Preparation method of biotinylation modified PTBP1 structural domain RRM3 / 4

Abstract

The invention relates to the technical field of gene engineering, in particular to a preparation method of biotinylation modified PTBP1 structural domain RRM3 / 4. According to the invention, after codon optimization is carried out on the RRM3 / 4 structural domain of PTBP1, efficient expression is carried out by using escherichia coli, and the active PTBP1 structural domain RRM3 / 4 is successfully obtained; during the expression, the synchronous biotinylation is realized by utilizing a specially modified expression host bacterium AVB101 (comprising a low-induction biotin ligase BirA gene); the expression quantity of the recombinant protein in each milliliter of bacterial liquid can reach more than 80 micrograms, so that the cost is saved, and the preparation efficiency is improved. The biotinylated PTBP1 structural domain RRM3 / 4 can be used for screening of PTBP1 inhibitors, development of drugs for treating various neurodegenerative diseases and nerve injuries, detection of kinetic parameters of interaction between the drugs and PTBP1, and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for preparing biotinylated modified PTBP1 domain RRM3 / 4. Background Art

[0002] At present, many neurological diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), still lack effective and safe therapeutic drugs. In the occurrence and development of neurological diseases, a key and common link is irreversible neuronal loss, which causes movement disorders, sensory loss, psychiatric sequelae and other symptoms. For example, a decrease in the number of neurons can be observed in different brain regions of AD patients, and the loss of neurons in the CA1 region of the hippocampus and the entorhinal cortex is positively correlated with the severity of memory deficits. Therefore, finding scientific means to supplement neurons, integrate neural networks and restore functions is of great significance to the clinical improvement of neurodegenerative diseases.

[0003] Glial cells and neurons are located close to each other and have the same lineage. Therefore, "glial cell-neuron" transdifferentiation has become a potential effective means to reverse neuronal loss due to its advantages of being in situ and direct. If the transdifferentiation of glial cells into neurons can be successfully achieved and integrated into the neural network, neuronal loss in the course of central nervous system diseases may be reversed, nervous system function may be improved, and new hope may be created for the clinical treatment of nervous system diseases.

[0004] PTBP1 is an RNA binding protein whose main function is to act as a splicing factor. PTBP1 selectively splices exons or introns to produce different mRNA isoforms by binding to specific sequences of target gene precursor mRNA, thereby playing an important regulatory role in multiple biological processes such as cell differentiation, apoptosis, cell movement, and cell metabolism. The PTBP1 gene is located in the 19p13.3 region of the genome. The coding region contains 16 exons and encodes a protein containing 531 amino acids. The PTBP1 protein structure contains 4 RNA recognition motifs (RRMs), 1 nucleocytoplasmic shuttling domain, and 1 nuclear export signal. These basic domains are highly conserved.

[0005] PTBP1 plays an important role in neuronal differentiation and is a "molecular switch" for a series of neuron-specific alternative splicing events. Knockout of PTBP1 can achieve transdifferentiation of glial cells into neuron-like cells; on the contrary, exogenous overexpression of PTBP1 can inhibit neuronal differentiation. For example, during the differentiation of human embryonic stem cells (ESC) into neuronal cells, PTBP1 promotes the skipping of exon 7 of the PBX1 gene in ESCs, increases the expression of the PBX1a isoform, and thus inhibits the expression of genes related to neuronal differentiation. Analysis of splicing events in neural progenitor cells (NPCs) purified from the developing human cerebral cortex found that PTBP1 maintains the morphology of some apical progenitor cells by splicing the cytotoxic exons of FLNA in NPCs; further studies also found that the occurrence of brain malformations is related to PTBP1 regulating the skipping of toxic exons of FLNA in NPCs. During neuronal differentiation, PTBP1 is involved in regulating the development of synapses. PTBP1 cooperates with PTBP2 to inhibit the splicing of exon 18 of PSD-95 pre-mRNA, resulting in the degradation of nonsense mRNA, downregulating the expression of PSD-95, inhibiting the maturation of synapses, and thus affecting the development of early nerves. PTBP1 is also involved in the regulation of brain development. For example, PTBP1 can interact with the evolutionarily conserved neural-specific lncRNA-Pnky, mediate the splicing of pre-mRNAs of genes such as Ntsr2, Igfbp5, Scrg1, and Ppp1r3c, produce subtypes that are beneficial to cell survival, reduce cell death, and inhibit the proliferation of neuronal progenitor cells and the differentiation of neural stem cells, thereby affecting the development of the brain.

[0006] RRM3 and RRM4 of PTBP1 interact extensively and have a fixed orientation with respect to each other. More than 20 amino acid side chains located in helices 1 and 2 of RRM3, helix 2 of RRM4, and the interdomain linker form a hydrophobic core between the two domains. Due to this orientation, RRM3 / 4 can bind to two pyrimidine tracts within the same RNA, causing the RNAs they bind to to be antiparallel. RNAs with more than 15 nucleotides between two (CU)3 hexamers have a higher affinity for PTBP1, which causes RRM3 / 4 of PTBP1 to have different properties compared to other proteins with two consecutive RRMs. Therefore, when PTBP1 binds to two separate pyrimidine tracts within the same RNA, RRM3 / 4 can bring the two pyrimidine tracts that are far apart closer together. The structural models of RRM3 / 4 highlight the key role of RRM4 in splicing repression, because the removal of RRM4 prevents RNA from forming loops around branch points or alternative exons, and once the RRM4 domain is inhibited, the function of the entire PTBP1 is inhibited accordingly.

[0007] At present, the relationship between PTBP1 domain RRM3 / 4 and diseases is still in the research stage, and its specific pathogenic mechanism has not been fully clarified. Further research will help to gain a deeper understanding of the occurrence and development mechanism of these diseases and provide a basis for the development of new diagnostic methods and treatment strategies. Biotinylated PTBP1 domain RRM3 / 4 can be used for the screening of PTBP1 inhibitors and the detection of kinetic parameters between inhibitors and PTBP1. However, the commonly used method for biotin modification of proteins is to first express the target protein with Escherichia coli, purify the recombinant protein, and then use a kit to biotinylate the recombinant protein, and finally use a desalting column to remove excess biotin. These methods have the disadvantages of complex processes, expensive kits, and random biotinylation sites. There is no data reported on the simultaneous expression of PTBP1 or its domains to achieve biotinylation. Summary of the invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing biotinylated PTBP1 domain RRM3 / 4.

[0009] The nucleic acid encoding the PTBP1 domain RRM3 / 4 provided by the present invention has:

[0010] I), the nucleic acid sequence shown in SEQ ID NO: 1 to 4;

[0011] II), a nucleic acid sequence encoding the PTBP1 domain RRM3 / 4 by substitution, deletion, addition and / or replacement of one or more bases in the nucleic acid sequences shown in SEQ ID NOs: 1 to 4;

[0012] III), a nucleic acid sequence having at least 80% identity with the nucleic acid sequence described in I) or II).

[0013] The present invention searches the NCBI database to obtain the annotations of the mRNA sequence and protein sequence of human PTBP1, and selects the RRM3 / 4 domain containing 200 amino acids at the C-terminus of PTBP1 for prokaryotic expression. The coding sequence of the RRM3 / 4 domain of PTBP1 is codon-optimized, and an Avi tag is added to its N-terminus. After optimization, multiple codons with fitness of almost 0 in Escherichia coli are optimized, and the distribution of GC content in the entire gene is more uniform. In a preliminary experiment, multiple nucleic acids encoding the PTBP1 domain RRM3 / 4 are optimized. Compared with the wild type, the nucleic acid of the sequence shown in SEQ ID NO:1 can significantly increase the expression amount of the protein and more effectively achieve biotinylation. Compared with the effect of expressing other optimized sequences, the nucleic acid of the sequence shown in SEQ ID NO:1 has a more significant effect on increasing the yield of the PTBP1 domain RRM3 / 4.

[0014] In the present invention, the at least 80% identity includes: at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity or at least 99.9% identity.

[0015] The present invention also provides an expression unit, which comprises a nucleic acid and a promoter as described above. Preferably, the promoter in the expression unit of the present invention is a prokaryotic promoter, for example, the promoter is a T5 promoter, a trp promoter, a lac promoter, a tac promoter, a PL promoter, a PR promoter, an araBAD promoter, a rhaPBAD promoter, a T7 promoter, a trc promoter or a pET promoter.

[0016] Furthermore, the expression unit also includes an Avi tag and / or a 6×His tag.

[0017] The Avi tag may be located at the 5' end or the 3' end of the encoding nucleic acid. In the embodiment of the present invention, the Avi tag is located at the 5' end of the encoding nucleic acid of the PTBP1 domain RRM3 / 4.

[0018] The 6×His tag may be located at the 5′ end or the 3′ end of the encoding nucleic acid. In an embodiment of the present invention, the 6×His tag is located at the 5′ end of the encoding nucleic acid of the PTBP1 domain RRM3 / 4.

[0019] Furthermore, the expression unit further comprises a Lac operator, an RBS site and / or a terminator. For example, the terminator is selected from Lambda t0 terminator, T7 bacteriophage terminator, SV40 terminator, CMV terminator, rrnB terminator, bGH terminator, hGH terminator, or rbGlob terminator.

[0020] In some specific embodiments, from the 5' end to the 3' end, a promoter, a 6×His tag, an Avi tag, and the aforementioned nucleic acid and terminator are sequentially connected, wherein the nucleic acid sequence encoding the Avi tag is GGCCTGAACGATATTTTTGAAGCGCAGAAAATTGAATGGCATGAA.

[0021] Furthermore, the present invention also provides a plasmid vector, which comprises a backbone vector and at least one of the following i) to ii):

[0022] i), a nucleic acid as described above;

[0023] ii) An expression unit as described above.

[0024] In the present invention, the plasmid vector is used for the storage, amplification or transcriptional expression of the nucleic acid or expression unit as described above, so it can be an amplification vector or an expression vector. For example, the amplification vector is selected from the pUC series vector or the pET series vector. The expression vector is selected from the pQE series vector.

[0025] As amplification vectors, the backbone vectors are pUC-SP, pUC18, pUC19, pUC9, pUC118, pUC119, pUC138, pUC139, pUC188, pUC189, pUC198, pUC199, pUC57, pUC86, pUC87, pUC103, pET50b(+), pET48b(+), pET21b(+), pET26b(+), pET32a(+), pET14b, pET20b(+), pET21a(+), pET27b(+), pET43.1c(+ ), pET44c(+), pET49b(+), pET32a(+), pET16bpET-21d(+), pET24a(+), pET28a(+), pET48b(+), pET21b(+), pET15b, pET21c(+), pET24d(+), pET30a(+), pET43.1a(+), pET37b(+), pET47b(+), pET22b(+), pET19b, pET21b(+), pET25b(+), pET42a(+) or pET44a(+).

[0026] As the expression vector, the backbone vector is pQE80L, pQE82L, pQE81L, pQE16, pQE9, pQE32, pQE60, pQE70, pQE40, pQE30 or pQE31.

[0027] In the present invention, expression vectors were screened. Compared with other vectors, the vector using pQE80L as protein expression vector can obtain higher expression efficiency, thereby obtaining a larger amount of biotinylated-modified PTBP1 domain RRM3 / 4.

[0028] Furthermore, the present invention also provides a host cell, which is transformed or transfected with the plasmid vector as described above, or has the nucleic acid as described above integrated into its genome, or has the expression unit as described above integrated into its genome.

[0029] In the present invention, the host cell is used for amplification of the plasmid or expression of the gene. In the present invention, the host is Escherichia coli. For amplification of the plasmid, the Escherichia coli is TOP10, DH5α, JM109, JM110, XL10-Gold, HB101 or Xl1-Blue. For expression of the protein as described above, the host is an Escherichia coli containing the biotin ligase BirA gene, for example, it can be AVB101, AVB99 or AVB100.

[0030] The present invention connects the synthesized PTBP1 domain RRM3 / 4 gene sequence between BamH I and Hind III of the pQE80L vector after double enzyme digestion, and the vector adds a 6-His tag at the N-terminus. The recombinant plasmid pQE80L-6His-Avi-PTBP1 is finally transformed into the expression host bacteria AVB101 (including the low-induction biotin ligase BirA gene). The present invention screens Escherichia coli containing biotin ligase genes, and finally determines that Escherichia coli AVB101 can most effectively connect biotin to obtain biotinylated PTBP1 domain RRM3 / 4. Some other strains have very low expression levels, and some cannot successfully connect biotin.

[0031] Furthermore, the present invention also provides a method for preparing biotinylated PTBP1 domain RRM3 / 4, which comprises culturing the host as described above in the presence of biotin to obtain a culture containing biotinylated PTBP1 domain RRM3 / 4.

[0032] In the method provided by the present invention, the recombinant protein is biotinylated while being expressed in Escherichia coli, and there is no need to purchase additional reagents and consumables such as biotinylation enzyme and desalting column, thus saving costs; there is no need to biotinylate the expressed protein again and remove the biotin through a column, thus saving a lot of time.

[0033] In the present invention, before the culture, the strain is activated, and the activated medium is an LB medium containing Amp and Chl. The activation package is performed three times, in order: activation in LB solid medium, activation in LB liquid medium, and activation again in LB liquid medium.

[0034] In the present invention, the culture medium is an LB liquid culture medium containing 100 mg / L Amp, 20 mg / L Chl, 0.2-1 mM IPTG and 50 μM biotin.

[0035] The culture comprises: adding IPTG and biotin to the activated bacterial solution, inducing culture at 37° C. and 150 rpm for 3 to 7 hours, and obtaining a culture containing biotinylated modified PTBP1 domain RRM3 / 4.

[0036] In the present invention, the step of separation and purification is also included after the culture, specifically: collecting the fermented bacteria, washing them, freezing and thawing them, ultrasonically disrupting them, and then collecting the eluate through a nickel column to obtain a solution of biotinylated PTBP1 domain RRM3 / 4.

[0037] The condition for collecting the fermented bacteria is centrifugation at 12000g for 2 minutes, and removing the culture medium. The cleaning reagent is a non-denaturing buffer, specifically 50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0. The number of times of cleaning is 2 times.

[0038] The ultrasonic crushing was performed twice, and the conditions for each crushing were as follows: The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0039] After the ultrasonic treatment, the mixture was centrifuged at 4°C and 12,000 g for 10 min, and the supernatant was collected and passed through a nickel column.

[0040] The equilibrium solution of the nickel column is Binding Buffer (BIB: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0).

[0041] The cleaning solution of the nickel column is Washing Buffer (WAB: 50 mM NaH2PO4, 300 mM NaCl, 60 mM imidazole, pH 8.0).

[0042] The eluent of the nickel column is Elution Buffer (ELB: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0).

[0043] The host of the present invention adds biotin while inducing with IPTG, and under the action of the co-expressed biotinylating enzyme BirA, the lysine K on the Avi tag is automatically biotinylated. Biotin -PTBP1 was purified using Ni-NTA6FF Sefinose prepacked columns (Shanghai Biotech). The purified 6His-Avi Biotin After the concentration of -PTBP1 was adjusted, it can be calculated that more than 75 μg of high-purity recombinant protein can be produced per milliliter of bacterial solution, with a purity greater than 99%. The biotinylation efficiency was verified by Western blot hybridization experiments using horseradish peroxidase-labeled Streptavidin as a secondary antibody and immunoprecipitation experiments using streptavidin magnetic beads, and the biotinylation rate reached 72.4%.

[0044] Furthermore, the present invention also provides a product obtained by the preparation method as described above.

[0045] In the present invention, the product includes biotinylated PTBP1 domain RRM3 / 4

[0046] After the codons of the RRM3 / 4 domain of PTBP1 are optimized, the active PTBP1 domain RRM3 / 4 is successfully obtained by using Escherichia coli for efficient expression; while expressing, the specially modified expression host bacteria AVB101 (including the low-induction biotin ligase BirA gene) is used to achieve synchronous biotinylation, saving costs and improving the efficiency of preparation. The biotinylated PTBP1 domain RRM3 / 4 can be used for the screening of PTBP1 inhibitors, as well as the detection of kinetic parameters of inhibitors and PTBP1. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Construction of recombinant vector of PTBP1 domain RRM3 / 4. A: The extracted pUC-SP-PTBP1 plasmid was detected by 1% Agar electrophoresis; B: The PCR amplification product of PTBP1 was recovered and detected by 1% Agar electrophoresis; C: The PCR recovery product of PTBP1 was double-digested with BamH I and Hind III, and the double-digested product was recovered and detected by 1% Agar electrophoresis;

[0048] Figure 2 Colony PCR test results after transformation of E. coli DH5α with pQE80L-6His-Avi-PTBP1 recombinant plasmid;

[0049] Figure 3Colony PCR test results after pQE80L-6His-Avi-PTBP1 recombinant plasmid was transformed into expression host bacteria AVB101;

[0050] Fig.4 Effect of different induction time on recombinant protein 6His-Avi Biotin - Effect of PTBP1 expression;

[0051] Fig.5 Effect of different IPTG concentrations on the recombinant protein 6His-Avi Biotin - Effect of PTBP1 expression;

[0052] Figure 6 Effects of different optimized sequences on recombinant protein 6His-Avi Biotin - Effect of PTBP1 expression;

[0053] Figure 7 Effects of different expression vectors on recombinant protein 6His-Avi Biotin - Effect of PTBP1 expression;

[0054] Figure 8 Recombinant protein 6His-Avi Biotin -Isolation, purification and induced expression of PTBP1. M: maker; Lane 1: sample at 0h of induction; Lane 2: sample at 4h of induction; Lane 3: supernatant sample after ultrasonic lysis of bacterial solution after 4h of induction; Lane 4: supernatant soluble protein after Ni-NTASefinose TM Samples after Resin purification;

[0055] Fig. 9 Recombinant protein 6His-Avi Biotin -PTBP1 WB detection, the antibody is HRP-labeled Streptavidin. M: maker; Lane 1: sample at 0h of induction; Lane 2: sample at 4h of induction; Lane 3: supernatant sample after ultrasonic lysis of bacterial liquid after 4h of induction; Lane 4: sample after supernatant soluble protein was purified by Ni-NTA SefinoseTM Resin;

[0056] Fig.10 Immunoprecipitation detection of recombinant 6His-Avi Biotin - Biotinylation rate of PTBP1 protein. M: maker; 1: purified 6His-Avi Biotin -PTBP1 protein, the sample volume is 5μl; 2: the same volume of 5μl 6His-Avi Biotin -PTBP1 was treated with 30 μl streptavidin magnetic beads for immunoprecipitation and dissolved in 10 μl 1*SDS-PAGE Loading buffer; the biotinylation rate was 72.4%;

[0057] Fig.11 Recombinant protein 6His-Avi Biotin -Binding and dissociation curves of PTBP1 and (CU)8;

[0058] Fig.12 Recombinant protein 6His-Avi Biotin - Steady-state fitting curve of the interaction between PTBP1 and (CU)8. DETAILED DESCRIPTION

[0059] The present invention provides a method for preparing biotinylated modified PTBP1 domain RRM3 / 4. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0060] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0061] In the present invention, "and / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone.

[0062] In the present invention, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0063] In the present invention, "include", "comprising" and "having" are used interchangeably, and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the descriptions of "include", "comprising" and "having" used in this article also provide "consisting of..." or "as shown in..." schemes.

[0064] In the present invention, "identity" can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, a gap can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. Taking into account the number of gaps that need to be introduced for optimal comparison of the two sequences and the length of each gap, the percentage of identity between the two sequences varies with the change in the same positions shared by the sequences. In the present invention, the above 80% identity includes: 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.

[0065] In the present application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'.

[0066] In the present application, the nucleic acid molecules encompass deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecules includes both sense strands and antisense strands, as well as single-stranded and double-stranded forms. Moreover, nucleic acid molecules as described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derived sugar or phosphate backbone bonding or chemically modified residues.

[0067] In the present invention, "expression unit" refers to a combination of a group of functional elements that can achieve specific gene expression, which usually includes a promoter and a nucleic acid fragment encoding an exogenous gene, and may also include other regulatory elements such as terminators, operators or enhancers.

[0068] In the present invention, the "Avi tag" is a short peptide consisting of 15 amino acid residues, and its amino acid sequence is Gly-Leu-Asn-Asp-Ile-Phe-Glu-Ala-Gln-Lys-Ile-Glu-Trp-His-Glu. The tag has a lysine residue that can be recognized by biotin ligase. In the presence of ATP, Bir A ligase can efficiently activate biotin (D-biotin) to form biotinyl-5' adenylate, and specifically connect biotin to the lysine residue of the Avi tag, thereby achieving biotin labeling of the target protein.

[0069] In the present invention, "plasmid vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced.

[0070] In the present invention, "host cell" refers to a cell into which an exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initially transformed cell are included herein.

[0071] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0072] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0073] The present invention will be further described below in conjunction with embodiments:

[0074] Example 1 Codon Optimization of Gene Sequence of PTBP1 Domain RRM3 / 4

[0075] 1. The C-terminal 200 amino acid sequence (332-531aa) of human PTBP1 (hPTBP1) was selected for prokaryotic expression. The C-terminal 200 amino acids are the RRM3 / 4 domain, and the amino acid sequence is as follows:

[0076] 1AGAGNSVLLV SNLNPERVTP QSLFILFGVY GDVQRVKILF NKKENALVQM ADGNQAQLAM

[0077] 61SHLNGHKLHG KPIRITLSKH QNVQLPREGQ EDQGLTKDYG NSPLHRFKKP GSKNFQNIFP

[0078] 121PSATLHLSNIPPSVSEEDLK VLFSSNGGVV KGFKFFQKDR KMALIQMGSV EEAVQALIDL

[0079] 181HNHDLGENHH LRVSFSKSTI(SEQ ID NO.9)

[0080] 2. Optimize the codons of the protein sequence to make it more suitable for E. coli expression. The restriction endonuclease sites that need to be avoided during codon optimization include: BamH I, Hind III.

[0081] 3. The codon-optimized nucleic acid sequence is as follows:

[0082] SEQ ID NO.1

[0083] 1 GCTGGTGCTG GCAACTCTGT TCTGCTGGTC TCCAACCTGA ACCCGGAACG CGTAACCCCG

[0084] 61 CAGTCTCTGT TCATCCTGTT CGGCGTTTAC GGTGACGTTC AGCGCGTAAA GATTCTGTTC

[0085] 121 AACAAGAAAG AGAACGCGCT GGTGCAGATG GCCGATGGTA ATCAAGCCCA GCTGGCTATG

[0086] 181 TCTCATCTGA ACGGCCACAA ACTGCACGGT AAACCAATCC GCATCACCCT GTCCAAACAT

[0087] 241 CAGAATGTCC AACTGCCACG TGAAGGCCAG GAAGACCAGG GCCTGACTAA AGATTACGGT

[0088] 301 AACTCCCCGC TGCACCGTTT TAAGAAAACCG GGCAGCAAAA ACTTCCAGAA CATCTTCCCA

[0089] 361 CCTTCTGCGA CGCTGCACCT GTCTAACATT CCGCCGTCCG TTTCCGAAGA AGACCTGAAA

[0090] 421 GTACTGTTCA GCAGCAACGG CGGTGTCGTA AAAGGTTTCA AGTTCTTCCA GAAAGACCGC

[0091] 481 AAAATGGCGC TGATTCAGAT GGGCTCTGTT GAGGAAGCTG TTCAGGCTCT GATTGATCTG

[0092] 541 CATAATCATG ATCTGGGCGA AAACCACCAC CTGCGTGTCT CCTTCTCTAA GAGCACCATC

[0093] SEQ ID NO.2

[0094] 1 GCCGGTGCTG GTAACTCTGT TCTGCTGGTA TCCAATCTGA ACCCGGAGCG CGTAACTCCG

[0095] 61 CAGTCCCTGT TCATCCTGTT CGGCGTTTAC GGTGATGTCC AGCGTGTAAA AATCCTGTTC

[0096] 121 AACAAGAAAG AGAACGCTCT GGTCCAGATG GCAGATGGTA ACCAGGCGCA ACTGGCGATG

[0097] 181 TCTCACCTGA ACGGTCACAA ACTGCACGGT AAACCGATCC GCATTACGCT GTCTAAACAT

[0098] 241 CAGAACGTAC AGCTGCCACG CGAAGGTCAG GAAGATCAAG GCCTGACCAA AGACTATGGT

[0099] 301 AATAGCCCGC TGCATCGTTT CAAAAAGCCT GGTAGCAAGA ACTTCCAGAA CATTTTTCCCG

[0100] 361 CCATCCGCGA CGCTGCACCT GTCTAACATC CCTCCGTCCG TTTCTGAAGA GGACCTGAAG

[0101] 421 GTGCTGTTTA GCTCTAACGG CGGTGTCGTC AAGGGTTTCA AATTTTTCCA GAAAGACCGT

[0102] 481 AAAATGGCAC TGATCCAGAT GGGTTCTGTG GAAGAGGCCG TACAGGCGCT GATTGACCTG

[0103] 541 CATAATCACG ACCTGGGTGA AAATCATCAC CTGCGTGTTT CTTTCAGCAA AAGCACCATC

[0104] SEQ ID NO.3

[0105] 1 GCTGGCGCTG GCAACTCTGT TCTGCTGGTA AGCAACCTGA ACCCGGAACG CGTGACCCCG

[0106] 61 CAGTCTCTGT TTATCCTGTT TGGCGTCTAC GGCGACGTGC AGCGTGTTAA AATCCTGTTC

[0107] 121 AACAAAAAAAG AGAACGCGCT GGTACAGATG GCTGATGGCA ACCAGGCTCA ACTGGCGATG

[0108] 181 TCCCATCTGA ACGGCCACAA ACTGCACGGC AAACCGATCC GTATCACCCT GTCTAAACAC

[0109] 241 CAGAACGTTC AGCTGCCGCG TGAAGGTCAG GAAGATCAGG GCCTGACTAA GGATTACGGC

[0110] 301 AACAGCCCGC TGCACCGCTT TAAAAAACCG GGCTCTAAAA ACTTCCAGAA CATCTTCCCG

[0111] 361 CCAAGCGCTA CCCTGCATCT GAGCAATATC CCGCCGTCTG TGTCCGAAGA GGACCTGAAA

[0112] 421 GTTCTGTTCT CCAGCAATGG CGGCGTTGTA AAAGGCTTTA AATTCTTCCA GAAAGACCGC

[0113] 481 AAGATGGCAC TGATCCAGAT GGGCTCTGTT GAGGAAGCTG TCCAGGCGCT GATTGACCTG

[0114] 541 CACAACCACG ACCTGGGGTGA AAATCACCAC CTGCGTGTGT CTTTTTCCAA ATCCACCATC

[0115] SEQ ID NO.4

[0116] 1 GCTGGCGCAG GTAACTCTGT ACTGCTGGTC TCTAACCTGA ACCCGGAACG TGTAACCCCG

[0117] 61 CAAAGCCTGT TTATTCTGTT CGGCGTATAC GGCGATGTTC AGCGTGTAAA AATTCTGTTC

[0118] 121 AACAAAAAAAG AGAACGCTCT GGTTCAGATG GCTGATGGCA ACCAGGCGCA GCTGGCTATG

[0119] 181 TCTCACCTGA ACGGTCATAA ACTGCATGGT AAACCTATCC GTATCACTCT GAGCAAGCAC

[0120] 241 CAGAATGTAC AGCTGCCACG TGAAGGTCAG GAAGATCAGG GTCTGACCAA GGACTATGGT

[0121] 301 AACTCTCCAC TGCACCGTTT CAAGAAACCG GGTAGCAAGA ACTTCCAGAA CATCTTTCCG

[0122] 361 CCATCTGCGA CTCTGCATCT GTCTAACATT CCTCCGTCTG TTTCCGAAGA GGACCTGAAA

[0123] 421 GTTCTGTTCA GCTCTAACGG TGGCGTAGTT AAAGGTTTCA AATTCTTCCA GAAGGATCGC

[0124] 481 AAAATGGCGC TGATCCAGAT GGGTTCTGTT GAGGAAGCGG TTCAGGCTCT GATCGATCTG

[0125] 541 CATAACCACG ATCTGGGCGA AAACCACCAC CTGCGTGTTA GCTTTAGCAA ATCCACCATC

[0126] 4. Add an Avi tag recognized by biotinidase to the N-terminus of the PTBP1 domain RRM3 / 4, and add BamH I and Hind III restriction sites at both ends. The further optimized nucleic acid sequence is as follows:

[0127] SEQ ID NO.5

[0128] 1 GGATCCGGCC TGAACGATAT TTTTGAAGCG CAGAAAATTG AATGGCATGA AGCTGGTGCT

[0129] 61 GGCAACTCTG TTCTGCTGGT CTCCAACCTG AACCCGGAAC GCGTAACCCC GCAGTCTCTG

[0130] 121 TTCATCCTGT TCGGCGTTTA CGGTGACGTT CAGCGCGTAA AGATTCTGTT CAACAAGAAA

[0131] 181 GAGAACGCGC TGGTGCAGAT GGCCGATGGT AATCAAGCCC AGCTGGCTAT GTCTCATCTG

[0132] 241 AACGGCCACA AACTGCACGG TAAACCAATC CGCATCACCC TGTCCAAACA TCAGAATGTC

[0133] 301 CAACTGCCAC GTGAAGGCCA GGAAGACCAG GGCCTGACTA AAGATTACGG TAACTCCCCG

[0134] 361 CTGCACCGTT TTAAGAAACC GGGCAGCAAA AACTTCCAGA ACATCTTCCC ACCTTCTGCG

[0135] 421 ACGCTGCACC TGTCTAACAT TCCGCCGTCC GTTTCCGAAG AAGACCTGAA AGTACTGTTC

[0136] 481 AGCAGCAACG GCGGTGTCGT AAAAGGTTTC AAGTTCTTCC AGAAAGACCG CAAAATGGCG

[0137] 541 CTGATTCAGA TGGGCTCTGT TGAGGAAGCT GTTCAGGCTC TGATTGATCT GCATAATCAT

[0138] 601 GATCTGGGCG AAAACCACCA CCTGCGTGTC TCCTTCTCTA AGAGCACCAT CTAAAAGCTT

[0139] SEQ ID NO.6

[0140] 1 GGATCCGGCC TGAACGATAT TTTTGAAGCG CAGAAAATTG AATGGCATGA AGCCGGTGCT

[0141] 61 GGTAACTCTG TTCTGCTGGT ATCCAATCTG AACCCGGAGC GCGTAACTCC GCAGTCCCTG

[0142] 121 TTCATCCTGT TCGGCGTTTA CGGTGATGTC CAGCGTGTAA AAATCCTGTT CAACAAGAAA

[0143] 181 GAGAACGCTC TGGTCCAGAT GGCAGATGGT AACCAGGCGC AACTGGCGAT GTCTCACCTG

[0144] 241 AACGGTCACA AACTGCACGG TAAACCGATC CGCATTACGC TGTCTAAACA TCAGAACGTA

[0145] 301 CAGCTGCCAC GCGAAGGTCA GGAAGATCAA GGCCTGACCA AAGACTATGG TAATAGCCCG

[0146] 361 CTGCATCGTT TCAAAAAGCC TGGTAGCAAG AACTTCCAGA ACATTTTTCC GCCATCCGCG

[0147] 421 ACGCTGCACC TGTCTAACAT CCCTCCGTCC GTTTCTGAAG AGGACCTGAA GGTGCTGTTT

[0148] 481 AGCTCTAACG GCGGTGTCGT CAAGGGTTTC AAATTTTTCC AGAAAGACCG TAAAATGGCA

[0149] 541 CTGATCCAGA TGGGTTCTGT GGAAGAGGCC GTACAGGCGC TGATTGACCT GCATAATCAC

[0150] 601 GACCTGGGTG AAAATCATCA CCTGCGTGTT TCTTTCAGCA AAAGCACCAT CTAAAAGCTT

[0151] SEQ ID NO.7

[0152] 1 GGATCCGGCC TGAACGATAT TTTTGAAGCG CAGAAAATTG AATGGCATGA AGCTGGCGCT

[0153] 61 GGCAACTCTG TTCTGCTGGT AAGCAACCTG AACCCGGAAC GCGTGACCCC GCAGTCTCTG

[0154] 121 TTTATCCTGT TTGGCGTCTA CGGCGACGTG CAGCGTGTTA AAATCCTGTT CAACAAAAAA

[0155] 181 GAGAACGCGC TGGTACAGAT GGCTGATGGC AACCAGGCTC AACTGGCGAT GTCCCATCTG

[0156] 241 AACGGCCACA AACTGCACGG CAAACCGATC CGTATCACCC TGTCTAAACA CCAGAACGTT

[0157] 301 CAGCTGCCGC GTGAAGGTCA GGAAGATCAG GGCCTGACTA AGGATTACGG CAACAGCCCG

[0158] 361 CTGCACCGCT TTAAAAAACC GGGCTCTAAA AACTTCCAGA ACATCTTCCC GCCAAGCGCT

[0159] 421 ACCCTGCATC TGAGCAATAT CCCGCCGTCT GTGTCCGAAG AGGACCTGAA AGTTCTGTTC

[0160] 481 TCCAGCAATG GCGGCGTTGT AAAAGGCTTT AAATTCTTCC AGAAAGACCG CAAGATGGCA

[0161] 541 CTGATCCAGA TGGGCTCTGT TGAGGAAGCT GTCCAGGCGC TGATTGACCT GCACAACCAC

[0162] 601 GACCTGGGTG AAAATCACCA CCTGCGTGTG TCTTTTTCCA AATCCACCAT CTAAAAGCTT

[0163] SEQ ID NO.8

[0164] 1 GGATCCGGCC TGAACGATAT TTTTGAAGCG CAGAAAATTG AATGGCATGA AGCTGGCGCA

[0165] 61 GGTAACTCTG TACTGCTGGT CTCTAACCTG AACCCGGAAC GTGTAACCCC GCAAAGCCTG

[0166] 121 TTTATTCTGT TCGGCGTATA CGGCGATGTT CAGCGTGTAA AAATTCTGTT CAACAAAAAA

[0167] 181 GAGAACGCTC TGGTTCAGAT GGCTGATGGC AACCAGGCGC AGCTGGCTAT GTCTCACCTG

[0168] 241 AACGGTCATA AACTGCATGG TAAACCTATC CGTATCACTC TGAGCAAGCA CCAGAATGTA

[0169] 301 CAGCTGCCACGTGAAGGTCAGGAAGATCAGGGTCTGACCAGGACTATGGTAACTCTCCA

[0170] 361 CTGCACCGTT TCAAGAAACC GGGTAGCAAG AACTTCCAGA ACATCTTTCC GCCATCTGCG

[0171] 421 ACTCTGCATC TGTCTAACAT TCCTCCGTCT GTTTCCGAAG AGGACCTGAA AGTTCTGTTC

[0172] 481 AGCTCTAACG GTGGCGTAGT TAAAGGTTTC AAATTCTTCC AGAAGGATCG CAAAATGGCG

[0173] 541 CTGATCCAGA TGGGTTCTGT TGAGGAAGCG GTTCAGGCTC TGATCGATCT GCATAACCAC

[0174] 601 GATCTGGGCG AAAACCACCA CCTGCGTGTT AGCTTTAGCA AATCCACCAT CTAAAAGCTT

[0175] 5. The codon-optimized PTBP1 gene sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The synthesized PTBP1 gene sequence was connected to the pUC-SP vector and transformed into Escherichia coli TOP10 for preservation.

[0176] Example 2 Construction of recombinant vector of PTBP1 domain RRM3 / 4

[0177] 1. Extract plasmid from TOP10 strain containing pUC-SP-PTBP1 plasmid, and detect the extracted plasmid by 1% Agar electrophoresis ( Figure 1 (A).

[0178] 2. Using pUC-SP-PTBP1 plasmid as template, amplify PTBP1 gene using specific primers.

[0179] 3. Primer: PTBP-S: CGCGGATCCGGCCTGAACGATA

[0180] PTBP-T:CCCAAGCTTTTTAGATGGTGCTCTTAG

[0181] 4. The PCR amplification system is as follows:

[0182] Components Volume (μl) pUC-SP-PTBP1 plasmid 0.5 PTBP-S 3 PTBP-T 3 dNTP 5 10*buffer 5 LA-Taq enzyme 0.5 <![CDATA[ddH2O]]> 33 Total volume 50

[0183] 5.PCR reaction conditions are as follows:

[0184]

[0185] 6. After the PCR amplification product is recovered, it is tested by electrophoresis using 1% Agar. The test results are as follows: Figure 1 As shown in B.

[0186] 7. The PCR product of PTBP1 was double-digested with BamH I and Hind III. After the double-digested product was recovered, it was detected by electrophoresis using 1% Agar. The detection results were as follows: Figure 1 As shown in C. The enzyme digestion reaction conditions are 30°C (1h)=>37°C (1h), and the enzyme digestion system is as follows:

[0187] Components Volume (μl) PTBP1 55 10*K buffer 7 BamH 4 Hind III 4 Total volume 70

[0188] 8. The pQE80L plasmid vector was double-digested with BamH I and Hind III. The digestion reaction conditions were 30°C (1h)=>37°C (1h). The digestion system was the same as in step 7.

[0189] 9. PTBP1 and pQE80L plasmids were double-digested with BamH I and Hind III, and then electrophoresed with 1% Agar and recovered.

[0190] 10. The double enzyme digestion products of PTBP1 and pQE80L plasmid were connected at 16°C for 2 hours. The recombinant expression plasmid obtained by connection is pQE80L-6His-Avi-PTBP1. The connection reaction system is as follows:

[0191] Components Volume (μl) QUR 5 PTBP1 5 Ligation A 10 Total volume 20

[0192] Example 3: Transformation of Escherichia coli DH5α with pQE80L-6His-Avi-PTBP1 recombinant plasmid

[0193] 1. Take out the DH5α competent cells from the -80℃ freezer and thaw them in an ice bath.

[0194] 2. Add all 20 μl of ligation solution into one DH5α competent cell, mix quickly and place in an ice bath for 30 minutes.

[0195] 3. Transfer the centrifuge tube to a constant temperature metal bath at 42°C, heat shock for 90 seconds and then transfer to an ice bath for 2 minutes.

[0196] 4. Add 900 μl of preheated LB medium (37°C) into the centrifuge tube, then transfer to a shaker at 37°C and culture at 200 rpm for 1 hour.

[0197] 5. The culture solution was evenly spread on LB medium containing 100 mg / L Amp in a volume gradient of 10 μl, 20 μl, 50 μl, 100 μl and 200 μl for screening. Each culture dish was placed in a 37°C incubator for overnight culture.

[0198] 6. Perform colony PCR detection on the colonies obtained by antibiotic screening. Rapid PCR detection uses Takara's EmeraldAmp MAX PCR Master Mix (2*Premix) kit. The detection primers are designed based on the sequences at both ends outside the multiple cloning site of the pQE80L vector, which are:

[0199] p80-S:CGAGGCCCTTTCGTCTTC

[0200] p80-T:GGATCTATCAACAGGAGTCC

[0201] The colony PCR reaction system is as follows:

[0202] Components Volume (μl) 2*EmeraldAmp Premix 10 p80-S 1 p80-T 1 <![CDATA[ddH2O]]> 8 Total volume 20

[0203] The reaction conditions are as follows

[0204]

[0205] 7. Colony PCR A total of 4 colonies were selected for testing. All four colonies were positive clones with obvious amplification bands, such as Figure 2 shown.

[0206] 8. Select colony No. 1 for expansion culture, and send the bacterial solution DH5α (pQE80L-6His-Avi-PTBP1) to Shanghai Sangon Biotechnology Co., Ltd. for sequencing to ensure that the PTBP1 gene has no mutations. The sequencing results were compared with the original sequence, and no mutations were found. Glycerol was used to preserve strain No. 1 of DH5α (pQE80L-6His-Avi-PTBP1), and the strains were backed up at -20℃ and -80℃.

[0207] Example 4: Transformation of pQE80L-6His-Avi-PTBP1 recombinant plasmid into expression host bacteria AVB101

[0208] 1. The strain AVB101 is a B strain of E. coli (hsdR, lon11, su1A1), which contains a recombinant plasmid pBirAcm constructed based on the pACYC184 plasmid and has chloramphenicol resistance. The plasmid contains the IPTG-induced Bir A gene and can overexpress biotin ligase.

[0209] 2. Preparation of competent cells of the expression host bacteria AVB101 The competent cells were prepared using the Competent Cell Preparation Kit (Code No. 9139) of Takara Company. The prepared competent cells were packaged at 100 μl / tube and stored at -80°C.

[0210] 3. Culture strain No. 1 of DH5α (pQE80L-6His-Avi-PTBP1) and extract the pQE80L-6His-Avi-PTBP1 recombinant plasmid.

[0211] 4. Take out the AVB101 competent cells from the -80℃ freezer and thaw them in an ice bath.

[0212] 5. Add 1 μl of pQE80L-6His-Avi-PTBP1 plasmid to one tube of AVB101 competent cells, mix quickly and place in an ice bath for 30 min.

[0213] 6. Transfer the centrifuge tube to a constant temperature metal bath at 42°C, heat shock for 90 seconds and then transfer to an ice bath for 2 minutes.

[0214] 8. Add 900 μl of preheated LB medium (37°C) into the centrifuge tube, then transfer to a shaker at 37°C and culture at 200 rpm for 1 hour.

[0215] 8. The culture solution was evenly spread on LB medium containing 100 mg / L Amp and 20 mg / L Chl in a volume gradient for screening, with the volume gradient being 5 μl, 10 μl, 20 μl, 30 μl and 40 μl. Each culture dish was placed in a 37°C incubator for overnight culture.

[0216] 9. Perform colony PCR detection on the colonies obtained by antibiotic screening, and the detection method is the same as Example 3.

[0217] 10. Colony PCR A total of 4 colonies were selected for testing. All four colonies were positive clones with obvious amplification bands, such as Figure 3 shown.

[0218] 11. Select colony No. 1 for expansion culture. Preserve strain No. 1 of AVB101 (pQE80L-6His-Avi-PTBP1) in glycerol and store the strain at -20℃ and -80℃ respectively.

[0219] Example 5 Inducible expression and synchronous biotinylation of AVB101 (pQE80L-6His-Avi-PTBP1)

[0220] 1. Strain 1 of AVB101 (pQE80L-6His-Avi-PTBP1) was streaked on LB solid medium (containing 100 mg / L Amp and 20 mg / L Chl) and cultured in a 37°C incubator overnight.

[0221] 2. Pick a single colony and place it in 30 ml of liquid LB medium (containing 100 mg / L Amp and 20 mg / L Chl) and culture it overnight in a shaker at 37°C and 150 rpm.

[0222] 3. Take 7.5ml of overnight cultured bacterial solution and inoculate it into 150ml new LB medium (containing 100mg / L Amp and 20mg / L Chl), and culture it in a shaker at 37℃ and 150rpm for 2h. Take 0.5ml bacterial solution into a 1.5ml centrifuge tube, centrifuge it at 12000g for 2min, remove the medium, add 250μl non-denaturing buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole, pH 8.0) to the bacterial sludge, blow and mix the bacteria, and then add 250μl 2*SDS-PAGE Loading buffer. After boiling at 95℃ for 5min, centrifuge it at 12000g for 2min, collect the supernatant, and this sample is the total protein sample of AVB101 (pQE80L-6His-Avi-PTBP1) strain 1 at 0 hours of induction (denoted as P0).

[0223] 4. Add 0.3-1.5ml IPTG (0.1M) and 0.3-1.5ml biotin (5mM) to the 150ml culture medium in the second round of activation, and induce culture in a shaker at 37℃ and 150rpm for 3-7h. Similarly, take 0.5ml bacterial solution in a 1.5ml centrifuge tube, centrifuge at 12000g for 2min, remove the culture medium, add 250μl non-denaturing buffer to the bacterial sludge, blow and mix the bacteria, and then add 250μl 2*SDS-PAGE Loading buffer. After boiling at 95℃ for 5min, centrifuge at 12000g for 2min, collect the supernatant, and this sample is the total protein sample of AVB101 (pQE80L-6His-Avi-PTBP1) strain 1 induced for 4 hours (denoted as P4).

[0224] 5. Centrifuge the remaining bacterial solution at 12000g for 2 minutes and remove the culture medium.

[0225] 6. Add 10 ml of non-denaturing buffer to wash the bacteria. After blowing the suspended bacteria, centrifuge at 12000g for 2 minutes and remove the supernatant.

[0226] 7. Finally, add a total of 40 ml of non-denaturing buffer, pipette to suspend the bacteria, and collect all the bacterial liquid in a 100 ml centrifuge tube.

[0227] 8. Freeze and thaw the bacterial solution, then place it in an ice bath for ultrasonic disruption. The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0228] 9. Repeat step 8 once until the bacterial solution is basically clear and the bacteria are fully broken.

[0229] 10. After ultrasonic disruption, the bacterial solution was centrifuged at 4°C and 12000g for 10 min, and the supernatant was collected, which was the crude soluble protein extract of the induced bacteria (denoted as PU).

[0230] Example 6 Recombinant protein 6His-Avi Biotin -Isolation and purification of PTBP1

[0231] 1. Isolation and purification of recombinant proteins using Ni-NTASefinose from Shanghai Biotech Biotechnology Co., Ltd. TM Resin 1ml gravity type pre-packed column. Before column purification, take the nickel column and various buffers out of the 4℃ refrigerator in advance and place them in a room temperature environment to equilibrate to room temperature (25℃).

[0232] 2. Take a 1 ml Ni-NTA pre-packed column according to the loading capacity and let the storage buffer flow out by gravity.

[0233] 3. Equilibrate the column with 2 ml of Binding Buffer (BIB: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0).

[0234] 4. Add 5 ml of crude soluble protein extract (PU) of the induced bacteria into the Ni-NTA prepacked column.

[0235] 5. Wash the column with Washing Buffer (WAB: 50 mM NaH2PO4, 300 mM NaCl, 60 mM imidazole, pH 8.0), 2 ml / time, for a total of 2 times.

[0236] 6. Elute the histidine-tagged protein on the column with Elution Buffer (ELB: 50mM NaH2PO4, 300mM NaCl, 250mM imidazole, pH 8.0), 2ml / time, for a total of 3 times. Collect all the eluates, which is the purified recombinant protein 6His-Avi Biotin -PTBP, denoted as PE.

[0237] Example 7 Effect of different induction times on the recombinant protein 6His-Avi Biotin -The effect of PTBP1 expression

[0238] 1. Strain 1 of AVB101 (pQE80L-6His-Avi-PTBP1) was streaked on LB solid medium (containing 100 mg / L Amp and 20 mg / L Chl) and cultured in a 37°C incubator overnight.

[0239] 2. Pick a single colony and place it in 50 ml of liquid LB medium (containing 100 mg / L Amp and 20 mg / L Chl) and culture it overnight in a shaker at 37°C and 150 rpm.

[0240] 3. Prepare 5 bottles of 150 ml LB liquid culture medium (containing 100 mg / L Amp and 20 mg / L Chl), inoculate 7.5 ml of overnight cultured bacteria in each bottle, and culture in a shaker at 37°C and 150 rpm for 2 h.

[0241] 4. Add 1.5 ml IPTG (0.1 M) and 1.5 ml biotin (5 mM) to the 150 ml culture medium for the second round of activation, and induce culture in a shaker at 37°C and 150 rpm for 3 to 7 h (a total of 5 time gradients).

[0242] 5. Centrifuge the bacterial solution at 12000g for 2 minutes and remove the culture medium.

[0243] 6. Add 10 ml of non-denaturing buffer to wash the bacteria. After blowing the suspended bacteria, centrifuge at 12000g for 2 minutes and remove the supernatant.

[0244] 7. Finally, add 40 ml of non-denaturing buffer, pipette to suspend the bacteria, and collect all the bacterial liquid in a 100 ml centrifuge tube.

[0245] 8. Freeze and thaw the bacterial solution, then place it in an ice bath for ultrasonic disruption. The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0246] 9. Repeat step 8 once until the bacterial solution is basically clear and the bacteria are fully broken.

[0247] 10. After ultrasonic disruption, centrifuge the bacterial solution at 4°C and 12000g for 10 min, and collect the supernatant, which is the crude soluble protein extract of the induced bacteria.

[0248] 11. Isolation and purification of recombinant protein was the same as in Example 6.

[0249] 12. The concentration of the purified recombinant protein was detected by Bradford method. The amount of induced protein obtained at different induction times is shown in the following table. As the induction time increases, the expression of the recombinant protein also increases. However, the expression increment of the recombinant protein reaches a maximum of 9.24 μg / ml·h after 6 hours of induction, and then the expression increment decreases sharply ( Figure 4 ).

[0250]

[0251] Example 8 Effect of different IPTG concentrations on the expression of recombinant protein 6His-Avi Biotin -The effect of PTBP1 expression

[0252] 1. Strain 1 of AVB101 (pQE80L-6His-Avi-PTBP1) was streaked on LB solid medium (containing 100 mg / L Amp and 20 mg / L Chl) and cultured in a 37°C incubator overnight.

[0253] 2. Pick a single colony and place it in 50 ml of liquid LB medium (containing 100 mg / L Amp and 20 mg / L Chl) and culture it overnight in a shaker at 37°C and 150 rpm.

[0254] 3. Prepare 5 bottles of 150 ml LB liquid culture medium (containing 100 mg / L Amp and 20 mg / L Chl), inoculate 7.5 ml of overnight cultured bacteria in each bottle, and culture in a shaker at 37°C and 150 rpm for 2 h.

[0255] 4. Add 0.3 ml, 0.6 ml, 0.9 ml, 1.2 ml and 1.5 ml IPTG (0.1 M) to the 150 ml medium for the second round of activation, respectively, to make the final concentrations 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1 mM. At the same time, add 1.5 ml biotin (5 mM) to make the final concentration 50 μM, and induce culture in a shaker at 37°C and 150 rpm for 6 h.

[0256] 5. Centrifuge the bacterial solution at 12000g for 2 minutes and remove the culture medium.

[0257] 6. Add 10 ml of non-denaturing buffer to wash the bacteria. After blowing the suspended bacteria, centrifuge at 12000g for 2 minutes and remove the supernatant.

[0258] 7. Finally, add 40 ml of non-denaturing buffer, pipette to suspend the bacteria, and collect all the bacterial liquid in a 100 ml centrifuge tube.

[0259] 8. Freeze and thaw the bacterial solution, then place it in an ice bath for ultrasonic disruption. The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0260] 9. Repeat step 8 once until the bacterial solution is basically clear and the bacteria are fully broken.

[0261] 10. After ultrasonic disruption, centrifuge the bacterial solution at 4°C and 12000g for 10 min, and collect the supernatant, which is the crude soluble protein extract of the induced bacteria.

[0262] 11. Isolation and purification of recombinant protein was the same as in Example 6.

[0263] 12. The concentration of the purified recombinant protein was detected by Bradford method. The amount of induced protein obtained under different IPTG concentrations is shown in the following table. As the IPTG concentration increases, the expression of the recombinant protein also increases. However, the expression increment of the recombinant protein reaches a maximum value of 6.19 μg / ml when the IPTG concentration is 0.8 mM, and then the expression increment begins to decrease ( Figure 5 ).

[0264]

[0265] Example 9 Effect of different PTBP1 optimized sequences on recombinant protein 6His-Avi Biotin -The effect of PTBP1 expression

[0266] 1. Four optimized sequences: SEQ ID NO. 5 to 8 were cloned into pQE80L vector respectively. The vector construction method was the same as in Example 2.

[0267] 2. The recombinant vector transformation method of DH5ɑ and AVB101 is the same as in Example 3 and Example 4.

[0268] 3. The antibiotic-selected AVB101 (pQE80L-6His-Avi-PTBP1) strain was streaked on LB solid medium (containing 100 mg / L Amp and 20 mg / L Chl) and cultured in a 37°C incubator overnight.

[0269] 2. Pick a single colony and place it in 50 ml of liquid LB medium (containing 100 mg / L Amp and 20 mg / L Chl) and culture it overnight in a shaker at 37°C and 150 rpm.

[0270] 3. Prepare 4 bottles of 150 ml LB liquid culture medium (containing 100 mg / L Amp and 20 mg / L Chl), inoculate 7.5 ml of overnight cultured bacteria in each bottle, and culture in a shaker at 37°C and 150 rpm for 2 h.

[0271] 4. In the second round of activation, 1.5 ml IPTG (0.1 M) was added to the 150 ml culture medium to make the final concentration 1 mM; at the same time, 1.5 ml biotin (5 mM) was added to make the final concentration 50 μM, and induced culture was carried out in a shaker at 37°C and 150 rpm for 6 h.

[0272] 5. Centrifuge the bacterial solution at 12000g for 2 minutes and remove the culture medium.

[0273] 6. Add 10 ml of non-denaturing buffer to wash the bacteria. After blowing the suspended bacteria, centrifuge at 12000g for 2 minutes and remove the supernatant.

[0274] 7. Finally, add 40 ml of non-denaturing buffer, pipette to suspend the bacteria, and collect all the bacterial liquid in a 100 ml centrifuge tube.

[0275] 8. Freeze and thaw the bacterial solution, then place it in an ice bath for ultrasonic disruption. The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0276] 9. Repeat step 8 once until the bacterial solution is basically clear and the bacteria are fully broken.

[0277] 10. After ultrasonic disruption, centrifuge the bacterial solution at 4°C and 12000g for 10 min, and collect the supernatant, which is the crude soluble protein extract of the induced bacteria.

[0278] 11. Isolation and purification of recombinant protein was the same as in Example 6.

[0279] 12. The concentration of the purified recombinant protein was detected by Bradford method. The amount of induced protein obtained by different optimized sequences is shown in the following table. Among them, the optimized sequence SEQ ID NO.5 can obtain the highest recombinant protein expression amount ( Figure 6 ).

[0280]

[0281] Example 10 Effects of different expression vectors on the expression of recombinant protein 6His-Avi Biotin -The effect of PTBP1 expression

[0282] 1. The optimized sequence SEQ ID NO.5 was cloned into expression vectors pQE80L, pQE30 and pET32a respectively. The vector construction method was the same as in Example 2.

[0283] 2. The recombinant vector transformation method of DH5ɑ and AVB101 is the same as in Example 3 and Example 4.

[0284] 3. The antibiotic-screened AVB101 (pQE80L-6His-Avi-PTBP1), AVB101 (pQE30-6His-Avi-PTBP1) and AVB101 (pET32a-6His-Avi-PTBP1) strains were streaked on LB solid medium (containing 100 mg / L Amp and 20 mg / L Chl) and cultured in a 37°C incubator overnight.

[0285] 2. Pick a single colony and place it in 50 ml of liquid LB medium (containing 100 mg / L Amp and 20 mg / L Chl) and culture it overnight in a shaker at 37°C and 150 rpm.

[0286] 3. Prepare three bottles of 150 ml LB liquid culture medium (containing 100 mg / L Amp and 20 mg / L Chl), inoculate 7.5 ml of overnight cultured bacteria into each bottle, and culture in a shaker at 37°C and 150 rpm for 2 h.

[0287] 4. In the second round of activation, 1.5 ml IPTG (0.1 M) was added to the 150 ml culture medium to make the final concentration 1 mM; at the same time, 1.5 ml biotin (5 mM) was added to make the final concentration 50 μM, and induced culture was carried out in a shaker at 37°C and 150 rpm for 6 h.

[0288] 5. Centrifuge the bacterial solution at 12000g for 2 minutes and remove the culture medium.

[0289] 6. Add 10 ml of non-denaturing buffer to wash the bacteria. After blowing the suspended bacteria, centrifuge at 12000g for 2 minutes and remove the supernatant.

[0290] 7. Finally, add 40 ml of non-denaturing buffer, pipette to suspend the bacteria, and collect all the bacterial liquid in a 100 ml centrifuge tube.

[0291] 8. Freeze and thaw the bacterial solution, then place it in an ice bath for ultrasonic disruption. The amplitude rod was 500W, the power was 500W, each ultrasonic treatment was 10s and then rested for 50s, for a total of 30 times.

[0292] 9. Repeat step 8 once until the bacterial solution is basically clear and the bacteria are fully broken.

[0293] 10. After ultrasonic disruption, centrifuge the bacterial solution at 4°C and 12000g for 10 min, and collect the supernatant, which is the crude soluble protein extract of the induced bacteria.

[0294] 11. Isolation and purification of recombinant protein was the same as in Example 6.

[0295] 12. The concentration of the purified recombinant protein was tested using the Bradford method. The amount of induced protein obtained by different expression vectors is shown in the following table. Among them, the expression vector pQE80L can obtain the highest amount of recombinant expression protein ( Figure 7 ).

[0296]

[0297] Example 11 Electrophoresis detection of recombinant protein

[0298] The recombinant protein was detected by SDS-PAGE electrophoresis, and the separation gel concentration was 12%. There was no obvious induced protein band in the sample after induction for 0 hours, but an obvious induced protein band appeared in the sample after induction for 6 hours. The induced band was between the 25kDa and 35kDa bands indicated by Maker. The preliminary detection was consistent with the theoretical calculation of the recombinant protein 6His-Avi Biotin -PTBP molecular weight (25.5kDa) is basically consistent. There is also an obvious induced band in the soluble protein crude extract (PU) after ultrasonic disruption, indicating that the induced recombinant protein can exist in the expression host bacteria in a soluble form. It is preliminarily inferred that the induced 6His-Avi Biotin -PTBP recombinant protein can be correctly folded to form an active protein. After the sample was purified by nickel column, a single band was formed after electrophoresis, and the band did not show tailing phenomenon, indicating that high-purity recombinant protein can be obtained by this method, and the recombinant protein will not be partially truncated. Figure 8 shown.

[0299] Example 12 Detection of recombinant protein 6His-Avi using Western blot Biotin -Biotinylation of PTBP1

[0300] 1. SDS-PAGE electrophoresis was performed according to the conditions of Example 11, with the separation gel concentration being 12%.

[0301] 2. After SDS-PAGE electrophoresis, transfer the protein to PVDF membrane.

[0302] 3. Wash the membrane with washing buffer TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.6) at room temperature (25°C) for 2 min.

[0303] 4. Block the membrane with blocking solution (TBS-T with 3% BSA) at room temperature (25°C) for 60 minutes.

[0304] 5. At room temperature (25°C), shake and react with horseradish peroxidase-labeled streptavidin (HRP-Streptavidin) antibody solution for 60 minutes. The dilution ratio of HRP-Streptavidin is 1 / 10000.

[0305] 6. Wash the membrane three times with TBS-T at room temperature (25°C), each time for 5 min.

[0306] 7. After washing, remove excess liquid with absorbent paper, develop with BeyoECLPlus colorimetric solution (1 ml of solution A + 1 ml of solution B), and take photos. The results of Western blot experiments showed that there was no hybridization band in the sample (Lane 1) induced for 0 hours; there was no hybridization band in the sample (Lane 2) induced for 4 hours, the supernatant sample after ultrasonic lysis after 4 hours of induction (Lane 3), and the supernatant sample after Ni-NTASefinose TM The samples purified by Resin (Lane 4) all showed specific hybridization bands, and the molecular weight of the bands was consistent with the induced bands shown by SDS-PAGE electrophoresis in Example 6. The above analysis can infer that all proteins of the AVB101 host bacteria themselves do not have biotinylation sites (Lane 1), while the induced recombinant protein PTBP1 can be labeled with biotin during induction, such as Fig. 9 However, the efficiency of biotinylation needs to be further determined.

[0307] Example 13 Detection of 6His-Avi using magnetic bead immunoprecipitation (IP) Biotin -Biotinylation efficiency of PTBP1

[0308] 1. Magnetic bead immunoprecipitation experiments using BeyoMag from Beyotime Biotechnology TM Streptavidin Magnetic Beads were used for detection.

[0309] 2. After gently blowing and mixing the streptavidin magnetic beads, aspirate 30 μl of streptavidin magnetic beads and place them in a 1.5 ml EP tube. After standing on the magnetic stand for 1 minute, remove the supernatant.

[0310] 3. Wash the magnetic beads 3 times with PBS-T buffer (PBS with 0.05% Tween), 1 ml each time.

[0311] 4. Add 1 ml PBS-T buffer to the washed magnetic beads and suspend them, then add 5 μl purified 6His-Avi Biotin -PTBP1 protein, shake and react for 60 min at room temperature (25°C). After precipitating on the magnetic stand for 1 min, remove the supernatant.

[0312] 5. Wash the magnetic beads 3 times with PBS-T buffer, 1 ml each time.

[0313] 6. After washing, add 200 μl PBS-T buffer to suspend the magnetic beads and transfer all of them into a 250 μl centrifuge tube. After standing on the magnetic stand for 1 minute, remove the supernatant.

[0314] 7. Add 10 μl 1*SDS-PAGE Loading buffer to the magnetic beads and boil at 95℃ for 5 min.

[0315] 8. Use SDS-PAGE electrophoresis to detect the immunoprecipitated samples, and the separation gel concentration is 12%. Fig.10 During electrophoresis, 5 μl of purified 6His-Avi Biotin -PTBP1 protein was used as a control (Lane 1), and the sample immunoprecipitated with streptavidin magnetic beads (Lane 2) was the biotinylated recombinant protein, with a biotinylation rate of 72.4%.

[0316] Example 14 Detection of recombinant 6His-Avi using biolayer interferometry (BLI) technology Biotin -PTBP1 protein activity and its kinetic parameters

[0317] 1. The instrument used in the biolayer interferometry experiment is Octect Red96 from Sartorius, and the sensor is streptavidin SA sensor.

[0318] 2. Polypyrimidine single-stranded RNA (CU) 8 was synthesized by Shanghai Shenggong Biological Company and prepared into a 20 μM concentration mother solution with DEPC water, stored at -80°C, and thawed in an ice bath before use.

[0319] 3. Purification of recombinant 6His-Avi Biotin -PTBP1 protein was diluted with PBS-T buffer at a ratio of 1 / 20, and the final concentration was about 26.4 μg / ml.

[0320] 4. The detection concentration gradient of polypyrimidine (CU) 8 is: 200nM, 100nM, 50nM, 25nM, 12.5nM and 0nM (Control).

[0321] 5. Since the molecular weight of polypyrimidine (CU)8 is much larger than that of small molecule compounds, its binding to the target PTBP1 does not belong to the fast binding and fast dissociation mode, so the standard operating procedure (SOP) for macromolecular interactions, which is closer, was selected, and 6 SA sensors were used for parallel detection in each test.

[0322] 6. After the sensor was pre-wetted in PBS-T for 10 minutes, the recombinant 6His-Avi Biotin -PTBP1 protein was immobilized onto the SA sensor, and the immobilization procedure was: Baseline (60 s), Loading (600 s), and Baseline (120 s).

[0323] 7. Recombinant 6His-Avi Biotin After the -PTBP1 protein was immobilized on the SA sensor, the kinetic parameters of the interaction between (CU)8 and PTBP1 were detected, and the analysis procedures were: Baseline (120 s), Association (180 s) and Dissociation (180 s). The experimental temperature was set at 30°C and the signal acquisition frequency was 5 Hz.

[0324] 8. The binding and dissociation curves of PTBP1 and (CU)8 are shown in Fig.11 As shown, K D , k on and k off Parameters such as table.

[0325] Con.(nM) <![CDATA[K D (M)]]> <![CDATA[K D Error]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k on Error]]> <![CDATA[K off (1 / s)]]> <![CDATA[K off Error]]> Full R^2 200 3.00E-08 2.75E-09 8.99E+05 7.85E+04 2.70E-02 7.35E-04 0.9594 100 3.05E-08 1.77E-09 8.26E+05 4.52E+04 2.52E-02 4.88E-04 0.9784 50 2.39E-08 1.06E-09 8.24E+05 3.46E+04 1.97E-02 2.87E-04 0.9835 25 5.15E-08 4.04E-09 3.94E+05 3.04E+04 2.03E-02 2.76E-04 0.9863 12.5 5.30E-08 6.39E-09 3.25E+05 3.90E+04 1.72E-02 1.88E-04 0.9888

[0326] 9. Steady-state fitting analysis results are as follows Fig.12 As shown in the figure, the steady-state fitting analysis shows that almost all of the five points obtained from the actual measurement values ​​fall on the theoretical curve, and the deviation from the theoretical curve is very small, indicating that the measured data has a high degree of credibility. The equilibrium dissociation constant KD is 38nM.

[0327] Chi^2 / DoF 2.4947E-05 R^2 0.9891 <![CDATA[R Max ]]> 0.1774 0.0062 <![CDATA[K D ]]> 3.80E-08 ±3.9E-09M

[0328] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid encoding the PTBP1 domain RRM3 / 4, which has: 1), the nucleic acid sequence shown in SEQ ID NO: 1; II), a nucleic acid sequence encoding the PTBP1 domain RRM3 / 4, in which one or more bases are substituted, deleted, added and / or replaced in the nucleic acid sequence shown in SEQ ID NO: 1; III), a nucleic acid sequence having at least 80% identity with the nucleic acid sequence described in I) or II).

2. An expression unit comprising the nucleic acid according to claim 1 and a promoter.

3. The expression unit according to claim 2, characterized in that An Avi tag and / or a 6×His tag may also be included.

4. The expression unit according to claim 2 or 3, characterized in that From the 5' end to the 3' end, it includes a promoter, a 6×His tag, an Avi tag, the nucleic acid according to claim 1 and a terminator connected in sequence.

5. A plasmid vector comprising a backbone vector and at least one of the following i) to ii): i) the nucleic acid according to claim 1; ii) The expression unit according to any one of claims 2 to 4.

6. A host cell transformed or transfected with the plasmid vector of claim 5, or having the nucleic acid of claim 1 integrated into its genome, or having the expression unit of any one of claims 2 to 4 integrated into its genome.

7. The host cell according to claim 6, characterized in that The host is Escherichia coli containing the biotin ligase BirA gene.

8. A method for preparing biotinylated PTBP1 domain RRM3 / 4, comprising culturing the host according to claim 6 or 7 in the presence of biotin to obtain a culture containing biotinylated PTBP1 domain RRM3 / 4.

9. The preparation method according to claim 8, characterized in that: The culture medium is an LB liquid culture medium containing 100 mg / L Amp, 20 mg / L Chl, 0.2-1 mM IPTG and 50 μM biotin.

10. The product obtained by the preparation method according to claim 8 or 9.

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