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LDLR-Lamp2b fusion gene, expression vector and application thereof

A technology of fusion gene and expression vector, applied in the field of genetic engineering, can solve the problem of untimely LDL clearance and other problems

Inactive Publication Date: 2019-02-01
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Endogenous PCSK9 can also competitively inhibit LDLR, leading to untimely clearance of LDL, which is an important mechanism for the development of atherosclerosis

Method used

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  • LDLR-Lamp2b fusion gene, expression vector and application thereof
  • LDLR-Lamp2b fusion gene, expression vector and application thereof
  • LDLR-Lamp2b fusion gene, expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Trizol extracts total RNA from 100mg mouse muscle tissue;

[0046] Using Promega M-MLV reverse transcriptase to reverse transcribe RNA to obtain mouse fibroblast cDNA;

[0047] Using reverse transcribed cDNA as a template and using SEQ ID NO.5-8 as primers to amplify Lamp2b5 and Lamp2b3 fragments;

[0048] Cloning Lamp2b5 into the pcDNA3.1 vector through restriction enzymes Nhe1 and Xho1 to obtain pcDNA3.1(-)-Lamp2b5;

[0049] Cloning Lamp2b3 into the pcDNA3.1(-)-Lamp2b5 vector through restriction enzymes Xho1 and BamH1 to obtain pcDNA3.1(-)-Lamp2b;

[0050] On the pUC57 vector backbone, directly synthesize the LDLR sequence (SEQ ID NO.9) without restriction sites, and clone the above-mentioned LDLR sequence into the pcDNA3.1-Lamp2b vector by restriction endonucleases Xho1 and BspE, as follows: figure 1 pcDNA3.1(-)-LDLR-Lamp2b indicated.

Embodiment 2

[0052] Transfect the exosome packaging cell AML12 with the pcDNA3.1-LDLR-Lamp2b vector obtained in Example 1, change the 10% serum DMEM culture medium after 6 hours after transfection, continue to cultivate and then change to serum-free conditions for 12 hours After culturing, exosomes were collected between 48 and 72 hours, and exosomes were extracted by exosome separation and extraction methods. The results of WB detection and IP experiments were as follows: figure 2 As shown, the target LDLR is loaded into the surface of exosomes, and the effect of exosomes is as follows image 3 shown.

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Abstract

The invention provides an LDLR-Lamp2b fusion gene, an expression vector and an application thereof, and relates to the field of genetic engineering technology. The nucleotide sequence of the LDLR-Lamp2b fusion gene is as shown in SEQ ID NO. 1. The extracellular segment of LDLR and the full length of Lamp2b are fused in a unique manner as a complete gene. After transfection of the gene to the exosome packaging cells with the expression vector, the LDLR can be presented on the surface of the exosome. The exosome can carry LDL into cells of tissues such as the liver, thereby promotiing the clearance of LDL and exerting effects similar to or even better than effects of PCSK9 inhibitors. The gene can be used for the prevention and treatment of atherosclerosis.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an LDLR-Lamp2b fusion gene, an expression vector and an application thereof. Background technique [0002] Cardiovascular disease ranks among the three major diseases that currently threaten human health, namely, cardiovascular disease, tumors and infectious diseases, and atherosclerosis is one of the most important pathological bases of cardiovascular disease. The main causes of death from cardiovascular and cerebrovascular diseases such as infarction, cerebral infarction and coronary heart disease are related to the occurrence and development of many diseases. Prevention and treatment of atherosclerosis is the fundamental measure to prevent and treat cardiovascular diseases. [0003] The prevention of atherosclerosis is divided into two levels: the first level of prevention is a light diet, no smoking, no drinking of strong alcohol, and a good mood; the...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C12N5/10A61K48/00A61K38/17A61P9/10
CPCA61K38/177A61K48/005A61P9/10C07K14/705C07K2319/00C12N15/85
Inventor 袁丽君李者龙杨薛康赵联璧邢长洋杨国栋
Owner FOURTH MILITARY MEDICAL UNIVERSITY