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Polypeptide based on human cell surface display technology and targeted HBV receptor and application of polypeptide

A surface display and cell technology, applied in the fields of molecular biology and biomedicine, can solve the problems of membrane proteins not forming a functional normal structure, membrane proteins are difficult to express and purify, and cannot be used, so as to improve the anti-HBV infection effect. Effect

Pending Publication Date: 2022-07-05
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, membrane proteins are difficult to express and purify from prokaryotic cells; on the other hand, membrane proteins may not form a normal structure with functions when detached from the membrane structure, so they cannot be used as actual screening targets

Method used

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  • Polypeptide based on human cell surface display technology and targeted HBV receptor and application of polypeptide
  • Polypeptide based on human cell surface display technology and targeted HBV receptor and application of polypeptide
  • Polypeptide based on human cell surface display technology and targeted HBV receptor and application of polypeptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Plasmid construction

[0052] Plasmids GFP-FRB, FRB-GFP, Cherry-FasL-FKBP, NTCP-GFP were constructed in the early stage, and other plasmids were constructed on this basis.

[0053] The plasmid GFP-FRB is a plasmid obtained by inserting the green fluorescent protein GFP gene into the expression frame between the CMV promoter and the SV40 terminator with RlucN-HBC as the backbone, wherein GFP is located at the N-terminus and FRB is located at the C-terminus.

[0054] The plasmid FRB-GFP is a plasmid obtained by inserting the green fluorescent protein GFP gene into the expression frame between the CMV promoter and the SV40 terminator with RlucN-HBC as the backbone, wherein FRB is located at the N-terminus and GFP is located at the C-terminus.

[0055] The plasmid Cherry-FasL-FKBP is a plasmid obtained by inserting the fusion gene of red fluorescent protein-FasL-FKBP into the expression frame between the CMV promoter and the SV40 terminator with RlucN-HBC as the b...

Embodiment 2

[0087] Example 2 Feasibility verification of flow sorting and enrichment

[0088] 1. Using the FKBP12-FRB system to verify the feasibility of flow sorting and enrichment

[0089] One of the keys to establishing the screening system in the technical solution of the present invention is that flow cytometry can be used to perform sorting and enrichment, and to isolate those polypeptide-displaying cells that can bind to NTCP. To verify the feasibility of this flow sorting method, we first performed a simulation test using rapamycin to modulate the FKBP12-FRB interaction system.

[0090] The plasmid GFP-FRB was transfected into HEK293 cells. After 48 hours of transfection, the cells were collected by trypsinization and resuspended in PBS, and then the cells were lysed by repeated freezing and thawing in liquid nitrogen and a 37°C water bath. The lysed cell debris was removed by centrifugation, and the supernatant containing the fusion protein GFP-FRB was obtained for use. The pla...

Embodiment 3

[0108] Example 3. Verification of the anti-HBV infection effect of Myr-217-3 in HepG2-NTCP cell model

[0109] The polypeptide 217-3 (N-terminal myristoylation modification) screened in Example 2 and the polypeptide Myr21 as a control were synthesized in vitro. HepG2-NTCP was pretreated with different concentration gradients of polypeptides for 2 hours, and then infected with hepatitis B virus. The medium was changed every 2 days, and the HBeAg and HBsAg in the cell culture supernatant were detected simultaneously on the seventh day. The results show( Figure 10 ), the IC50 of Myr-217-3 (N-terminal myristoylation-modified polypeptide 217-3) in inhibiting HBV infection was improved by about 8 times compared with Myr-21.

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Abstract

The invention discloses a human cell-based surface display technology which comprises the following steps: S1, fusing receptor protein with fluorescent protein to obtain membrane fragments containing receptor-fluorescent protein fusion protein for later use; s2, fusing to-be-screened polypeptide with the transmembrane sequence of FasL and fluorescent protein, constructing fluorescent protein-FasL transmembrane region-polypeptide fusion protein plasmids, and obtaining a stable expression cell line as a cell surface display polypeptide library; s3, carrying out mixed incubation on the receptor-fluorescent protein fusion protein membrane fragments and the stable expression cell line obtained in the step S2, then sorting display cells with high affinity with the receptor by using a flow cytometry, and screening and enriching to obtain target polypeptide with high affinity with the receptor; the invention further discloses polypeptide 217-3 or 217-4 of a targeted HBV receptor, and experiments prove that IC50 of Myr-217-3 for inhibiting HBV infection is improved by about 8 times compared with that of Myr-21.

Description

technical field [0001] The present invention relates to the technical fields of molecular biology and biomedicine, in particular to a human cell-based surface display technology, a polypeptide targeting HBV receptors, and applications thereof. Background technique [0002] The process of enveloped virus infection of cells depends on the binding of viral envelope proteins to cell surface receptors. Therefore, interfering with the binding of viral envelope proteins to receptors can be an effective antiviral strategy. To achieve this strategy, the corresponding viral envelope protein can be blocked, or the corresponding receptor on the cell surface can be blocked, thereby preventing the binding of the two. Molecules that can interact with receptors to prevent them from binding to viral proteins can be compound molecules or polypeptide molecules. Compared with compound molecules, polypeptide molecules are composed of amino acid modules, and huge molecular diversity can be obta...

Claims

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Application Information

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IPC IPC(8): C12N15/85C07K19/00C07K14/02A61K38/16A61P1/16A61P31/20
CPCC07K14/005C12N15/85A61P1/16A61P31/20C12N2730/10122C07K2319/03C07K2319/60C12N2800/107C12N2800/90A61K38/00
Inventor 胡接力王珮耘黄爱龙
Owner CHONGQING MEDICAL UNIVERSITY
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