Intracellular probing and use thereof

Abstract

To provide application of a tumor suppressor gene.SOLUTION: Two antibodies capable of binding to a mutated P53 protein are created to sandwich the P53 protein from both sides for detection. The entire molecule thus sandwiched constitutes a transcription factor. The transcription factor expresses a toxin.

Description

[Technical Field]

[0001] Genetic Engineering [Background technology]

[0002] Current technology distinguishes cells by their surface molecules. Cellular intervention is achieved by using inhibitors. This method cannot be used for tumor suppressor genes. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0003] I want to somehow use these useless suppressor genes. [Means for solving the problem]

[0004] This method involves detecting the intracellular condition and then causing cell death. To induce this type of action, I think the old method of gene therapy would be a good choice.

[0005] The intracellular situation is thought to be the accumulation of mutant P53 protein. This can be detected using methods such as the two-hybrid assay. This method involves creating two types of antibodies that bind to P53 protein, and flanking P53 on both sides to detect it.

[0006] One possible way to carry out cell death behavior is through the expression of toxins.

[0007] There is a transcription factor that expresses the toxin. The activation site and DNA binding domain of this transcription factor are fused to a P53-binding antibody. These two fusion proteins sandwich P53. TIFF2025122605000001.tif23166The complex acts as a transcription factor, causing the expression of the toxin. [Effects of the Invention]

[0008] The cells undergo necrosis. BEST MODE FOR CARRYING OUT THE INVENTION

[0009] First, the original transcription factor for expressing the toxin was identified, the activation domain and DNA binding domain of the transcription factor gene were isolated, and a nuclear localization signal gene was added to each of the isolated genes.

[0010] Next, a monoclonal antibody that binds to any part of p53 is created. The light chain mRNA of the p53-binding antibody is extracted from a mouse B cell hybridoma. DNA that hybridizes with the target mRNA is synthesized and immobilized, and the target mRNA is then hybridized to it. Next, the variable heavy chain and constant heavy chain 1 mRNAs are extracted. Both are converted to DNA using enzymes.

[0011] Furthermore, a second antibody is made that binds to a different site than p53.

[0012] The gene for the light domain of the binding antibody is attached downstream of the viral promoter region. Next, another promoter is attached, and the gene for the heavy domain is attached downstream of that. The gene for the activation site of the toxin transcription factor is attached downstream of that. This is then inserted into an adenovirus.

[0013] In the above example, a DNA binding sequence for a constitutively expressed transcription factor is placed immediately before the second promoter. The constitutively expressed transcription factor binds to this. The first polymerase encounters this and transcription stops. If the light and heavy polymerases are connected in series, this binding sequence is not necessary.

[0014] The gene for the second binding antibody is fused to the gene for the DNA binding domain of the transcription factor responsible for toxin expression, and this is then inserted into a second adenovirus.

[0015] The toxin gene is placed intact into the third adenovirus.

[0016] Mix adenovirus, No. 1, No. 2, and No. 3.

[0017] This infects cancer cells, attaches to integrins, and is drawn into the cancer cells. The viral genes are released into the nucleus.

[0018] The viral promoter transcribes the gene for the light portion of the binding antibody and the gene for the heavy portion of the binding antibody and the fusion gene for the activation domain. The light portion protein of the antibody and the fusion protein for the heavy portion and activation domain are synthesized. The light and heavy portions bond with disulfides. Eventually, the antibody and activation domain are fused. This is transported to the nucleus. The same process is carried out for the second virus.

[0019] Mutant P53 proteins are swarming in cancer cells. Two types of fusion proteins bind to P53. This triplet forms a structure similar to that of the two-hybrid method, forming a single transcription factor.

[0020] This triple complex acts as a transcription factor on the third viral gene, resulting in the expression of a toxin. Eventually, the cell undergoes necrosis. [Example]

[0021] The toxin is likely ricin, and possibly a ricin analogue.

[0022] In the above-mentioned 0006, if there are multiple enhancers of the final execution gene, multiple activation regions are connected in series, like a turn, and connected in a staggered manner.

[0023] The double-tube method involves a flexible thin tube and a slightly thicker tube. The thin tube has many fin-like structures growing from it. This thin tube is inserted into the thicker tube. Eventually, a concentric double tube is created. This is then inserted into the cancer tissue. It penetrates through normal cells and is stopped just after reaching the cancer cell. A small amount of viral fluid is released from the thin inner tube. The virus is taken up by the cancer cell. The cell undergoes necrosis. The thin tube is moved forward along the necrosis. The remains of the necrosis are absorbed by the outer organ.

[0024] The above-mentioned p53-binding antibody may be replaced with the p53-binding domain of the large T protein of simian virus, or the Mdm protein.

[0025] Two-type adenovirus method. First, there is adenovirus A. One protein is made by fusing the DNA binding domain of normal P53 with an antibody that binds to a region other than the transcription activation region of the P53 protein. This gene is inserted into adenovirus A and expressed using the viral promoter. There is also adenovirus B, which contains the gene for the toxin ricin. A DNA binding sequence controlled by P53 is connected upstream of this gene. The adenovirus infects cancer cells. A protein fusion of the P53-binding antibody and the DNA binding region of normal P53 is expressed. Mutant P53 accumulates in cancer cells. The above-mentioned fusion protein binds to the mutant P53. This results in the normal activation domain, the mutant DNA-binding domain, the antibody, and the normal DNA-binding domain being arranged in this order. The normal activation domain and the normal DNA-binding domain are present in this complex. This complex has properties equivalent to those of the normal P53 protein. The mutant P53 has transformed into normal P53. Another adenovirus B also infects. A gene appears. The above-mentioned transformation complex acts on this gene to generate ricin.

[0026] In the above item 0009, the gene encoding the activation site of the transcription factor that causes the expression of the toxin may be replaced with a gene encoding the activation site of any transcription factor expressed in cancer cells. [Brief explanation of the drawings]

[0027] [Figure 1] This represents the complex of number 0007. [Figure 2] This is the complex number 0025 above.

Claims

1. Mutant p53 accumulates in cancer cells, a condition that can be detected using a method similar to the two-hybrid assay.

2. Using a method similar to the two-hybrid method, cells are directly induced to undergo necrosis.

3. Double pipe system.

4. Two types of expression viruses.

Images