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Rhesus monkey NY-ESO-1 protein, coding gene, and applications thereof

A technology of NY-ESO-1 and rhesus monkeys, which is applied in the fields of molecular biology and genetic engineering, can solve the problems of no serious adverse reactions and achieve broad application prospects

Inactive Publication Date: 2014-01-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic and foreign reports on immunotherapy clinical trials of NY-ESO-1 vaccine in various tumors include: peptide vaccine and full-length protein vaccine; for example, Odunsi K et al. The combination of adjuvants was used as a vaccine. It was found that the vaccine could induce humoral immunity and cellular responses in most patients, and some patients could still detect specific T cell responses one year after vaccination. In addition to redness, swelling and pain, no serious adverse reactions related to the experimental vaccine have been found (Odunsi, PNAS USA, 2007, 104:12837-12842)

Method used

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  • Rhesus monkey NY-ESO-1 protein, coding gene, and applications thereof
  • Rhesus monkey NY-ESO-1 protein, coding gene, and applications thereof
  • Rhesus monkey NY-ESO-1 protein, coding gene, and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, detect and clone the rhesus monkey NY-ESO-1 protein gene in the rhesus monkey tissue

[0056] 1. Extraction of total RNA from rhesus monkey tissues

[0057] Take the isolated rhesus monkey brain, bone marrow, skeletal muscle, cardiac muscle, lung, liver, stomach, spleen, small intestine, kidney, prostate, thymus and testis tissue, and process them according to conventional methods, such as liquid nitrogen treatment, aseptic treatment, etc. , using QIAGEN RNeasy Plus Mini Kit (Catalog No.74104) to extract tissue RNA.

[0058] 2. Synthesis of rhesus monkey tissue cDNA fragments and detection of NY-ESO-1

[0059] use III First-Strand Synthesis System (Catalog No.18080-051) synthesizes the first strand of cDNA from each tissue of rhesus monkey as a PCR template for NY-ESO-1 gene detection; according to the genome sequence of rhesus monkey published by Genebank, the primers are designed as follows :

[0060] rESO-F: 5'-GGGATGCAGGCTGCAGGCCAGGGC-3'

[0061]...

Embodiment 2

[0070] Embodiment 2, construction and identification eukaryotic expression vector

[0071] 1. Construction of eukaryotic expression vector pCMVTNT-rESO

[0072] design primers

[0073] rESO-F 1: 5'-CCCTCTAGATCACCATGCAGGCTGCAGGCCAG-3'

[0074] rESO-R1a:

[0075] 5'-CTTGTAGTCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCGCGCCTTTGTCCTGAGGG-3'

[0076] rEOS-R1b:

[0077] 5'-CCCGGATCCTCATTACTCGAGCCCGGGCTTGTCATCGTCATCCTTGTAGTCGATGTCATGATCTTT-3'

[0078] Using pMD19T-rNY-ESO-1 as a template, the rhesus monkey NY-ESO-1 fragment linked to the 3FLAG tag was amplified, cloned into the eukaryotic expression vector pCMVTNT (Promega), sequenced and identified, using the EndoFree Plasmid Giga Kit from QIAGEN ( Catalog No.12391) to extract endotoxin-free plasmids for transfection of cells and animal immunization; use Thermo’s Nanodrop 1000 to determine the concentration and purity of the plasmids, and dilute the plasmids to 1 μg / μl with PBS; obtain the plasmids, whose map is as follows figur...

Embodiment 3

[0084] Example 3. Analysis of splenocytes secreting interferon-γ after immunization of BALB / c mice with eukaryotic expression vector pCMVTNT-rESO

[0085] 1. Immunization of BALB / c mice with eukaryotic expression vector

[0086] The BALB / c mice were divided into two groups, 3 mice in the empty vector group and 5 mice in the experimental group. The endotoxin-removed plasmid pCMVTNT-rESO and the pCMVTNT empty vector were injected into the tibialis anterior muscle of the mouse hindlimb at 0, 2, 4, and 7 weeks respectively according to the amount of 100 μg DNA per mouse.

[0087] 2. Isolation of mouse splenocytes

[0088] At the 10th week, the mice were killed by cervical dislocation after blood was collected from eyeballs, and spleen lymphocytes were isolated under sterile conditions. Resuspend with 1640 complete medium (10% calf serum, 1% PS antibiotics).

[0089] 3. ELISPOT detection of mouse splenocytes secreting interferon-γ

[0090] Refer to the instructions of BD Mouse ...

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Abstract

The invention belongs to the fields of molecular biology and gene engineering, and relates to tumor antigen NY-ESO-1 protein originated from rhesus monkeys, coding gene, and applications thereof. The mentioned rhesus monkey NY-ESO-1 protein can be used for preparing antitumor vaccines, the gene which codes the NY-ESO-1 protein can be used for preparing antitumor nucleic acid vaccine; the antitumor vaccine or nucleic acid vaccine mentioned above can be merged into one or more cell factors or microbial components such as granular leukocyte-macrophage colony stimulating factor, gamma interferon, interleukin 2, transforming growth factor beta4 to made into immunity auxiliary agents; and the rhesus monkey NY-ESO-1 protein can be used with tumor-testicle antigen to have a combined immunization function or be made into polyvalent subunit vaccines. Furthermore, the invention also provides an immunogenicity composition or vaccine, and provides a novel method, which has a wide application prospect, for tumor immunity treatment.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetic engineering, and relates to a protein derived from rhesus monkey tumor antigen NY-ESO-1, its coding gene and its application, in particular to an antigen derived from rhesus monkey tumor antigen NY-ESO-1 peptides and carriers. Background technique [0002] NY-ESO-1 was discovered in 1997 by Chen et al. using recombinant cDNA library serological analysis (Serological analysis of recombinant cDNA expression libraries, SEREX) to screen the cDNA expression library from esophageal cancer tissue; NY-ESO-1 belongs to Tumor-testis antigen (Cancer-Testis Antigen, CT) family, its expression in normal people is limited to testis, ovary and embryonic trophoblast cells, not expressed in other adult somatic tissues (Simpson, Nat. Rev. Cancer, 5 (8):615-625 (2005); Chen, PNAS USA, 1997, 94(5):1914-1918; Lethe, Int J Cancer, 1998, 76(6):903-908). [0003] Studies have shown that human (Homo sapiens...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/12C12N15/63C12N5/10C12N5/0784A61K39/00A61K48/00A61P35/00A61P35/02G01N33/68C12Q1/68C07K16/30
CPCC07K14/4748A61K39/0011A61K48/005C07K2319/43C12N5/0639C12N2501/998
Inventor 徐建青曾刚周明哲徐军
Owner FUDAN UNIV
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