Method for producing endomorphin 2 via high-efficiency expression, and application for same

A high-efficiency expression technology of endomorphin, applied in the direction of biochemical equipment and methods, application, botany equipment and methods, etc., to achieve good effect and convenient administration route

Inactive Publication Date: 2013-09-25
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no relevant literature report on the use of human-derived nerve growth factor leader peptide to achieve high-efficiency secretion of endomorphin-2 at home and abroad

Method used

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  • Method for producing endomorphin 2 via high-efficiency expression, and application for same
  • Method for producing endomorphin 2 via high-efficiency expression, and application for same
  • Method for producing endomorphin 2 via high-efficiency expression, and application for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Acquisition of Fusion Gene and Construction of Adenovirus

[0031] 1. Acquisition and identification of target genes

[0032] The target gene was amplified from the plasmid pTCNE by PCR method, and the primers used for the amplification were sequenced as follows:

[0033] Forward sequence: 5'-ACGCGAATTCACCATGTCCCAT-3' (SEQ ID NO: 4);

[0034] Reverse sequence: 5'-CAAGAGCAAGCGCTACCCCTTCTTCTAAGGATCCATAT-3' (SEQ ID NO: 5);

[0035] PCR system:

[0036]

[0037] Melting at 94°C for 5 minutes; 32 cycles of 94°C for 50s, 56°C for 50s, and 72°C for 90s; elongation at 72°C for 10 minutes. Take 5 μl and do 1% agarose gel electrophoresis (containing EB 0.5 μg / ml; voltage: 80V) for identification; the rest is used for recovery and purification of target fragments.

[0038] The target gene and pUC19 plasmid were digested by EcoRI and BamHI respectively, then ligated into pUC19-NEM2 with T4 DNA ligase, and transformed into E.coli.DH5α competent cells (purchased from ...

Embodiment 2

[0047] Example 2: Expression of Endomorphin 2

[0048] The adenovirus Ad-hPNEM2 constructed in Example 1 was transfected into host cells: HEK293 cells (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences), and the culture condition of HEK293 cells was DMEM with 10% fetal bovine serum (purchased from GIBICO Company) , 37°C, 5% CO 2 .

[0049] On the 7th day after virus infection, the cells were broken by high-speed centrifugation to remove cell fragments, and centrifuged again to collect the cell supernatant culture medium, which contained a large amount of EM-2 protein, and the concentration of endomorphin 2 was detected by EIA method to reach 1873.56± 225.17ng / ml.

[0050] The expression product endomorphin 2 was collected, and endomorphin 2 was purified by silica gel column chromatography, followed by gradient elution with dichloromethane, dichloromethane:methanol=19:1 to obtain a yellow oil, which was dried in vacuum A yellow blocky solid was o...

Embodiment 3

[0051] Example 3: Analysis of the secretion and expression of endomorphin-2 in vitro

[0052] The six-well plate NIH3T3 cell adenovirus Ad-hPNEM2 infection test was carried out. The control groups were the Ad-EGFP group (constructed in the previous experiment), the blank group without virus infection, and Ad-rPNEM2 with mouse-derived nerve growth factor as the leader peptide. Control group (preliminary experimental construction). On the 7th day after the virus infection, collect the cell culture fluid on the 1st, 3rd, 7th, and 10th day after the infection, centrifuge to remove the cells, and measure the concentration of EM-2 protein.

[0053] The results found that the expression of endomorphin-2 in Ad-hPNEM2 group and Ad-rPNEM2 group was significantly increased, and the content of endomorphin-2 in Ad-hPNEM2 group was significantly higher than that of Ad-rPNEM2 group (Pfigure 1 ). The results show that the DNA sequence inserted into the viral genome can correctly translate th...

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Abstract

The invention belongs to the technical field of biological genes, wherein the related literature reports of realizing high-efficiency secretion for endomorphin 2 by virtue of a human-original nerve growth factor leader peptide are inexistent at present. The invention provides a method for producing endomorphin 2 via high-efficiency expression. The method comprises the following steps of: designing a fused gene including a human-original nerve growth factor signal peptide sequence and an endomorphin 2 expression sequence; then inserting the fused gene in an expression carrier; then converting host cells, screening and authenticating positive recombinants, performing induced culture, and externally secreting expression protein; and finally collecting the product via purification. The expression product, namely, endomorphin 2, prepared by the method, is applied to preparation for medicines treating chronic pain and cancer pain. The product is an endogenous opiate peptide, thus being free form the reactions of habituation, tolerance and the like, as well as capable of being expressed for a long term in a body, so as to achieve the purpose of treating pain for a long time.

Description

technical field [0001] The invention belongs to the technical field of biological genes, and relates to the high-efficiency expression and production of endomorphin 2 and its application, in particular to the large-scale expression and high-efficiency exocrine of biologically active endomorphin 2, and its use in the preparation and treatment of chronic pain and Application in cancer pain medicine. Background technique [0002] Chronic pain is one of the manifestations of neuronal plasticity changes, and its physiological characteristics are increased pain responsiveness, mainly manifested as hyperalgesia and allodynia. The treatment of this type of pain is difficult, mainly because its mechanism is unknown. At present, in treatment, opioid receptor blockers are mainly represented by morphine, but there are many side effects, addiction, tolerance and other reactions, which limit its long-term use. [0003] Endomorphin-2 is an endogenous ligand with high affinity and high se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/867C12N15/869C12N15/864C12N15/85A61K38/07A61P25/04
Inventor 吴飞翔俞卫锋孙玉明
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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