Novel E3 ubiquitin ligase TcpC and application thereof
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A technology of ubiquitin ligase and protein, applied in the fields of molecular biotechnology and medicine
Inactive Publication Date: 2020-07-14
ZHEJIANG UNIV CITY COLLEGE
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Problems solved by technology
Urinary tract infection is currently treated with antibiotics, but with the increase of drug resistance of pathogenic bacteria, it has brought great challenges to the treatment of urinary tract infection
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Embodiment 1
[0032] Example 1: Nucleotide (SEQ ID No. 1) and amino acid sequence (SEQ ID No. 2) of GenBank (GQ903014.1) TcpC.
Embodiment 2
[0033] Example 2: Construction and validation of pyelonephritis model mice and detection of MyD88 protein level in renal macrophages.
[0034] We have secreted TcpC CFT073 wild strain (CFT073 wt ) on the basis of gene homologous recombination to construct the CFT073 mutant strain (CFT073 Δtcpc ).
[0035] 1. Construction and verification of pyelonephritis model mice
[0036] 1) The CFT073 wt 、CFT073 Δtcpc Cultivate in LB liquid medium to the logarithmic growth phase, centrifuge at 5500rpm / min at 4°C for 30min, discard the supernatant, wash the bacteria three times with LB medium and resuspend to 1×10 10 CFU / mL.
[0037] 2) 24 female C57BL / 6 mice of SPF grade, 6-8 weeks old were selected and divided into blank control group, CFT073 wt group with CFT073 Δtcpc groups, with 8 mice in each group.
[0038] 3) Anesthetize the mouse with Avertin (40mg 2,2,2-tribromoethanol dissolved in 1mL tert-butanol) (intraperitoneal 0.01mL / g), and instill 0.1mL from the urethra to the blad...
Embodiment 3
[0078] Example 3: CFT073 wt Inhibition of MyD88 protein levels in macrophages.
[0079] 1. Preparation of mouse bone marrow macrophages (BMDM): SPF grade C57BL / 6 mouse bone marrow was aseptically taken, and bone marrow cell suspension was prepared with DMEM medium. Centrifuge with the density gradient of lymphocyte separation medium, absorb the mononuclear cell layer, wash with PBS and suspend in DMEM medium containing M-CSF (10ng / mL), adjust the cell concentration to 1×10 7 / mL, inoculated into 6-well plates. 37°C, 5% CO 2 After 3 days of culture, the adherent cells were supplemented with fresh medium, and the adherent cells were collected on the 7th day. BMDM were obtained by CD11b immunomagnetic bead sorting, and then CD11b was detected by flow cytometry + and F4 / 80 + Cell Purity.
[0080] 2. BMDM, macrophage cell line (J774A.1, RAW264.7) (5 × 10 5 ) respectively with CFT073 wt or CFT073 Δtcpc The co-cultivation was separated by Transwell (MOI=100) for 4, 8, and 12...
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Abstract
The invention discloses a novel E3 ubiquitin ligase TcpC and an application thereof. According to the invention, the TcpC has a ubiquitination function, and not only can cause ubiquitination in vitro,but also can cause ubiquitination in vivo. A research of the invention shows that the TIR containing protein encoded by E.coliprotein (TcpC) which contains a Toll/interleukin 1 receptor structural domain and is secreted by a uropathogenic E. coli CFT073 strain can enhance ubiquitination degradation of myeloid differentiation factor 88 (MyD88) in a toll-like receptor (TLR) signal pathway, block the TLR signal pathway, inhibit macrophage-mediated inherent immune function, and is closely related to occurrence of urinary tract infection such as pyelonephritis and the like caused by the uropathogenic E. coli (UPEC). The invention finds that a prokaryotic expression recombinant protein rTcpC can cause reduction of the MyD88 protein of macrophages in a dose-dependent manner, but does not influence transcription of myd88 mRNA, and the rTcpC can obviously enhance the ubiquitination level of the MyD88 in cells and in vitro. According to the invention, the TcpC can be used as a target point forpreparing medicaments for treating urinary tract infection, and is also a molecular marker for clinical laboratory examination of patients with urinary tract infection.
Description
technical field [0001] The invention belongs to the fields of molecular biotechnology and medicine, and specifically relates to a new E3 ubiquitin ligase TcpC and its application. Background technique [0002] Urinary tract infection (UTI) is inflammation of the urinary tract caused by various pathogens invading the urinary system, urinary tract mucosa or urinary tract tissue, and some severe patients can lead to renal failure. It is estimated that there are approximately 150 million UTI patients worldwide each year, with medical costs exceeding $6 billion. Therefore, if the occurrence of urinary tract infection can be controlled, it is of great significance to human health and social and economic development. Uropathogenic Escherichia coli (uropathogenic E.coli, UPEC) can specifically adhere to and implant in the epithelial cells of the urinary tract mucosa, leading to ascending urinary tract infection. Urinary tract infection is currently treated with antibiotics, but wi...
Claims
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