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HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine

A recombinant vector and gene technology, applied in gene therapy, genetic engineering, plant gene improvement, etc., can solve the problems of uncontrollable expression, the influence of the second gene expression efficiency, and the impossibility of controlling the infection efficiency of two adenoviruses. To achieve the effect of improving the therapeutic effect and good application prospects

Active Publication Date: 2012-09-12
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Double gene transfer usually uses two vectors to carry two different genes, but it is almost impossible to control the infection efficiency of the two adenoviruses in vivo studies, and it is also difficult to control the expression of the two genes
Or insert an internal IRES site between two genes to share the same promoter, but often the expression efficiency of the second gene is affected by the expression of the first gene, and the expression level is difficult to control

Method used

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  • HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine
  • HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine
  • HBx and human IL-12 double-gene recombinant vector and liver caner-resistant vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction and screening of HBx and hIL-12 double-gene recombinant adenoviral vector

[0044] The construction process of HBx and hIL-12 double-gene recombinant adenoviral vector is as follows: on the basis of the double-gene shuttle vector pAdV-hcERB2 / hIL-12 (the nucleotide sequence of its expression frame is shown in Seq ID NO.6) using Age I digestion makes it linearized, DNA fragments are blunt-ended, and then Nhe I is digested to generate sticky ends. Plasmid pcDNA3.1-HBx (with the nucleotide sequence shown in Seq ID NO.7) was subjected to a double enzyme digestion reaction with Pme I and Xba I to obtain a -HBx- fragment, one end was blunt and the other end was digested by Xba I resulting sticky ends. -pAdV-hIL-12-large fragment and -HBx-small fragment were ligated by T4DNALigase, recombined into pAdV-HBx / hIL-12 shuttle plasmid, and pAdV-HBx / hIL-12 was recombined by two-step bacterial homologous recombination The HBx / hIL-12 gene on the shuttle plasmid ...

Embodiment 2

[0095] Example 2 Construction and Screening of HBx and mIL-12 Double Gene Recombinant Adenoviral Vector

[0096] The construction process of the HBx and mIL-12 double-gene recombinant adenoviral vector is as follows: on the basis of the recombinant HBx and hIL-12 double-gene shuttle vector pAdV-HBx / hIL-12 in Example 1, pAdV-HBx / hIL-12 was digested with PmeI hIL-12 shuttle plasmid, recover large fragment (pAdV-HBx-), use T4 DNA Ligase to connect mIL-12 PCR product with Pme I restriction site to pAdV-HBx-large fragment, recombine into pAdV-HBx / mIL-12 shuttle plasmid, the HBx / mIL-12 gene on the pAdV-HBx / mIL-12 shuttle plasmid was recombined into the adenovirus backbone vector pAdenoVator ΔE1 / E3 using a two-step homologous recombination method in bacteria to obtain an adenovirus plasmid pAd-HBx / mIL-12, and then packaged in 293A cells into HBx and mIL-12 double-gene recombinant adenocarcinoma (Ad-HBx / mIL-12).

[0097] The specific process of constructing HBx and mIL-12 double-gen...

Embodiment 3

[0106] Example 3 In vivo efficacy experiment of the double-gene recombinant adenovirus vaccine of the present invention

[0107] 1. Preventive immunization experiments

[0108] Ad-HBx / IL-12 intramuscularly injected into 6-8 week old C57 mice 10 7 vp / 100μl / time, Ad-HBx 10 7 vp / 100μl / time, Ad-IL-12 10 7 vp / 100μl / time, Ad-null 10 7 vp / 100 μl / time or normal saline 100 μl / time, once a week, 3 times in total, 7 days after the last immunization, subcutaneously inoculate 2×10 6 A Hepa1-6 / HBx mouse liver cancer cell, when the tumor was palpable under the mouse skin, the length and width of the tumor were measured with a vernier caliper every 3 days, according to the formula: tumor volume (mm 3 )=0.52×length×width 2 Calculate the tumor volume, record the death time of the mice, and draw the tumor growth curve and survival curve.

[0109] Figure 12 A and 12B show: In the preventive immunization test, the tumor growth rate of Ad-HBx / IL-12 group was significantly slower than that o...

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PUM

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Abstract

The invention belongs to the field of gene therapy and aims at providing a novel targeted gene vaccine for treating HBV (Hepatitis B Virus)-relevant liver cancer and application of the novel targeted gene vaccine in preparation of a targeted gene immunotherapeutic drug for the HBV-relevant liver cancer. The main active component of the liver caner-resistant vaccine disclosed by the invention is a recombinant vector of a gene coded with an HBx protein and a gene for coding a human IL-12 protein; and the recombinant vector can be used for simultaneously expressing the HBx protein and the human IL-12 protein in a eukaryotic cell. Provided by the experiment, a recombinant adenovirus vaccine disclosed by the invention can be used for selectively killing HBx-electropositive liver cancer cell and expresses a favorable liver cancer-resisting effect. The recombinant adenovirus vaccine provides a new selection for targeted gene immunization therapy of the HBV-relevant liver cancer and has a favorable application prospect.

Description

technical field [0001] The invention belongs to the field of disease gene immunotherapy. It relates to a double-gene recombination vector of hepatitis B virus X protein (HBx) and human interleukin-12 (hIL-12) and an anti-hepatoma vaccine prepared with the main active ingredient. Background technique [0002] Liver cancer is one of the common malignant tumors in humans. There are 626,000 new patients with liver cancer every year in the world, and its death rate ranks third in the death rate of malignant tumors. Liver cancer is an extremely aggressive malignant tumor. The average 6-month survival rate of patients after diagnosis is less than 50%, the 1-year survival rate is 24%, and the 5-year survival rate is only 5%. my country is an area with a high incidence of liver cancer, and the incidence of liver cancer in China is increasing year by year. The incidence of liver cancer in my country accounts for 45% of the world, ranking second in the incidence of cancer in my count...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10A61K48/00A61P35/00
Inventor 魏于全程平杨莉李玉华文艳君
Owner SICHUAN UNIV
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