Preparation and application of prostate cancer targeting adenovirus

A technology for prostate cancer and adenovirus, applied in the fields of biotechnology and gene therapy, can solve the problem of weak immune response

Inactive Publication Date: 2010-12-29
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the gene carried by the oncolytic adenovirus is an immunoregulatory gene, and its activa

Method used

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  • Preparation and application of prostate cancer targeting adenovirus
  • Preparation and application of prostate cancer targeting adenovirus
  • Preparation and application of prostate cancer targeting adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of the TE vector containing the prostate cancer-specific promoter PPTp

[0042] The method is to use human genomic DNA as a template, design primers to amplify the prostate-specific antigen enhancer AREc3 region (PSAe), prostate membrane-specific antigen enhancer (PSMAe) and TARPp, and then use PCR to connect them into a chimeric The promoter PPTp replaces Tertp in TE-SV-tertp-E1A-GM-IRES-E1B55K (TE-Tertp-SV-IR-55K) to obtain the target plasmid TE-SV-PPT-E1A-GM-IRES-E1B55K ( TE-PPT-SV-IR-55K). (if attached figure 1 shown)

[0043] (1) Amplification of the prostate-specific antigen enhancer AREc3 region (PSAe)

[0044] Design primers for PSA enhancer sequence (AF243527:38200-38900) AREc3 region: see Sequence List 1 for the upstream of the outer primer, see Sequence List 2 for the downstream; see Sequence List 3 for the upstream of the inner primer, and see Sequence List 3 for the downstream For the PSMAe sequence, see Sequence Listing 4. Using...

Embodiment 2

[0054] Example 2 Construction of the shuttle plasmid pShuttle-cmv-PL-PPT-E1A-E1Bp-GM-IR-E1B55K

[0055] The method uses the cDNA of human prostate cancer cell LNCaP as a template to amplify prostate cancer-specific antigen (PSA); uses the cDNA of human leukemia cell Juarket as a template to amplify CD40L; the method of gene synthesis is used to synthesize PSA and CD40L connector. The above three fragments were connected by PCR to obtain the fusion expression cassette of PSA and CD40L, which was then cloned into the shuttle plasmid pShuttle-cmv to obtain pShuttle-cmv-PL. MfeI digested and ligated pShuttle-cmv-PL and TE-PPT-SV-IR-55K to obtain the target plasmid pShuttle-cmv-PL-PPT-E1A-E1Bp-GM-IR-E1B55K. (if attached figure 2 shown)

[0056] (1) PCR amplification of prostate cancer specific antigen (PSA)

[0057] Design primers for the PSA sequence (NM_001648.2, GI: 22208990): the upstream primer has a KpnI restriction site, such as sequence 14 in the sequence listing, and ...

Embodiment 3

[0071] Example 3 Preparation of recombinant adenovirus Ad-pSh-cmv-PL-PPT-E1A-E1Bp-GM-IR-E1B55K

[0072] The method is to digest the shuttle plasmids pshuttle-cmv-PL-PPT-E1A and pShuttle-cmv-PL with PmeI, electroporate BJ5183 competent cells, and homologously recombine with Adeasy-1 to obtain the recombinant adenovirus plasmid Ad-pSh-cmv-PL- PPT-E1A-E1Bp-GM-IR-E1B55K (Ad-pSh-PL-PPT-E1A-GM) and Ad-pSh-cmv-PL. Recombinant adenovirus plasmid was digested with PacI and transfected into HEK293 cells to prepare virus. After correct identification, proceed to amplification, purification and titer determination. (if attached Figure 4 shown)

[0073] (1) Production of recombinant adenovirus plasmid:

[0074] The shuttle plasmids pshuttle-cmv-PL-PPT-E1A and pShuttle-cmv-PL were digested with PmeI and dephosphorylated, and transformed into BJ5183-Adeasy-1 competent cells by electroporation under the conditions of "200Ω, 2.5KV, 25μF" (purchased from Stratagene), homologous recombinat...

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Abstract

The invention discloses preparation and an application of a prostate cancer targeting adenovirus and relates to the construction of a prostate cancer specific immunity and oncolytic dual-functional recombinant adenovirus carrier system and the expression of a target gene in a prostate cancer cell, the virus specificity replication and the verification of the oncolytic function, which belong to the fields of biotechnology and gene therapy. The adenovirus can be replicated massively in the prostate cancer cell, thus destroying the tumor cell; and the continuously replicated recombinant adenoviruses can express PSA-IZ-CD40L and GM-CSF efficiently, thus activating the specificity anti-tumor immune response of an organism. The immune response can not only cooperate with partial oncolytic effect of the tumor effectively but also kill and wound transferred prostate cancer cells through systemic immune response.

Description

technical field [0001] The invention relates to the fields of biotechnology and gene therapy, in particular to the establishment of a prostate cancer-targeted immuno-oncolytic bifunctional adenovirus vector system and its application in the treatment of prostate cancer. Background technique [0002] Prostate cancer is one of the most common malignant tumors in men. In 2005, prostate cancer ranked first in incidence and second in mortality among male cancers in the United States. In my country, with the process of population aging, the incidence of prostate cancer is also increasing year by year, seriously affecting the quality of life of elderly men. Traditional treatment methods such as surgery, hormones, radiotherapy and chemotherapy, and cryotherapy are often insensitive to patients with recurrence, metastasis, and hormone resistance. Therefore, exploring a new and more effective immune gene therapy program for prostate cancer has important theoretical and practical sig...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/861C12N15/62A61K48/00A61P35/00
Inventor 王立生杨月峰王华吴祖泽
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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