Construction of tRNA m1A modification tool based on aminoacyl-tRNA synthetase

Abstract

The invention relates to the technical field of biology, and discloses construction of a novel fusion protein based on aminoacyl-tRNA synthetase. The expression plasmid vector of the fusion protein provided by the invention is pSL4, a MetRS gene and tRNA m1A modified methylase TRMT61A are connected to obtain a MetRS-TRMT61A fusion gene, and the MetRS-TRMT61A fusion gene is integrated into a pSL4 eukaryotic expression plasmid, so that the fusion protein has the application potential of targeting specific tRNA methylation modification. Meanwhile, the fusion protein expression plasmid vector provided by the invention can be expressed in eukaryotic cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein expression plasmid vector and a construction method thereof. Background Art

[0002] tRNA (Transfer RNA), also known as transfer RNA, is a carrier that carries out polypeptide chain extension to the specific ribosome site by complementary pairing of codon-anticodon bases with mRNA during protein translation. tRNA is the most widely modified RNA, with an average of 13 modifications per tRNA molecule, such as mRNA. 1 A.m 3 C.m 5 C et al. Posttranscriptional modifications are required for tRNA maturation and can greatly affect its biogenesis and function. m 1 A modification is a reversible modification that is written or removed by "Writer" and "Eraser". Writer is the methyltransferase TRMT61A / TRMT6 complex, while Eraser includes demethylase ALKBH1 and ALKBH3.

[0003] Aminoacyl-tRNA synthetases are a class of key enzymes in the process of protein synthesis in organisms. They can specifically recognize amino acid side chains and their corresponding tRNAs, and catalyze the binding of specific amino acids to specific tRNAs with the participation of ATP to form aminoacylated tRNA molecules. Different aminoacyl-tRNA synthetases have different structures, sizes, and subunit compositions, which are important guarantees for their recognition of specific tRNA structures, specific binding to tRNAs, and reduction of tRNA and amino acid mismatches during aminoacyl-tRNA synthesis. Fusion proteins constructed using aminoacyl-tRNA synthetases can recognize tRNAs and perform modification-directed manipulation. They are potential tools for tRNA epigenetic research and have broad application prospects. Summary of the invention

[0004] The present invention provides a fusion protein expression plasmid vector and a construction method thereof. The fusion protein expression plasmid vector provided by the present invention can target the methylation modification of a specific tRNA by integrating the MetRS-TRMT61A fusion gene obtained by connecting MetRS and TRMT61A into the eukaryotic expression plasmid pSL4.

[0005] The specific technical scheme of the present invention is: The present invention provides a fusion protein expression plasmid vector, which is constructed by the following method: The MetRS-TRMT61A fusion gene is integrated into the eukaryotic expression plasmid to obtain the fusion protein expression plasmid vector.

[0006] MetRS, i.e., methylthionyl-tRNA synthetase, is an aminoacyl-tRNA synthetase responsible for the activation of methylthionyl-tRNA, which can specifically recognize and bind to methylthionyl-tRNA, and promote methylthionyl-tRNA to participate in protein translation of ribosomes. The fusion protein expression plasmid vector pSL4 provided by the present invention integrates the MetRS-TRMT61A fusion gene obtained by connecting the MetRS gene and TRMT61A into the eukaryotic expression plasmid pSL4, which can target the methylation of specific tRNA.

[0007] In the above technical solution of the present invention, preferably, the nucleotide sequence of the fusion protein expression plasmid vector is as shown in SEQ ID NO.1 in the sequence table.

[0008] In another aspect, the present invention provides a method for constructing the fusion protein expression plasmid vector, comprising the following steps: (1) Using pSL4 MetRS-ALKBH3 as a template, reverse amplification was performed to obtain the pSL4 MetRS vector; (2) amplifying the TRMT61A gene fragment to obtain the TRMT61A fragment; (3) Integrate the pSL4 MetRS fragment described in step (1) and the TRMT61A gene fragment described in step (2) into the pSL4 vector described in step (1) by homologous recombination technology to obtain the fusion protein expression plasmid vector.

[0009] In the above technical solution of the present invention, preferably, the nucleotide sequence of the pSL4 vector is as shown in SEQ ID NO.2 in the sequence listing.

[0010] The fusion protein expression plasmid vector of the present invention uses pSL4 as a vector skeleton, and its expression region is a MetRS-TRMT61A fusion protein sequence with a BsmBI restriction site added to the 5' end and an XbaI restriction site added to the 3' end; the fusion protein sequence is in the reading frame of the CMV promoter on the pSL4 vector. The connection sequence of the MetRS-TRMT61A fusion gene is 5'-MetRS gene-TRMT61A gene-3'.

[0011] The MetRS-TRMT61A fusion gene was amplified from the gene vector pSL4 MetRS-ALKBH3 using the primer pair of nucleotide sequences SEQ ID NO.11 and SEQ ID NO.12, and from the gene vector pSL4-TRMT61A using the primer pair of SEQ ID NO.13 and SEQ ID NO.14. The nucleotide sequence of the gene vector pSL4-TRMT61A is SEQ ID NO.15.

[0012] In the above technical solution of the present invention, preferably, the nucleotide sequence of the MetRS gene is as shown in SEQ ID NO.4 in the sequence listing.

[0013] In the above technical solution of the present invention, preferably, the nucleotide sequence of the TRMT61A gene is as shown in SEQ ID NO.5 in the sequence table.

[0014] Preferably, the amplification method includes PCR technology and E. coli amplification. In the above technical solution of the present invention, the amplification technology is further preferably PCR amplification.

[0015] Compared with the prior art, the present invention has the following technical effects: (1) The fusion protein expression plasmid vector provided by the present invention can achieve methylation modification of methylthionyl-tRNA by targeting the methylthionyl-tRNA of MetRS with TRMT61A.

[0016] (2) The fusion protein expression plasmid vector provided by the present invention can be expressed in eukaryotic cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a schematic diagram of the structure of a fusion protein expression plasmid vector of the present invention; Figure 2 Schematic diagram of the components of the plasmid vector in Example 4 of the present invention; Figure 3 The plasmid vector map drawn in Example 4 of the present invention; Figure 4 Detection of fusion protein gene expression in Example 5 of the present invention; Figure 5 This is a diagram showing the expression of the fusion protein detected in Example 5 of the present invention. DETAILED DESCRIPTION

[0018] like Figure 1 The figure is a schematic diagram of the structure of a fusion protein expression plasmid vector provided by the present invention. The present invention is further described below in conjunction with the embodiments.

[0019] Example 1 Synthesis of pSL4 MetRS linear vector Select appropriate sites on the pSL4-MetRS ALKBH3 plasmid vector to design specific primers, the downstream primer sequence is SEQ ID NO.9, the upstream primer sequence is SEQ ID NO.10, reverse PCR amplification is performed on the pSL4 MetRS-ALKBH3 plasmid vector, the original template is digested with DpnI enzyme, and the PCR product is detected by 1% agarose gel electrophoresis and then recovered with a gel recovery kit to obtain a linear vector for subsequent use. The nucleotide sequence is shown in SEQ ID NO.8. Table 1 shows the components and amounts of the PCR amplification reaction system. The PCR reaction conditions (30 cycles) are: pre-denaturation at 98°C for 3min; denaturation at 98°C for 30s; annealing at 55°C for 15s; extension at 72°C for 3min; final extension at 72°C for 5min.

[0020] Table 1 Components Reaction system PrimeSTAR Max Premix (2x) Upstream Primer Downstream Primer Templated dH2O 25ul1ul1ul100ngto 50ul Example 2 TRMT61A fragment amplification A suitable PCR site was selected on the pSL4 TRMT61A eukaryotic overexpression plasmid vector, and corresponding specific homology arm primers were designed therefrom. The upstream primer is shown in SEQ ID NO.13 in the sequence list, and the downstream primer sequence is shown in SEQ ID NO.14 in the sequence list. The PCR reaction amplification system was the same as in Example 1. The PCR product was subjected to 1% agarose electrophoresis to detect the amplification result and the TRMT61A fragment was recovered. The nucleotide sequences of MetRS and TRMT61A were SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0023] Example 3: Ligation of linear vector and multigene fragment The linear vector and the obtained TRMT61A gene fragment were connected by homologous recombination, and the connection order of the fusion gene part was 5'-MetRS-TRMT61A-3'. The reaction system is shown in Table 3. After the system was mixed and centrifuged, 37°C for 30min, 4°C ∞, the nucleotide sequence was SEQ ID NO.3. The reaction system is shown in Table 2.

[0024] Table 2 Components Reaction system <![CDATA[5x CE II BufferExnase IILinear VectorInsertddH2O]]> 4ul2ul1ul3ulto 20ul Example 4 Screening and Purification of Recombinant Fusion Protein MetRS-TRMT61A Expression Plasmid Take 10μl of the ligation product to transform 100μL E. coli DH5α competent cells, heat shock at 42℃ for 70s, cool on ice for 2mins, shake the bacteria in 900ul LB for 1h, and take 100μL of the bacterial solution to apply on an LB plate containing 100μg / mL ampicillin for screening. Pick the positive transformants after 12-16h, transfer to 1mL LB liquid medium containing 100μg / mL ampicillin, culture at 37℃ for 7h, take part of the bacterial solution for sequencing analysis, the sequence analysis is as SEQ ID NO.1, the plasmid components are as Figure 2 As shown, draw the vector map as Figure 3 shown.

[0025] Example 5 Fusion protein plasmid transfection The adherent HEK-293T cells were digested and resuspended, plated into 6-well plates, and 5x10 5 Cells were pre-cultured in the culture plate in an incubator for 24 h. When the cell density reached 50% to 60% confluence, Lipofectamine 8000 (Invitrogen) was used for transfection. The transfected cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 24 h, and the cell RNA was extracted by Trizol method. The mRNA level of TRMT61A was detected by RT-qPCR. Figure 4 After the fusion protein plasmid was transfected into 293T cells, RT-qPCR was used to detect the RNA expression of the fusion protein gene. Figure 5 This is the result of Western blot detection of fusion protein.

[0026] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0027] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A fusion protein expression plasmid vector, characterized in that: The fusion protein expression plasmid vector is constructed by the following method: the MetRS gene and the TRMT61A gene are fused and integrated into the eukaryotic expression plasmid pSL4 to obtain the fusion protein expression plasmid vector.

2. A fusion protein expression plasmid vector according to claim 1, characterized in that: The nucleotide sequence of the fusion protein expression plasmid vector is shown in SEQ ID NO.1 in the sequence table.

3. A method for constructing a fusion protein expression plasmid vector as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Using pSL4 MetRS-ALKBH3 as a template, reverse amplification was performed to obtain the pSL4 MetRS vector; (2) Using cDNA as a template, amplify the TRMT61A fragment; (3) Integrate the pSL4 MetRS fragment described in step (1) and the TRMT61A gene fragment described in step (2) into the pSL4 vector described in step (1) by homologous recombination technology to obtain the fusion protein expression plasmid vector.

4. The method according to claim 3, characterized in that: The nucleotide sequence of the pSL4 vector is shown as SEQ ID NO.2 in the sequence listing.

5. The method according to claim 3, characterized in that: The nucleotide sequence of the MetRS gene is shown as SEQ ID NO.4 in the sequence listing.

6. The method according to claim 3, characterized in that: The nucleotide sequence of the TRMT61A gene is shown in SEQ ID NO.5 in the sequence listing.

7. The method according to claim 3, characterized in that: The amplification adopts homologous recombination technology.

8. The fusion protein expression plasmid vector according to any one of claims 1 to 2 or the fusion protein expression plasmid vector constructed by the method according to any one of claims 3 to 8 is expressed in tRNA m 1 A modification application.

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