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Use of PNAS-4 gene in preparing antineoplastic and antineoplastic auxiliary medicament

An anti-tumor drug and anti-tumor technology, applied in the direction of anti-tumor drugs, drug combinations, recombinant DNA technology, etc., can solve the problem of lack of comprehensive search for homologous genes and functional research, few genes for tumor growth, and damage to the health of the body and other problems, to achieve the effect of overcoming toxic and side effects, good market prospects, and inhibiting growth

Inactive Publication Date: 2010-11-03
SICHUAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Among them, the urgent problem to be solved is that there are too few genes currently used for tumor gene therapy, and there are not many genes that can inhibit tumor growth, and there is an urgent need to provide more genes that can be used
At the same time, traditional chemotherapy and radiotherapy will also have a negative impact on the patient's immune system, which will damage the body's health and reduce the quality of life. Therefore, looking for anti-tumor adjuvant drugs, especially radiotherapy and chemotherapy adjuvant drugs is also a research hotspot in the field of cancer treatment
[0005] The human PNAS-4 (h PNAS-4) gene is one of the novel genes identified in large-scale sequencing of the human genome (Strausberg, RL, Feingold, EA, Grouse, LH, et al. Generation and initial analysis of more than 15,000full -length human and mouse cDNA sequences.Proc.Natl.Acad.Sci.U.S.A.2002.99(26):16899-16903), registered in the database of NIH in the United States, and the search and functional research of its homologous genes have not yet been fully carried out

Method used

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  • Use of PNAS-4 gene in preparing antineoplastic and antineoplastic auxiliary medicament
  • Use of PNAS-4 gene in preparing antineoplastic and antineoplastic auxiliary medicament
  • Use of PNAS-4 gene in preparing antineoplastic and antineoplastic auxiliary medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0062] The acquisition of embodiment one PNAS-4 gene

[0063] 1. Cloning of human PNAS-4 (hPNAS-4) gene cDNA

[0064] According to the full-length human PNAS-4 gene sequence (NM-016076) submitted in NCBI, primers were designed to amplify the coding sequence of PNAS-4 gene. In order to further clone into the pGEX-6p-1 vector, BamH I and Xho I restriction sites were respectively introduced into the upper and lower primers of the coding sequence.

[0065] Upstream primer (SEQ ID NO.7):

[0066] 5′-GC GGATCC AAGATGGCAGATTTTTTGAAAGG-3′;

[0067] Downstream primer (SEQ ID NO.8):

[0068] 5′-CGC GATATC TTAAGTGTCCTC GTGCATGTCTG-3' (the enzyme cleavage site is underlined).

[0069] PCR amplification primers were synthesized by Shanghai Boya Company. To enhance the expression level of the PNAS-4 gene, the following primers were used when constructing the pcDNA3.1-hPNAS-4 recombinant plasmid:

[0070] Upstream primer (SEQ ID NO.9): 5'-GC GGATCC AAGATGGCAGATTTTTTGAAAGG-3' (t...

Embodiment 2

[0160] Embodiment two can be used for the construction of the PNAS-4 recombinant vector of gene therapy

[0161] 1. Construction of recombinant plasmid PNAS-4-pVAX1:

[0162] The PNAS-4 cloned on pcDNA3.1-hPNAS-4 in Example 1 was excised with BamH I and Xho I enzymes according to conventional techniques in the art, and inserted into the BamH I and Xho of the pVAX1 plasmid purchased on the market.

[0163] It was obtained at the I site, and the agarose electrophoresis and sequencing verifications were correct.

[0164] 2. Preparation of liposome complexes of recombinant plasmid PNAS-4-pVAX1

[0165] Lipofectamine (lipofectamine2000) was purchased from Invitrogen.

[0166] The liposome complex of the recombinant plasmid PNAS-4-pVAX1 was prepared according to the instructions of lipofectamine2000, the amount of liposome was screened, and liposome:plasmid=1:3 (weight ratio) was finally determined. 3. Construction of PNAS-4 recombinant adenovirus Construction, production and pur...

Embodiment 3

[0192] Example three recombinant adenovirus pAd-DEST-hPNAS-4 and pAd-DEST-mPNAS-4 experimental results

[0193] In vitro experiments

[0194] pAd-DEST-hPNAS-4 was selected for in vitro experiments.

[0195] DNA ladder (DNA ladder strip) test results

[0196] The results of DNA ladder showed that LL / 2 cells were treated with liposome-wrapped pAd-DEST-hPNAS-4, and after 24 hours of treatment, obvious DNA ladder phenomenon was found in this group of cells, while liposome-wrapped pAd-DEST-Null and other control groups did not. The phenomenon of DNA ladder can be observed, which indicates that liposome-wrapped pAd-DEST-hPNAS-4 can obviously induce apoptosis of LL / 2 cells.

[0197] Apoptosis PI staining experiment

[0198] The results of apoptosis PI staining experiment are shown in ( Figure 11) showed that LL / 2 cells were treated with liposome-wrapped pAd-DEST-hPNAS-4 for 24 hours and found that this group of cells had obvious apoptosis, while liposome-wrapped pAd-DEST-Null tr...

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Abstract

The invention relates to a use of PNAS-4 gene in aspects of preparing antineoplastic in the tumour gene treatment field. The invention with PNAS-4 recombinant vector can limit the growth and the migration of the endocytosis effectively, which can limit the growth of various tumors and extend the survive time of mouse. The antineoplastic of the invention with PNAS-4 recombinant vector liposome andchemotherapy drug cisplatin and magnolol as the active component has better effect than the single application and can reduce the usage of both parties. The product has the appreciable effect, the little toxic and side effect and the easy preparing method, which compensates the defect of protein infusion drug, increases the time interval of medicine, reduces the usage, reduces the economic burdenof the patient, improves the life quality of patient and has the wide market prospect.

Description

technical field [0001] The invention belongs to the field of tumor gene therapy, and specifically relates to the application of PNAS-4 gene in the preparation of antitumor drugs and antitumor auxiliary drugs. Background technique [0002] Tumor cells are a group of cells that proliferate out of control and express original traits, and the occurrence of tumors is related to the imbalance of "proliferation-apoptosis" of tumor cells. Apoptosis is closely related to the occurrence and development of tumors, as well as cell death in tumor treatment. Apoptosis is an important part of the cell surveillance system. When the cell genome is damaged, the cell first starts the repair mechanism, and once the damage is completely repaired, the cell will re-enter the normal growth state; if the repair fails, the apoptosis mechanism will be activated, and the damaged cells will enter apoptosis and be eliminated, thereby avoiding Genomic damage is passed on to progeny cells, eliminating th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K45/00A61P35/00C12N15/79
Inventor 魏于全邓洪新赵霞梁淑芳陈俐娟杨寒朔
Owner SICHUAN UNIV
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