阅读说明：本技术 一种豆状带绦虫SUMO化修饰系统基因TpUBC9及其用途 (Sumo modification system gene TpUBC9 of taenia pisiformis and application thereof ) 是由 张少华 才学鹏 骆学农 郭爱疆 王帅 侯俊玲 梁盼红 于 2019-09-30 设计创作，主要内容包括：本发明提供了一种豆状带绦虫SUMO化修饰系统基因TpUBC9及其用途,涉及基因工程技术领域。本发明所述基因TpUBC9的核苷酸序列如SEQ ID NO.1所示；所述基因TpUBC9编码蛋白质的氨基酸序列如SEQ ID NO.2所示。本发明通过酵母双杂交技术首次筛选到豆状带绦虫亮氨酸氨基肽酶TpLAP的互作蛋白基因TpUBC9,并克隆了TpUBC9基因的全长cDNA,并验证了TpUBC9-TpLAP相互作用,证实带绦虫虫体内也存在精细的SUMO化修饰系统；为进行后续带绦虫SUMO化修饰相关研究提供了新的线索。将其制备成TpUBC9抗体免疫磁珠,具有特异性好,亲和性高,操作便捷,结果可靠等特点。(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.)

1. A taenia pisiformis SUMO modification system gene TpUBC9 is characterized in that 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.

2. A primer pair for cloning the gene TpUBC9 of claim 1, wherein the primer pair comprises 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.

3. A method for amplifying the TpUBC9 gene of claim 1, comprising the steps of: (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 of claim 2 as a primer to carry out rapid amplification of the cDNA end to obtain a PCR target gene; 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.

4. The method of claim 3, wherein Clontech is used in step (1)RACE cDNA Amplification kit was used for the reverse transcription.

5. A recombinant expression vector pET28a-TpUBC9 comprising the gene TpUBC9 of claim 1, wherein the gene TpUBC9 is cloned between cleavage sites Nde I and XhoI of the expression vector using pET-28a (+) as an expression vector.

6. A recombinant bacterium comprising the gene TpUBC9 of claim 1 or the recombinant expression vector pET28a-TpUBC9 of claim 5.

7. 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;

(b) immunizing rabbits with the His-TpUBC9 fusion protein to obtain polyclonal antibody;

(c) and coating Dynabeads M-280 Tosylactivated magnetic beads with the polyclonal antibody to obtain the TpUBC9 immunomagnetic beads.

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-280 Tosylactivated 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.

Drawings

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 ClontechThe RACE cDNA Amplification kit performs the reverse transcription to obtain a first strand cDNA with a 3 'linker and a 5' linker.

After obtaining a first cDNA chain, carrying out rapid amplification on the terminal of the cDNA by taking the first cDNA chain as a template and the primer pair as a primer; 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 ClontechThe 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-280 Tosylactivated 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.