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PIMT (protein isoaspartyl-methyltransferase) expression plasmid, amplification primer, construction method and application thereof

A technology for expressing plasmids and amplification primers, which is applied in the field of genetic engineering and achieves the effects of wide application prospects, improved anti-invasion ability, and improved detection amount

Inactive Publication Date: 2018-10-02
江苏天瑞精准医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there has not been any report on the recombinant construction of PIMT expression plasmids for the treatment of rheumatoid arthritis

Method used

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  • PIMT (protein isoaspartyl-methyltransferase) expression plasmid, amplification primer, construction method and application thereof
  • PIMT (protein isoaspartyl-methyltransferase) expression plasmid, amplification primer, construction method and application thereof
  • PIMT (protein isoaspartyl-methyltransferase) expression plasmid, amplification primer, construction method and application thereof

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Embodiment 1

[0035] Construction of PIMT expression plasmid

[0036] Select Homo sapiens protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1), mRNA, (NCBI Reference Sequence: NM_005389.2)

[0037] Amplified CDs: 74~757

[0038]

[0039]

[0040] According to the restriction site provided by Homo sapiens protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1), mRNA (NCBI Reference Sequence: NM_005389.2) and pcDNA3.1 (-)-Flag-GFP plasmid, carry out Restriction site selection, only XhoI and BamHI can be used as restriction sites for PCMT1, respectively design 3 pairs of primers for PCR amplification, and send the products for sequencing, see figure 1 and figure 2 shown.

[0041] Wherein, primer set sequence 1 is respectively:

[0042] PCMT1-XhoI-684F: TAA CTCGAG ACCATGGCCTGGAAATCCGGCG

[0043] PCMT1-BamHI-684R: TAA GGATCC TCACTTCCACCTGGACCACTGC

[0044] Primer set sequences 2 are:

[0045] PCMT-XhoI-858-F: TAT CTCGAG ATGCCGGGAGCGCGCAGT

[0046] PCMT-B...

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Abstract

The invention discloses PIMT (protein isoaspartyl-methyltransferase) expression plasmid, an amplification primer, a construction method and the application thereof. The sequence of the PIMT expressionplasmid is as shown in SEQ ID NO:1. The construction method of the PIMT expression plasmid comprises the steps of performing enzyme cutting site selection according to enzyme cutting sites provided by humanized PCMT, mRNA and pcDNA3.1(-)-Flag-GFP plasma to obtain the PIMT expression plasmid, wherein the enzyme cutting sites of the PCMT comprise XhoI and BamHI. The sequences of an amplification primer for the PIMT expression plasmid are as shown in SEQ ID NO:2 to SEQ ID NO:7. The PIMT expression plasmid is constructed by a gene technology, can increase the expression quantity of the PIMT in RA-FLS (rheumatoid arthritis-fibroblast-like synoviocytes) and increase the detection quantity, can serve as a medicine for regulating and controlling apoptosis and proliferation of the RA-FLS, and realizes treatment of rheumatoid arthritis.

Description

technical field [0001] The present invention relates to a PIMT (protein isoaspartyl-methyltransferase, protein isoaspartyl-methyltransferase, referred to as PIMT) expression plasmid, in particular to a method for constructing a PIMT expression plasmid and used amplification primers, and its use in The application in the preparation of medicines for treating rheumatoid arthritis belongs to the technical field of genetic engineering. Background technique [0002] Under physiological conditions, L-asparaginyl residues (L-asparaginyl residues) and L-aspartyl residues (L-aspartyl residues) in proteins will spontaneously deaminate or isomerize to form abnormal L -Aspartyl (abnormal l-isoaspartyl residues, L-isoAsp), the accumulation of this abnormal L-isoAsp in the protein can change the structure of the protein and affect the function of the protein. Protein isoaspartyl-methyltransferase (PIMT) is a repair enzyme ubiquitous in prokaryotic and eukaryotic cells, which can recogniz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/54C12N15/11C12N9/10A61K38/45A61K48/00A61P29/00A61P19/02
CPCA61K38/45A61K48/005A61P19/02A61P29/00C12N9/1007C12N15/85C12Y201/01077
Inventor 周洁陆英张慧杨曹骅
Owner 江苏天瑞精准医疗科技有限公司
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