adult segments into a precooled RNase free centrifugal tube, adding liquid nitrogen for quick freezing, immediately placing the tube into a liquid nitrogen grinder for full grinding, and setting parameters as follows: 50Hz, 120 s. After grinding is finished, taking out the centrifuge tube, adding Trizol (1mL/100mg) into the insect powder immediately, and mixing uniformly; extracting adult total RNA according to Trizol reagent instruction, and measuring the concentration and integrity of the total RNA by using Agilent Bioanalyzer 2100 system and a Qubit2.0 Fluorometer; immediately after quality qualification, mRNA was purified for library preparation according to Oligo d (T)25Magnetic Beads instructions.
1.2 construction of adult three-box Yeast cDNA library
Respectively synthesizing a first cDNA chain and a second cDNA chain according to the operation steps of a CloneMiner II cDNA library construction kit, and connecting the cDNA with a three-frame attB1 recombinant joint; separating and collecting cDNA; connecting the cDNA to a vector pDONR222 by using BP clone II enzyme Mix, and electrically transforming Escherichia coli DH10B to prepare a primary library bacterial liquid; after 10. mu.L of the transformed bacterial suspension was diluted 1000-fold, 50. mu.L of a LB-coated plate (containing 50. mu.g/mL kanamycin, kan) was taken out of the diluted bacterial suspension+) Counting the next day and calculating the library capacity; at the same time, 24 clones were randomly picked for colony PCR to identify library recombination rate and insert length. Ligating the qualified primary library plasmid to the AD library vector pGADT7-DEST with LR clone II Mix, and electro-transforming DH 10B; preparing a secondary library bacterial liquid. Identifying the library capacity, recombination rate and insert length as above; and obtaining the yeast double-hybrid cDNA library after qualification.
1.3 results
The results of the yeast secondary library identification indicated that the primary library plasmid was successfully recombined with pGADT7-DEST vector LR. The library titer was determined to be 675/50 μ L × 100 × 1000 μ L ═ 1.35 × 10 at a plate dilution of 1:1006(ii) a Total clone number (CFU) of the transformation plate was 1.35X 106×4＝5.4×106. Randomly selecting 24 clones, amplifying the inserts, with recombination rate up to 100%, and average length of cDNA inserts>1kb, which meets the requirements of a standard cDNA library.
Example 2
Bait vector BD-TpLAP construction and yeast two-hybrid screening
2.1 construction of BD-TpLAP
Based on the TpLAP complete ORF sequence (GenBank: MG366593) and pGBKT7 vector plasmid maps, the following primers (underlined as restriction sites) were designed and synthesized using Oligo6.0 software:
TpLAP-BDF：5'-GGAATTCCATATGATGGGGGACCAGGGCATCAATT TCG-3'(Nde I，SEQ ID NO.9)
TpLAP-BDR：5'-AACTGCAGTCAGAGACGAGGGAGGACGTACATGC-3'(Pst I，SEQ ID NO.10)
PCR reaction (50. mu.L): LATaq Mix 25. mu.L, 10. mu.M of each of TpLAP-BDF and TpLAP-BDR 2. mu.L, adult cDNA 2. mu.L, and sterilized water 19. mu.L.
And (3) PCR reaction conditions: 5min at 94 ℃, 1min at 94 ℃, 30s at 56 ℃, 30s at 72 ℃, 35 cycles, and 10min at 72 ℃. The gel recovery target band is subjected to Nde I and Pst I double enzyme digestion, recovered and connected to a pGBKT7-T vector, transformed into a DH5a competent cell and subjected to sequencing verification; the bait vector is named BD-TpLAP; in LB (kan)+) Increasing bacteria in culture medium to logarithmic phase, extracting bait plasmid and determining concentration, and storing at-20 deg.C for use.
2.2 preparation of Yeast competent cells
(1) Streaking the Y2HGold yeast on a YPDA plate, and carrying out inverted culture at 30 ℃ for 2-3 d;
(2) selecting yeast with the diameter of 2-3 mm, inoculating the yeast single clone into a 3mLYPDA culture medium, and performing shaking culture at 30 ℃ and 230r/min for 8 hours; sucking 5 μ L of bacterial liquid into 50mLYPDA, shaking at 30 deg.C and 230r/min for 16-20 hr to OD6000.15 to 0.3; centrifuging at room temperature of 700g for 5min, and removing supernatant;
(3) resuspending yeast with 100ml LYPDA; carrying out shaking culture at 30 ℃ and 230r/min for 3-5 h until the OD600 reaches 0.4-0.5; centrifuging at room temperature of 700g for 5min, and removing supernatant; resuspending yeast with 60mL sterile water; centrifuging at room temperature of 700g for 5min, and removing supernatant; resuspending yeast cells in 3mL of 1.1 XTE/LiAc solution;
(4) equally dividing the yeast suspension into 2 centrifugal tubes with the volume of 1.5 mL; centrifuging at 12000g for 15 s;
(5) the supernatant was discarded and the yeast was resuspended in 600. mu.L of 1.1 XTE/LiAc solution, the resuspension solution being the yeast competent cells.
2.3 BD-TpLAP plasmid self-activation and toxicity detection
(1) Heating Carrier DNA at 100 deg.C for 8min, and performing pre-denaturation for 2 times in ice bath for 3 min; placing on ice for later use;
(2) taking 50 mu L of Y2HGold yeast competent cells, adding 100ng BD-TpLAP plasmid, 20 mu L of pre-denatured Carrier DNA and 500 mu L of 1.1 xTE/LiAc/PEG 4000, and uniformly mixing; adding 100ng of PGBKT7-Lam and 200ng of PGADT7-T plasmid into the negative control group; 100ng of PGBKT7-p53 and 200ng of PGADT7-T plasmid are added into the positive control group; respectively adding 20 mu L of pre-denatured Carrier DNA and 500 mu L of 1.1 × TE/LiAc/PEG4000, and uniformly mixing;
(3) water bath at 30 deg.C for 1h, and mixing for 1 time every 10 min; adding 160 mu L DMSO into each tube, and gently inverting and mixing;
(4) heating in 42 deg.C water bath for 20min, and mixing 1 time every 10min by turning up and down gently; centrifuging at 700g for 5min, and discarding the supernatant; resuspending the cells in 3mL YPD Plus broth; placing in 30 deg.C shaking table for 90 min; centrifuging at 700g for 5 min; discarding the supernatant, and resuspending the cells with 30mL of 0.9% NaCl; washing the thalli for 1 time by deionized water; resuspending the cells with 0.9% NaCl;
(5) coating 100 mu L of thallus suspension on an SD auxotroph culture plate; wherein BD-TpLAP is plated on SDO/X (SD/-Trp/X-. alpha. -Gal) plates; negative control group (pGBKT7-Lam + pGADT7-T) and positive control group (pGBKT7-p53+ pGADT7-T) were plated on DDO/X (SD/-Trp/-Leu/X-. alpha. -Gal) plates; and (5) placing the culture box at 30 ℃ for inverted culture for 3-5 days, and observing the result.
2.4 Yeast two-hybrid Screen
(1) Co-transforming prey and bait plasmids by the steps of: adding 10 mu g of yeast library plasmid, 3 mu g of BD-TpLAP plasmid and 50 mu L of pre-denatured Carrier DNA into 8mL of Y2HGold yeast competent cells, and gently mixing the mixture; adding 60mL of 1 × TE/LiAc/PEG, and uniformly mixing by vortex; shaking and culturing at 30 deg.C and 220r/min for 45 min; adding 7mL of DMSO, and gently mixing; heating in 42 deg.C water bath for 20min, and slightly and uniformly mixing 1 time every 10 min; performing ice bath for 5 min; centrifuging at 700g for 5 min; adding 1000mLYPDA, and culturing at 30 deg.C under shaking for 60 min; centrifuging for 5min at 1000g, and collecting thallus; water bath at 30 deg.C for 1h, and mixing for 1 time every 10 min;
(2) adding 1 × TE suspended bacteria, taking bacteria liquid, coating the bacteria liquid on a QDO/X/A (SD/-Ade-His-Leu-Trp/X-alpha-Gal/AbA) plate, and keeping the bacteria liquid at 300 μ L/block; culturing in an electric heating constant-temperature incubator for 3-5 days; the growth state of the colonies was observed, and the colonies turned blue were positive.
2.5 Positive plasmid amplification and sequencing
(1) Selecting a blue yeast single colony to 5mL of QDO culture medium, carrying out shaking culture at 30 ℃ for 2d, centrifuging at 12000g for 1min, collecting thalli, and extracting yeast plasmids by using a small rhizopus yeast plasmid extraction kit;
(2) 5 mul of yeast plasmid was transformed into DH5a competent cells; after single colony enrichment is selected, plasmid is extracted by utilizing a plasmid miniextraction kit of Omega company, false positive clones are eliminated by sequencing, and Blast analyzes the sequence similarity of a positive plasmid sequence and known genes of various species in GenBank;
(3) selecting an interaction plasmid for reversion verification, and co-transforming the BD-TpLAP plasmid and the interaction plasmid into Y2HGold competent cells; the thallus is coated on a QDO/X/A plate; culturing at 30 ℃ for 3-5 days, and determining that the bacterial colony turns blue.
2.6 results
The bait plasmid BD-TpLAP grows white colonies on the SDO/X plate, the size is uniform, and blue clones do not appear, which shows that the BD-TpLAP has no toxicity and self-activation effect on yeast cells. Screening library and sequencing comparison analysis show that 4 TpUBC9 positive clones are obtained, and the reversion hybridization result is positive.
Example 3
Full-length cDNA clone of taenia pisiformis TpUBC9 gene
3.1 extraction of Total RNA and first strand cDNA Synthesis of Taenia pisiformis adults
Reference to Clontech
RACE cDNA Amplification kit instruction book, with 1.1 extraction of adult RNA as template, respectively synthesis of 3 'joint and 5' joint of cDNA first chain.
3.2 cloning of the full-Length cDNA of the TpUBC9 Gene
3 'was designed in a conserved region of the gene using Oligo6.0 software based on the sequencing results of 4 TpUBC9 positive plasmids'And 5' specific primers, and amplifying the full-length cDNA of TpUBC9 by respectively taking 3' cDNA and 5' cDNA of taenia pisiformis imagoes as templates; specific amplification procedures are referenced to Clontech
RACE cDNA Amplification kit instructions, primer information is shown in Table 1 above.
PCR reaction (50. mu.L): 2 XSeqAmp PCR buffer 25. mu.L, 10 XUPM 5. mu.L, 3'GSP/5' GSP (25. mu.M) 1. mu.L, 3'cDNA/5' cDNA 2.5. mu.L, SeqAmp DNA Polymerase 1. mu.L, sterile water 15.5. mu.L.
PCR amplification procedure: 30s at 94 ℃; 3min at 72 ℃ for 5 cycles; 30s at 94 ℃, 30s at 70 ℃, 3min at 72 ℃ and 5 cycles; 30s at 94 ℃, 30s at 68 ℃, 3min at 72 ℃ and 25 cycles.
Purifying the PCR target band by using a gel recovery kit, connecting the PCR target band to a pMD19-T vector and transforming E.coli DH5a competent cells; the monoclonal colonies were picked and the clones that were positive by PCR of the bacterial solution were sequenced by GenScript, Nanjing. The 3 'and 5' amplified sequences were spliced using DNAStar and SeqMan (version 5.0) software to obtain the full-length cDNA sequence of TpUBC9 gene. In order to further verify the accuracy of the TpUBC9 Open Reading Frame (ORF) sequence, the LD-PCR primers designed in Table 1 are used to amplify target fragments, and the target fragments are connected with a pMD19-T vector, DH5a competent cells are transformed, and after positive clone sequencing, the obtained sequence is compared with the TpUBC9 full-length cDNA sequence, so that the correct TpUBC9 sequence is determined.
Ligation system (10 μ L): solution I5. mu.L, PCR-recovered fragment 4.5. mu.L, pMD19-T vector 0.5. mu.L.
LD PCR System (50. mu.L): 5 XQ 5 Reaction buffer 10. mu.L, 2.5mM dNTP 4. mu.L, upstream and downstream primers 1. mu.L, 5' cDNA template 2. mu.L, Q5 DNA polymerase 1. mu.L, 5 XQ 5 high GC Enhancer 10. mu.L, and sterile water 21. mu.L.
LD PCR reaction procedure: 30s at 98 ℃, 10s at 98 ℃, 30s at 56 ℃, 30s at 72 ℃ and 35 cycles; further extension was carried out at 72 ℃ for 2 min.
3.3 results
Sequencing shows that the total length of the TpUBC9 gene cDNA is 608bp, the 3' UTR is 90bp long, and contains 28bp polyA tail; the ORF size is 516bp, codes 171 amino acids, and has a predicted molecular weight of about 19.59kDa and an isoelectric point of 8.25. BLAST alignment shows that TpUBC9 is a homologous sequence of UBCc gene superfamily, and the conserved structural domain is located between 43-480 nucleotides.
Example 4
Preparation of His-TpUBC9 recombinant protein
4.1 design and Synthesis of expression primers
Based on the Taenia pisiformis TpUBC9 gene ORF sequence (SEQ NO.1) obtained in example 1, the Oligo6.0 software was used to design the TpUBC9 prokaryotic vector expression primers (the restriction sites are underlined) as follows:
TpUBC9F：5ˊ-GGAATTCCATATGATGGGGGGAGTAATGGGTGATT-3ˊ(Nde I，SEQ ID NO.7)
TpUBC9R：5ˊ-CCGCTCGAGAGATAGATTTGGGTTACGAAAAAG-3ˊ(Xho I，SEQ ID NO.8)
4.2 construction of expression vector pET-28a (+) -TpUBC9
PCR amplification of TpUBC9 ORF is carried out by taking positive plasmid pMD19T-TpUBC9 as a template; the recovered product of the gel and the pET-28a plasmid were digested with Nde I/Xho I, respectively, and the recovered product was ligated to Escherichia coli Transetta (DE3) competent cells. Screening positive clones, extracting plasmids in small quantity by an alkaline lysis method, and generating 516bp DNA fragments which are consistent with the size of target fragments after PCR amplification and restriction enzyme Nde I/Xho I double digestion identification; after sequencing verification, the positive plasmid was designated as pET-28a (+) -TpUBC 9.
4.3 inducible expression of recombinant protein of Taenia pisiformis TpUBC9
The recombinant pET-28a (+) -TpUBC9/Transetta (DE3) was inoculated into LB medium (100. mu.g/mL, Kan+) And (4) carrying out enrichment culture until the logarithmic phase is reached, and adding 0.5mM IPTG to induce and express for 24h at 20 ℃. After the induced bacteria are repeatedly frozen and thawed and ultrasonically broken, the supernatant is collected, and the target protein is purified according to the operation of the Ni-NTA specification.
4.4 results
SDS-PAGE electrophoretic analysis shows that after the recombinant bacteria are induced, a soluble expressed His-TpUBC9 fusion protein is obtained, the size of a band is about 23kDa and is basically consistent with the size of the presumed protein; the induced empty vector did not show the band of interest. The induced protein is purified by Ni affinity chromatography, and the recombinant protein with higher purity is eluted under the concentration of 200-300 mM imidazole (figure 1).
Example 5
Preparation of polyclonal Rabbit anti-TpUBC 9 antibody
5.1 animal immunization
Diluting the purified His-TpUBC9 protein to 1.0mg/mL with PBS (pH7.2), mixing with Freund's complete adjuvant in equal volume, and emulsifying; inoculating rabbit, immunizing for the first time at 100 mug/rabbit, and performing subcutaneous multi-point injection; mixing the antigen II and Freund's incomplete adjuvant in equal volume, emulsifying, and inoculating 200 μ g/vaccine; then boost 2 more times, immunization interval 21 d. Rabbit serum was collected 10d after the last immunization.
5.2 purification and characterization of polyclonal antibodies
The antibody purification adopts a saturated ammonium sulfate precipitation method and a protein A affinity chromatography for purification step by step, and comprises the following specific steps: taking 5mL of immune serum with highest titer, diluting the immune serum to 10mL by PBS, dropwise adding 10mL of saturated ammonium sulfate (pH7.2) on a magnetic stirrer, uniformly stirring, and standing at 4 ℃ for precipitation overnight; centrifuging at 4 deg.C and 10000g for 30min, and discarding supernatant; re-dissolving the precipitate in 10mL PBS, and precipitating with 33% saturated ammonium sulfate for 1 time in the same way; the precipitate was then reconstituted in 3mL PBS and the antibody was further purified by reference to GenScript Protein A affinity chromatography procedure; measuring the concentration of the antibody by spectrophotometry; the indirect ELISA and Westernblot methods are adopted to detect the serum titer and reactivity.
5.3 results
The ELISA can detect the rabbit hyperimmune serum titer to 1:5.12 multiplied by 105(Table 2); western blot detection shows that a specific positive reaction band appears at about 23kDa, and serum does not react before immunization. The result shows that the prepared His-TpUBC9 fusion protein has high purity; the anti-His-TpUBC 9 polyclonal antibody has high titer, good immunoreactivity and specificity (figure 1).
TABLE 2 anti-His-TpUBC 9 polyclonal antibody ELISA titer detection results
Example 6
Preparation of TpUBC9 antibody immunomagnetic beads
6.1 magnetic bead coating
Taking 10mg Dynabeads M-280Tosylactivated magnetic beads, suspending the magnetic beads by using 0.1M boric acid solution (pH 9.5), placing the magnetic beads on a magnetic frame for 3min, and discarding the supernatant; repeatedly washing for 3 times; mu.L rabbit anti-TpUBC 9 polyclonal antibody (0.5mg/mL) and 200. mu.L coupling buffer (0.1M boric acid, with a final concentration of 1.2M (NH) were added to the beads4)2SO4pH 9.5), and mixing; slowly rotating at 37 ℃ for 20h, collecting magnetic beads by a magnetic frame, and reserving supernatant; the content of the residual antibody in the supernatant was measured by spectrophotometry, and the antibody coupling rate [ antibody coupling rate (%) ═ content of antibody bound to magnetic beads (. mu.g)/total amount of antibody added (. mu.g) was calculated]. Adding washing solution 1(0.01M PBS containing 0.15M NaCl and 0.5% BSA, pH 7.4) into the magnetic beads, placing at 37 ℃ for slow rotation and combination for 1h, and collecting the magnetic beads by a magnetic frame; washing the immunomagnetic beads with washing solution 2(0.01M PBS containing 0.15M NaCl and 0.1% BSA, pH 7.4) repeatedly for 3 times; storing at 4 deg.C for use.
6.2 Immunomagnetic bead purification of His-TpUBC9 protein Effect analysis
Mixing 0.5mL of the ultrasonication supernatant containing TpUBC9 in 4.3 with 1mg of immunomagnetic beads, incubating at 37 deg.C for 1h, placing on a magnetic frame for 2min, and discarding the supernatant; washing the magnetic beads 5 times with washing buffer 2; the MPCs collected the magnetic beads, added 1 Xprotein loading buffer 50. mu.L, analyzed the proteins by 12% SDS-PAGE and identified with Anti-His-HRP as a Westernblot.
6.3 results
The TpUBC9 antibody immunomagnetic beads are successfully prepared, the antibody is completely combined, and the coupling rate reaches 100%; the soluble His-TpUBC9 protein is obtained by separating from 1mL of inducing bacteria liquid supernatant by using immunomagnetic beads (figure 2), the operation is convenient, and the protein purification effect is good.
Example 7
Validation of TpUBC9-TpLAP interaction
7.1 plasmid construction
According to the ORF sequences of TpLAP (GenBank: MG366593) and TpUBC9 genes (SEQ NO.1), respectively constructing AD-TpUBC9, Flag-TpUBC9 and Myc-TpLAP vectors; oligo6.0 software designed and synthesized primers (Table 3, underlined cleavage sites). Amplifying a target band with a restriction enzyme site by a conventional PCR method, and carrying out double restriction, recovery and purification, ligation and transformation and sequencing verification. Completing the construction of prey vectors and eukaryotic label expression vectors; plasmids were extracted and the concentration was measured by Nanodrop 2000, respectively, according to the instructions of the small endotoxin-free plasmid extraction kit of Omega.
TABLE 3 vector construction primers
7.2 one-to-one verification of the TpUBC9-TpLAP interaction by Yeast
AD-TpUBC9 self-activation and toxicity testing were performed according to the protocol 2.3. Performing one-to-one verification of AD-TpUBC9 and BD-TpLAP yeast by referring to the operation method of 2.4, and co-transforming 100ng of BD-TpLAP and 200ng of AD-TpUBC9 plasmid into competent cells of Y2HGold yeast; the cell suspension is coated on DDO and QDO/X/A plates; culturing in an incubator at 30 ℃ for 3-5 days, observing the growth condition of colonies, and obtaining positive blue spots on a QDO/X/A plate.
7.3 immunomagnetic bead validation of TpUBC9-TpLAP interaction
(1) HEK293T cell culture and transfection conventional culture HEK293T cell, when the cell growth density reaches 70% and the form is good, DMEM complete culture solution (containing 10% fetal calf serum) is replaced; according to XfectTMInstructions for Transfection of Transfection Reagent, co-Transfection of Myc-TpLAP and Flag-TpUBC9 plasmids into HEK293T cells; HEK293T cells not transfected with the plasmid were also used as negative controls. The specific transfection steps are as follows: taking 10 mu g of Myc-TpLAP and Flag-TpUBC9 plasmids respectively, adding the Myc-TpLAP and Flag-TpUBC9 plasmids into an Xfect Reaction Buffer (the final volume is 300 mu L), and fully mixing; then adding 6.5 mu L of Polymer, fully and uniformly mixing, and incubating for 10min at room temperature to form a nano compound; dropping 300 μ L of the compound into the culture solution of the cell flask after instantaneous centrifugation, mixing gently, and adding CO2Culturing overnight in a constant temperature incubator; the next day, fresh DMEM complete culture solution is replaced, and the culture is continued for 48 h.
(2) Co-immunoprecipitation (CoIP) was performed to collect cells, add cell lysate (100. mu.L cell lysate + 10. mu.L LPMSF), shake the flask for 30min, and centrifuge at 12000g for 20min at 4 ℃; adding 1mg of immunomagnetic beads into 500 mu L of cell lysis supernatant, and performing rotary incubation at 4 ℃ overnight; placing on a magnetic frame for 2min, and discarding the supernatant; washing with pre-cooled PBS for 3 times; adding 50 mu L of 1 Xprotein loading buffer solution into the magnetic beads, boiling for 10min, centrifuging, and taking the supernatant for electrophoresis; performing 12% SDS-PAGE electrophoresis on untransfected cell whole protein lysate, cotransfected cell whole protein lysate (Input) and an IP sample, and transferring an eBlot system to a PVDF membrane; reacting with mouse anti-Myc antibody and rabbit anti-Flag antibody respectively, and standing overnight at 4 deg.C; rinsing with TBST for 3 times; then respectively incubating the two antibodies with corresponding HRP labeled secondary antibodies at 37 ℃ for 1 h; rinsing with TBST for 3 times and 5 min/time; and adding ECL substrate luminescent solution to avoid light for color development, and observing the result.
7.3 results
The reporter gene in the Y2HGold can not be activated under the condition that two full-length genes carried by BD-TpLAP and AD-TpUBC9 exist independently, and the self-activating activity is avoided, the colony growth state is good, and no toxicity is caused. One-to-one analysis of yeast showed: after co-transformation, blue spots were grown on QDO/X/A plates, confirming the interaction between the two (FIG. 3). The CoIP results show that Myc-TpLAP and Flag-TpUBC9 can form an interactive protein complex in cells, and TpUBC9 can directly recognize TpLAP target protein molecules (fig. 4); the prepared TpUBC9 antibody immunomagnetic beads can be used for analyzing and identifying TpUBC9 direct interaction protein.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> taenia pisiformis SUMO modification system gene TpUBC9 and application thereof
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gaagaaagga aggcatggcg tagggaccat ccctttggat tcgttgctaa acccacaaaa 120
aatgccgatg gcactctcga cctcatgacc tgggattgta gtattcctgg taaaaaagga 180
actctttggg agggtgggct ttttcatctt cgtatgtact ttaagcctga ataccctaca 240
actccgccta aatgcaaatt cgaaccgcct ctgttccacc cgaacatttt tccttcaggg 300
acggtttgtc tttcgcttct agatgaggaa aaacattggc gccccgctgt taccattaag 360
cagatccttc tggggattca agatctttta gatcacccta accccaagga tccggcgcag 420
gcggatgcat atacattgtt cattcagaat cggaaggact atgactacag aataaagaaa 480
caagctgaac tttttcgtaa cccaaatcta tcttaa 516
<210> 2
<211> 171
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<213> Taenia pisiformis
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Met Gly Gly Val Met Gly Asp Phe Ser Glu Ala Asp Gly Ile Ala Leu
1 5 10 15
Lys Arg Leu Ala Glu Glu Arg Lys Ala Trp Arg Arg Asp His Pro Phe
20 25 30
Gly Phe Val Ala Lys Pro Thr Lys Asn Ala Asp Gly Thr Leu Asp Leu
35 40 45
Met Thr Trp Asp Cys Ser Ile Pro Gly Lys Lys Gly Thr Leu Trp Glu
50 55 60
Gly Gly Leu Phe His Leu Arg Met Tyr Phe Lys Pro Glu Tyr Pro Thr
65 70 75 80
Thr Pro Pro Lys Cys Lys Phe Glu Pro Pro Leu Phe His Pro Asn Ile
85 90 95
Phe Pro Ser Gly Thr Val Cys Leu Ser Leu Leu Asp Glu Glu Lys His
100 105 110
Trp Arg Pro Ala Val Thr Ile Lys Gln Ile Leu Leu Gly Ile Gln Asp
115 120 125
Leu Leu Asp His Pro Asn Pro Lys Asp Pro Ala Gln Ala Asp Ala Tyr
130 135 140
Thr Leu Phe Ile Gln Asn Arg Lys Asp Tyr Asp Tyr Arg Ile Lys Lys
145 150 155 160
Gln Ala Glu Leu Phe Arg Asn Pro Asn Leu Ser
165 170
<210> 3
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<400> 3
gcctgaatac cctacaactc cgcct 25
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
cggatccttg gggttagggt gatc 24
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acatgggggg agtaatgggt gat 23
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttggggacga atgcacatat tgg 23
<210> 7
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ggaattccat atgatggggg gagtaatggg tgatt 35
<210> 8
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ccgctcgaga gatagatttg ggttacgaaa aag 33
<210> 9
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ggaattccat atgatggggg accagggcat caatttcg 38
<210> 10
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
aactgcagtc agagacgagg gaggacgtac atgc 34
<210> 11
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ccggaattca tggggggagt aatgggtg 28
<210> 12
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cacgagctct taagatagat ttgggttacg 30
<210> 13
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gcccaagctt atggggggag taatgggt 28
<210> 14
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccggaattct taagatagat ttgggttacg 30
<210> 15
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cggaattcaa atgggggacc agggcatc 28
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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acgcgtcgac tcagagacga gggaggacg 29
Claims (1)
1. A preparation method of TpUBC9 immunomagnetic beads comprises the following steps: (a) transforming the recombinant expression vector pET28a-TpUBC9 into escherichia coli, inducing with IPTG, and purifying with Ni-affinity column to obtain His-TpUBC9 fusion protein; the recombinant expression vector pET28a-TpUBC9 takes pET-28a (+) as an expression vector, and clones the taenia pisifera SUMO modification system gene TpUBC9 between enzyme cutting sites Nde I and Xho I of the expression vector; the nucleotide sequence of the gene TpUBC9 is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene TpUBC9 is shown in SEQ ID NO. 2;
(b) immunizing rabbits with the His-TpUBC9 fusion protein to obtain polyclonal antibody;
(c) and coating Dynabeads M-280Tosylactivated magnetic beads with the polyclonal antibody to obtain the TpUBC9 immunomagnetic beads.
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