NUCLEIC ACID CONSTRUCT ENCODING Trk FRAGMENT AND USE THEREOF
A nucleic acid construct and encoding technology, applied in DNA/RNA fragments, medical preparations containing active ingredients, applications, etc., can solve the problems of lack of truncated forms of tyrosine kinase domains and functions that have not been fully elucidated
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Embodiment 1
[0120] [Example 1] Production of constitutive active TrkB (constitutive active TrkB (hereinafter, CA-TrkB)) vector
[0121] (method)
[0122] An AAV vector expressing the intracellular region of the TrkB molecule (referred to as AAV-CA-TrkB) was constructed as follows. The AAV vector into which the gene fragment encoding CA-TrkB was inserted was pAAV-CAG-shuttle-WPRE manufactured by Applied Viromics. A DNA fragment encoding the following protein was prepared as a gene fragment encoding CA-TrkB: a myc-tag was added to the N-terminus of the intracellular region of TrkB, and a farnesylation (Farnecyl) signal sequence was added to the C-terminus of the region (see also figure 1 ). That is, CA-TrkB does not contain the extracellular region and transmembrane region of TrkB, but is constructed by using only the intracellular region of TrkB, and adding a myc-tag and a farnesylation signal sequence to the intracellular region.
[0123] In addition, the farnesylation signal sequence...
Embodiment 2
[0129] [Example 2] Pathological Analysis of Optic Nerve Trauma Model
[0130] (method)
[0131] =Evaluation method of neuroprotective effect=
[0132] For mice (system name C57BL / 6: purchased from Charles River Corporation), 2 μL of AAV-DsRed (control group) or AAV-CA-TrkB was administered into the eye 14 days before the optic nerve crush . In addition, AAV-DsRed refers to an adeno-associated virus vector in which a gene fragment encoding a fluorescent protein DsRed is inserted instead of a gene fragment encoding CA-TrkB compared to the structure of AAV-CA-TrkB.
[0133] Next, the optic nerve of the mouse was exposed, and the optic nerve was physically pinched about 1 mm behind the eyeball (for details of the method, please refer to Non-Patent Documents 6 and 7). Then, 14 days after the optic nerve crush, the retinal specimens were expanded, and the number of residual retinal ganglion cells was quantitatively counted. In addition, 10 days before optic nerve collection (sam...
Embodiment 3
[0142] [Example 3] Protective effect of CA-TrkB on retinal ganglion cells (glaucoma model mice)
[0143] (method)
[0144] GLAST knockout mice as glaucoma model mice (reference paper: Harada T, et al. The potential role of glutamate transporters in the pathogenesis of normaltension glaucoma. The Journal of Clinical Investigation 117:1763-1770, 2007.) in 3 - Loss of retinal ganglion cells during 5 weeks of age, causing decreased visual function. To 10-day-old GLAST knockout mice, 1 µL of AAV-DsRed (control group) or AAV-CA-TrkB was administered into the eyeball (see also Example 2). Next, developed retinal specimens of these knockout mice at 12 weeks of age were prepared, and the number of remaining retinal ganglion cells was counted. Immunostaining using an RBPMS antibody (anti-RBPMS antibody manufactured by Merck Millipore) was used for detection of retinal ganglion cells. RBPMS is a marker of retinal ganglion cells.
[0145] (result)
[0146] Such as Figure 7 As shown...
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