| Autor: |
Shiohama, Yasuo, Tadasuke Naito, Matsuzaki, Toshio, Tanaka, Reiko, Tomoyose, Takeaki, Takashima, Hiroshi, Fukushima, Takuya, Yuetsu Tanaka, Mineki Saito |
| Rok vydání: |
2017 |
| DOI: |
10.6084/m9.figshare.c.3829054_d1 |
| Popis: |
Preparation of His-NY-ESO-1 recombinant protein a. Sequence of NY-ESO-1 gene (NCBI Reference Sequence: NC_000023.11). b. Scheme of construction of pET-14b-NY-ESO-1 plasmid. The full-length NY-ESO-1 gene was amplified by PCR using the MT2 cell genome as a template. The three exons of NY-ESO-1 were separately amplified using primers matching the exon-termini. The DNA fragments of each exon were amplified by PCR using the following primer set: exon1: 5′-GGA ATT CCA TAT GCA GGC CGA AGG CCG GGG-3′ and 5′-AAC TCA AGC AGG CGG CTC TCC GGC CC-3′, exon2: 5′-CTA CCT CGC CAT GCC TTT CGC GA-3′ (exon2-for primer) and 5′-ATA GTC AGT ATG TTG CCG GAC ACA-3′, exon3: 5′- CCG ACT GAC TGC TGC AGA CCA-3′ and 5′-TCG CGG ATC CTT AGC GCC TCT GCC CTG AGG GAG G-3′ (exon3-rev primer). To combine exon2 and exon3 fragments, the joined exon2–3 DNA fragment was amplified by an overlap PCR using exon2-for and exon3-rev as primers and the generated each two DNA fragments as template. Exon1 and exon2–3 DNA fragments were phosphorylated with T4 polynucleotide kinase (Takara Bio Inc., Shiga, Japan). The exon1 fragment was digested with NdeI, and the exon2–3 fragment was digested with BamHI, and cloned into NdeI- and BamHI-digested pET-14b. This plasmid was used for transformation of Escherichia coli BL21 (DE3). c. Amplified three exons of NY-ESO-1 were subjected to agarose gel electrophoresis. d. His-NY-ESO-1 recombinant protein was purification using Ni Sepharose 6 Fast Flow (GE Healthcare Japan, Tokyo, Japan). (PDF 57 kb) |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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