Immunity inducing agent
a technology of immune-inducing agents and agents, which is applied in the field of immuno-inducing agents, can solve the problems of ineffective cancer treatment, inability to effectively treat cancer in dogs or cats, and already too late, so as to achieve cancer therapy, prophylaxis and/or the like, and cancer therapy and/or prophylaxis.
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example 1
Obtaining Novel Cancer Antigen Protein by SEREX Method
(1) Preparation of cDNA Library
[0075]Total RNA was extracted from testis of a dog by the acid-guanidium-phenol-chloroform method, and poly(A) RNA was purified using Oligotex-dT30 mRNA purification Kit (manufactured by Takara Shuzo Co., Ltd.) in accordance with the protocol attached to the kit.
[0076]Using the obtained mRNA (5 μg), a cDNA phage library was synthesized. For the preparation of a cDNA phage library, cDNA Synthesis Kit, Zap-cDNA Synthesis Kit, and ZAP-cDNA Gigapack-III Gold Cloning Kit (manufactured by STRATAGENE) were used in accordance with the protocols attached to the kits. The size of the prepared cDNA phage library was 1×106 pfu / ml.
(2) Screening of cDNA Library with Serum
[0077]Using the thus prepared cDNA phage library, immunoscreening was carried out. More specifically, the host E. coli (XL1-Blue MRF′) was infected with the library such that 2340 clones appeared on an NZY agarose plate with a size of 90 mm dia.×...
example 2
Analysis of Cancer Antigenicity of KATNAL1 In Vivo
(1) Preparation of Recombinant Vector that Expresses KATNAL1 In Vivo
[0084]Based on the base sequence of SEQ ID NO:5, a recombinant vector that expresses KATNAL1 in vivo was prepared. PCR was prepared from the mouse cancer cell line N2a (purchased from ATCC), which showed the expression in Example 1. The reagents and the attached buffer were mixed such that 1 μl of the cDNA, 0.4 μM and each of two kinds of primers having the HindIII and XbaI restriction sites (shown in SEQ ID NOs:25 and 26), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) were contained in the resulting mixture in a final volume of 50 μl, and PCR was carried out by 30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 4 minute using a Thermal Cycler (manufactured by BIO RAD). The above-described two kinds of primers were those for amplification of the region encoding the full-length of the amino acid sequence ...
example 3
Preparation of Human Recombinant KATNAL1 Protein and Evaluation of its Immunity-Inducing Ability
(1) Preparation of Human Recombinant KATNAL 1 Protein
[0089]Based on the base sequence of SEQ ID NO:3, a recombinant protein of human KATNAL1 was prepared. The regents and the attached buffer were mixed such that 1 μl of the cDNA prepared in Example 1 whose expression could be confirmed for cDNAs from various tissues and cells by the RT-PCR method, 0.4 μM each of two kinds of primers having the EcoRI and XhoI restriction sites (shown in SEQ ID NOs:27 and 28), 0.2 mM dNTP and 1.25 U PrimeSTAR HS polymerase (manufactured by Takara Shuzo Co., Ltd.) were contained in the resulting mixture in a final volume of 50 μl, and PCR was carried out by 30 cycles of 98° C. for 10 seconds, 55° C. for 15 seconds and 72° C. for 4 minute using a Thermal Cycler (manufactured by BIO RAD). The above-described two kinds of primers were those for amplification of the region encoding the full-length of the amino a...
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