Intein proteins and uses thereof
An internal protein and protein technology, applied in the fields of constructs, vectors, related host cells and pharmaceutical compositions, can solve the problems of low transgene expression efficiency and so on
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Embodiment 1
[0904] Example 1: AAV-EGFP protein in the full length protein reconstituted in vitro
[0905] The present inventors tested the efficiency of the retina-mediated protein trans-splicing; formation of two AAV vectors, each vector encoding point are candidiasis [ figure 1 N-terminus or C-terminus and C-terminus of the N-terminal half of the halves of Npu A] of the split-type DnaE protein fused to EGFP reporter protein. EGFP protein amino acids in the cleavage (a.a.) at C71. Each AAV vectors comprise appropriate regulatory elements (i.e., promoter and bovine growth hormone polyadenylation signal (bGHpA) and triple flag tag (3xflag), to allow detection of the two halves and the reconstructed full-length EGFP protein ( figure 1 A).
[0906] Generated using the E protein plasmid transfected human embryonic kidney 293 (HEK293) cells and evaluated single N and C-terminal halves of the full-length EGFP protein and AAV-EGFP Dna. Detected in the cell pellet and the AAV-protein EGFP, rather tha...
Embodiment 2
[0911] AAV-EGFP more effective than double the protein of AAV vectors in vitro: Example 2
[0912] To confirm the protein from the reconstructed EGFP protein AAV vectors, so that HEK293 cells infected with AAV2 / 2-CMV-EGFP or the DnaE protein comprising a single and dual expression cassette same AAV vectors. Multiplicity of infection (m.o.i) 5x10 ^ 4 genome copies (GC) / vector per cell, which means assuming a double dose of between similar proteins or recombinant DNA vector, three experienced complete system. In order to accurately quantify the amount of EGFP, were harvested at 72 hours post-infection cell lysates. By ELISA and WB assay (ELISA) to evaluate both the EGFP expression: using the AAV protein EGFP expression vector was expressed in about half of AAV obtained using a single (single AAV = 0.735 ± 0.2ng EGFP / μg total lysate, n = 5 separate experiments; the AAV protein = 0.403 ± 0.04ng EGFP / μg total lysate, n = 5 separate experiments) and dual expression than AAV vect...
Embodiment 3
[0913] Example 3: the subretinal AAV-EGFP fusion expression vectors results in both mice and pigs administered retina effectively reconstructed full-length protein.
[0914] To the AAV-mediated trans-splicing whether the reconstructed full-length protein expressed in the retina, for the injection of 4-week-old C57BL / 6J mice retinal AAV2 / 8-CMV-EGFP Dna E protein carrier (each carrier Study dose / eye: 5.8x10 ^ 9GC). Harvest eye and analyzed by microscopy analysis after 1 month. In all ocular retinal pigment epithelium, most importantly, the detection of EGFP fluorescence in photoreceptors ( figure 1 D). To transgene expression and transgene expression from AAV single and double inner AAV proteins are compared in a photoreceptor, in the injection of G protein coupled receptor photoreceptor-specific human retina at 4-week-old mice C57BL / 6J kinase 1 (GRK1) under the control of a promoter AAV2 / 8 vector encoding EGFP (each carrier / eye dose: 5x10 ^ 9GC). 1 month after injection...
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