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Preparation and application of a drug for overexpressing PCNP gene

A GV230-PCNP, overexpression technology, applied in the direction of drug combination, gene therapy, anti-tumor drugs, etc., can solve the problems of lack of research reports

Active Publication Date: 2021-02-26
吴东栋 +8
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the correlation between PCNP and tumors needs to be further studied, and there is a lack of relevant research reports on the role of regulating PCNP overexpression in tumor treatment.

Method used

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  • Preparation and application of a drug for overexpressing PCNP gene
  • Preparation and application of a drug for overexpressing PCNP gene
  • Preparation and application of a drug for overexpressing PCNP gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The present embodiment provides a kind of construction method of the plasmid of overexpression PCNP gene, it comprises the following steps:

[0052] 1. First, according to the PCNP gene sequence (NM_020357), design primers for PCR amplification of the target gene. The specific sequence is as follows:

[0053] Forward primer:

[0054] 5'-TACCGGACTCAGATCTCGAGCGCCACCATGGCGGACGGGAAGGCGGGAGAC-3' (as shown in SEQ ID NO: 1);

[0055] Reverse primer:

[0056] 5'-GATCCCGGGCCCGCGGTACCGTaTTGTCTTGGTCATGGACATTTC-3' (shown in SEQ ID NO: 2).

[0057] Digest the restriction enzyme sites XhoI and KpnI in the GV230 vector (carrier sequence: CMV-MCS-EGFP-SV40-Neomycin) to form a linearized GV230 vector;

[0058] 2. Acquisition of target gene fragments

[0059] The primers synthesized by Jinweizhi Biotechnology (Beijing) Co., Ltd. were dissolved in double distilled water to prepare a solution with a concentration of 100 μM. The 50 μL reaction system was designed as follows:

[0060] 5...

Embodiment 2

[0097] This example provides the application of the plasmid overexpressing the PCNP gene prepared in the above example in the preparation of a drug for treating human neuroblastoma. The plasmid overexpressing the PCNP gene was transfected into human neuroblastoma cells as a drug for the treatment of human neuroblastoma, a stable strain was obtained by G418 screening, and finally the PCNP gene was overexpressed. The specific operation process was introduced as follows:

[0098] 1. First determine the optimal concentration of geneticin G418, that is, the lowest concentration of G418 that can completely kill cells, specifically:

[0099] Human neuroblastoma cells SH-SY5Y and SK-N-SH were digested and inoculated in 96-well culture plates with an inoculation amount of 1×104 cells / well;

[0100] After the tumor cells were inoculated in the 96-well culture plate, 100 μL of medium containing G418 was added to each well the next day, and the G418 gradient setting was as follows: 100 μg...

Embodiment 3

[0132] In order to detect the influence of the overexpressed PCNP gene on tumor cell proliferation in the above-mentioned embodiment 2, the inventor has done further detection experiments, and the relevant process is introduced as follows:

[0133] 1. First, the MTS method was used to determine the effect of PCNP overexpression on tumor cell proliferation. The specific process was as follows:

[0134] The tumor cells of the negative control and PCNP gene overexpression in Example 2 were digested and counted respectively, spread to a 96-well culture plate, and the inoculation amount was 8×10 3 Each well, 6 auxiliary wells for each group, add 100 μL medium to each well, and incubate in an incubator at 37°C;

[0135] After 24 hours, add 20 μL and 5 mg / mL MTS solution to each well, and continue to culture in the incubator for 2 hours;

[0136] Then the cells were taken out, and the absorbance value of each well was measured at 490 nm using a microplate reader, and the inhibition ...

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Abstract

The invention provides the preparation and use of a drug overexpressing PCNP gene. The medicine includes a plasmid overexpressing PCNP gene and PCNP protein obtained by overexpressing PCNP gene. The construction method of the plasmid overexpressing PCNP gene, which comprises the following steps: according to the PCNP gene sequence, design primers, PCR amplify the target gene; carry out restriction enzyme cutting site Xho I and Kpn I in the GV230 vector. , to form a linearized GV230 vector; the target gene is connected with the linearized GV230 vector to construct a GV230-PCNP vector, which is a plasmid that overexpresses the PCNP gene. When the drug overexpressing PCNP gene is used in human neuroblastoma, the overexpression of PCNP gene in human neuroblastoma can promote cell apoptosis, inhibit cell growth, inhibit cell migration, and inhibit the growth of human neuroblastoma. grow.

Description

technical field [0001] The application belongs to the technical field of biopharmaceuticals, and in particular relates to the preparation and application of a drug that overexpresses the PCNP gene. Background technique [0002] Neuroblastoma (neuroblastoma, NB) is an embryonal tumor, mostly located in the cerebral hemispheres. It is the most common extracranial tumor in children and the most common tumor in infants and young children. Nearly half of neuroblastomas occur in infants and young children under the age of 2. Neuroblastoma accounts for about 6-10% of childhood tumors and 15% of childhood cancer mortality. [0003] PCNP (PEST-containing nuclear protein) is a newly discovered nuclear protein containing PEST sequence. PCNP as the substrate of ubiquitin ligase NIRF (Np95 / ICBP90-like RING figer protein) was first reported by Japanese scientists in 2004 , PCNP and NIRF together constitute a new signal transduction pathway, which is closely related to the regulation of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/85C12N15/52A61K48/00A61P35/00
Inventor 吴东栋李涛高荧苒王大勇陆丹刘诗语洪亚魏建设姬新颖
Owner 吴东栋
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