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Recombinant adenovirus carrier and application thereof

A recombinant adenovirus, vector plasmid technology, applied in the direction of virus/bacteriophage, recombinant DNA technology, introduction of foreign genetic material using vectors, etc., can solve the problems of low survival rate of malignant tumor patients and poor prognosis of solid tumors.

Inactive Publication Date: 2010-11-24
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Although the level of medical diagnosis and treatment technology has been significantly improved in recent years, including radiotherapy and chemotherapy, the prognosis of most solid tumors is still poor, and the five-year survival rate of malignant tumor patients is still very low, such as pancreatic cancer (4%) ), liver cancer (7%), and lung cancer (15%), there is an urgent need for new treatment methods to relieve the suffering of patients and improve the prognosis (Cross, D. and J.K.Burmester, Gene therapy for cancer treatment: past, present and future. Clin Med Res, 2006.4(3): p.218-27.)

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  • Recombinant adenovirus carrier and application thereof
  • Recombinant adenovirus carrier and application thereof
  • Recombinant adenovirus carrier and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1, the preparation of Ad-DENND2D recombinant adenovirus

[0053] pShuttle-CMV shuttle vector (see figure 1 ): Nosy Genetics.

[0054] pAdxsi adenoviral backbone vector (see figure 2 ): Nosy Genetics.

[0055] 1. Construction of pAdxsi-DENND2D recombinant plasmid

[0056] 1. Construction of DENND2D recombinant shuttle vector

[0057] The primers of the DENND2D gene with the SfiI restriction endonuclease site were designed as follows:

[0058] DENND2D Sfi I up: 5'-AAAAAAGGCCGCTGCGGCCCACTCCAGGGGCCATGGATG-3';

[0059] DENND2D Sfi I dw: 5'-AAAAAAGGCCTGTTTGGCCGTCATTCTTATTCACCACAGCTC-3'.

[0060] ①Use the above primers to obtain the full-length coding region of the target DENND2D gene by PCR from the human lung tissue cDNA library (purchased from Clontech Company). 3-4 hours. A 1.4kb fragment was recovered.

[0061] ② The pShuttle-CMV shuttle vector was digested with restriction endonuclease SfiI at 37°C for 3-4 hours, and then the vector was dephosphoryla...

Embodiment 2

[0105] Example 2, Induction of apoptosis in vitro by Ad-DENND2D recombinant adenovirus

[0106] Infect the human lung cancer cell line H1299 cells with Ad-DENND2D (or Adv) according to the dose of moi 100 or moi 200 respectively, collect the cells after 48 hours for Annexin V-FITC and PI double staining, and detect cell apoptosis by flow cytometry Happened. The experiment was repeated three times, and the results were averaged.

[0107] see results Figure 5 (The figure shows the results of one of the experiments). After infection with a dose of moi100, the apoptosis rate of Ad-DENND2D group was 17.09±3.22%, which was significantly higher than that of Adv group (11.36±1.25) (P<0.05). After infection with a dose of moi200, the apoptosis rate of the Ad-DENND2D group was 21.76±1.99%, which was significantly higher than that of the Adv group (14.06) (P<0.05). For cells infected with Ad-DENND2D, the apoptosis rate of the moi200 dose group was higher than that of the moi100 dose...

Embodiment 3

[0108] Example 3, In vivo anti-tumor effect of Ad-DENND2D recombinant adenovirus

[0109] 1. Select 24 female BALB / c nude mice (purchased from Victoria Lihua Company) aged 3 to 4 weeks; collect H1299 cells in the logarithmic growth phase (purchased from ATCC), and adjust the cell concentration to 2.5×10 7 / ml.

[0110] 2. Take 200 μl of cell suspension and inoculate it subcutaneously in the right armpit of nude mice. After about 10 days, when the tumor grows to a size of 5×5 mm, divide them into four groups, with 6 mice in each group, and perform the following treatments:

[0111] Normal saline injection group (NS): injected with normal saline, once every three days at multiple points in the tumor, 50 μl each time, three times in total.

[0112] Adv virus group (Adv): adjust the Adv virus titer to be 2×10 10 pfu / ml, intratumoral multi-point injection once every three days, 50 μl each time, a total of three times, the cumulative virus injection volume was 3×10 9 pfu.

[011...

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Abstract

The invention discloses a recombinant adenovirus carrier and application thereof. A plasmid of the recombinant adenovirus carrier is a pAdxsi plasmid carrying a DNA fragment (a) or (b): (a) a DNA fragment of a coding gene which contains a CMV promoter and DENND2D protein from the upstream to the downstream, and (b) a DNA fragment of a coding gene which contains a tetracycline reactive factor, a promoter, and a DENND2D protein from the upstream to the downstream. Two recombinant adenoviruses both have inhibiting effects on various tumor cell lines in vitro and in vivo and have a certain genetic therapy value.

Description

technical field [0001] The present invention relates to recombinant adenovirus vector and its application. Background technique [0002] Malignant tumors are currently the number one cause of death in my country, seriously endangering people's health. Although the level of medical diagnosis and treatment technology has been significantly improved in recent years, including radiotherapy and chemotherapy, the prognosis of most solid tumors is still poor, and the five-year survival rate of malignant tumor patients is still very low, such as pancreatic cancer (4%) ), liver cancer (7%), and lung cancer (15%), there is an urgent need for new treatment methods to relieve the suffering of patients and improve the prognosis (Cross, D. and J.K.Burmester, Gene therapy for cancer treatment: past, present and future. Clin Med Res, 2006.4(3): p.218-27.). In the past 30 years, the occurrence and development of malignant tumors have been deeply understood. At present, most scholars believ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N7/01A61K48/00A61P35/00C12R1/93
Inventor 付国斌马大龙程书钧高燕宁郑宏伟石太平郭金海
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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