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Lentivirus for specifically inhibiting DCUN1D1 gene and construction method and application thereof

A construction method and lentiviral technology, applied in the fields of molecular biology and oncology, can solve the problems of unknown protein expression, function and mechanism

Pending Publication Date: 2019-05-31
信雅生物科技(苏州)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, unfortunately, there are no reports on the related functions of DCUN1D1 gene, and its protein expression and its role and mechanism in tumors and other diseases are still unknown

Method used

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  • Lentivirus for specifically inhibiting DCUN1D1 gene and construction method and application thereof
  • Lentivirus for specifically inhibiting DCUN1D1 gene and construction method and application thereof
  • Lentivirus for specifically inhibiting DCUN1D1 gene and construction method and application thereof

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Embodiment Construction

[0079] A method for constructing a lentivirus that specifically inhibits DCUN1D1 gene expression, comprising the steps of:

[0080] (1) Using the NCBI (https: / / www.ncbi.nlm.nih.gov / gene) database, obtain the coding (CDS) region sequence of the human DCUN1D1 gene, and mark its start codon coding sequence (ATG) and termination Codon coding sequence (TAG):

[0081] ATG ATCTTCACACAATCTAGTGAAAAAACAGCAGTAAGTTGTCTTTCTCAAAATGACTGGAAGTTAGATGTTGCAACAGATAATTTTTTCCAAAATCCTGAACTTTATATACGAGAGAGTGTAAAAGGATCATTGGACAGGAAGAAGTTAGAACAGCTGTACAATAGATACAAAGACCCTCAAGATGAGAATAAAATTGGAATAGATGGCATACAGCAGTTCTGTGATGACCTGGCACTCGATCCAGCCAGCATTAGTGTGTTGATTATTGCGTGGAAGTTCAGAGCAGCAACACAGTGCGAGTTCTCCAAACAGGAGTTCATGGATGGCATGACAGAATTAGGATGTGACAGCATAGAAAAACTAAAGGCCCAGATACCCAAGATGGAACAAGAATTGAAAGAACCAGGACGATTTAAGGATTTTTACCAGTTTACTTTTAATTTTGCAAAGAATCCAGGACAAAAAGGATTAGATCTAGAAATGGCCATTGCCTACTGGAACTTAGTGCTTAATGGAAGATTTAAATTCTTAGACTTATGGAATAAATTTTTGTTGGAACATCATAAACGATCAATACCAAAAGACACTTGGAATCTTCTTTTAGACTTCAGTACGATGATT...

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Abstract

A construction method of a lentivirus capable of specifically inhibiting a DCUN1D1 gene comprises the following steps: S1, interfering target design and primer synthesis: S2, annealing primers to formdouble-stranded fragments with sticky ends: S3, synthesizing a sequence containing a restrictive endonuclease recognition sequence and a DCUN1D1 gene interference site by PCR reaction. S4, preparinga linearized expression vector; S5, connecting ane interference fragment of the DCUN1D1 with a double-digested pLKD-CMV-eGFP-U6-shRNA vector to form a recombinant plasmid: S6, carrying out competent cell transformation, culture, screening and sequencing verification on the recombinant plasmid, and selecting a correctly cloned recombinant bacterial solution; S7, extracting plasmids in the correctlycloned recombinant bacterial solution by using a plasmid extraction kit; S8, carrying out lentivirus production for specifically inhibiting DCUN1D1 gene expression by using a Swiss virus packaging system;S9, verifying the expression of the DCUN1D1 protein after the lentivirus produced in the previous step is infected with the lung squamous cancer cells by Western blot.

Description

technical field [0001] The invention belongs to the fields of molecular biology and oncology, and specifically relates to a lentivirus that specifically inhibits the DCUN1D1 gene and its construction method and application. Background technique [0002] DCUN1D1 is also known as RP42; SCRO; Tes3; DCNL1; SCCRO; DCUN1L1, its gene is located on human chromosome 3q26.33, has a sequence of 259 amino acids, and a molecular weight of about 30.1KDa. [0003] DCUN1D1 is an essential component of E3 ubiquitin ligase during ubiquitination, and promotes ubiquitination of E3cullin-RING ubiquitin ligase complex. It binds to the cullin-RBX1 complex in the cytoplasm and promotes the nuclear translocation of the complex, promotes the binding of E2-NEDD8 to the complex, and efficiently transfers NEDD8 from E2 to the cullin substrate protein. It is involved in the assembly and activity of the inhibitory CAND1 on the E3cullin-RING ubiquitin ligase complex. It is also an oncogene that promotes ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/113C12N15/66C12N5/10A61K31/7088A61P35/00
Inventor 宋鑫赵留芳孟旭东葛春蕾
Owner 信雅生物科技(苏州)有限公司
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