Synthetic gene encoding human epidermal growth factor 2/neu antigen and uses thereof
a technology of epidermal growth factor and synthetic gene, which is applied in the field of cancer treatment, can solve the problems of hindering the development and commercialization of many vaccines, and achieve the effect of high level expression and enhanced immunity
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example 1
Human HER2 Optimized Codon Sequence.
[0102] The entire hHER2.opt coding sequence was synthesized and assembled by BIONEXUS (Bionexus Inc. Oakland Calif.) and cloned into the pCR-blunt vector (Invitrogen, The Netherlands). The hHER2.opt cDNA was constructed using oligonucleotides and assembled by PCR. For many experiments described herein, the hHER2.opt nucleotide sequence used carried an optimized Kozak sequence at its 5′-end, the complete nucleotide sequence as set forth in SEQ ID NO:11.
[0103] In addition, the ATP binding Lysine residue 753 was substituted with Alanine (K753A) by replacing codon AAA with GCA. This mutation abrogates tyrosine kinase activity of the corresponding protein and eliminates the downstream signaling events and resulting oncogenic activity of human (Messerle et al. Mol Cell Endocrinol 105(1): 1-10 (1994)) or rat HER2 (Ben Levi et al., supra). In addition, the kinase-deficient K756A mutant can inactivate the signaling activity of a co-expressed oncogenic h...
example 2
Plasmid Constructs
[0104] pV1J-hHER2.wt: The human HER2 wild type coding sequence was amplified by PCR from plasmid pLTR-2 / erb-B2 (kindly provided by P. Di Fiore, European Institute of Oncology, Milan, Italy; Di Fiore et al. Science 237 (4811): 178-82 (1987)) using primers hNeu.for1 (5′-CCAGTTTAA ACATTTAAATGCCGCCACCATGGAGCTGGCGGCCT-3′; (SEQ ID NO:3 coding sequence is underlined) and hNeu.rev2 (5′-GCCGTCGACTTTACACTGGCACGT CCAGACCCA-3′ (SEQ ID NO:4) and TaKaRa LA Taq polymerase (TaKaRa Otsu, Shiga, Japan). The amplification product, which incorporates an optimized translation start site (Kozak, M., J Mol Biol 196(4): 947-50 (1987); Kozak, M., Nucleic Acids Res 15(20): 8125-48 (1987)), was digested with PmeI and SalI restriction enzymes and cloned into the EcoRV and SalI sites of mammalian expression plasmid pV1JnsA (see Montgomery et al. DNA Cell Biol 12(9): 777-83 (1993)). The plasmid pV1J-hHER2 thus generated contained the full-length wild type human HER2 sequence under the transcr...
example 3
Codon Optimized hHER2 cDNA
[0106] A synthetic human HER2 gene (hHER2.opt, FIG. 1) was designed to incorporate human-preferred (humanized) codons for each amino acid (hereinafter aa) residue. During assembly of the gene, PCR amplification consistently deleted an 86 bp sequence starting from position 3642, due to the high GC content of the sequence in this region. To overcome this problem, a less stringent optimization design was adopted for the hHER2 sequence between position 3601 and 3805, which reduced GC content while preserving the same aa composition.
[0107] The codon optimized cDNA was modified to maintain 83.9% nucleotide identity to the original clone. The codon optimized cDNAs were cloned into the pV1J vectors (Montgomery et al., supra), placing in front a Kozak optimized sequence (5′-GCCGCCACC-3′, SEQ ID NO:8) and under the control of the human cytomegalovirus (CMV) / intron A promoter plus the bovine growth hormone (BGH) termination signal. The construct was named pV1J-hHER...
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