Compounds for therapy and diagnosis for lung cancer and method for their use
A lung cancer and enhancer technology, applied in the field of peptides, can solve the problem of difficult treatment of lung cancer
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Embodiment 1
[0089] Example 1 Preparation of Lung Tumor-Specific cDNA Sequences Using Differential RT-PCR
[0090] This example illustrates the preparation of cDNA molecules encoding lung tumor-specific polypeptides using differential screening.
[0091] Tissue samples were prepared from breast tumor tissues and normal tissues of lung cancer patients, and the determination of lung cancer was proved by pathological examination of tissue samples from the patients. Normal RNA and tumor RNA were extracted from samples, mRNA was isolated, and analyzed by using a (dT) 12AG (SEQ ID NO: 47) anchored 3' primer was converted into cDNA. Differential PCR was performed by using a randomly selected primer (SEQ ID NO: 48). Amplification conditions: containing 1.5mM MgCl 2 , 20 pmol primer, 500 pmol dNTP and 1 unit Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ) standard buffer. Denaturation at 94°C for 30 seconds, annealing at 42°C for 1 minute, and extension at 72°C for 30 seconds completed 40 amp...
Embodiment 2
[0094] Identification of DNA sequences encoding lung tumor antigens using patient serum
[0095] This example exemplifies the isolation of cDNA sequences encoding lung tumor antigens by screening lung tumor samples by expression using autologous patient serum.
[0096] A human lung tumor-targeted cDNA library was constructed using the Lambda ZAP Express Expression System (Stratagene, La Jolla, CA). All RNA used for the library was obtained from human squamous cell lung cancer passaged from a dead SCID mouse, and poly A+ RNA was isolated by using the INFO labeling kit (Gibco BRL, Gaithersburg, MD). The resulting library was screened using autologous patient sera absorbed from E. coli as described in Sambrook et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989), and the secondary antibody was alkaline phosphatase-conjugated Goat anti-human IgG-A-M (H+L), developed with NBT / BCIP (Gibco BRL). Positive plaques express...
Embodiment 3
[0100] Identification of DNA sequences encoding lung tumor antigens using mouse antisera
[0101] This example illustrates the isolation of cDNA sequences encoding lung tumor antigens by screening a lung tumor cDNA library with mouse antitumor serum.
[0102] A directed cDNA lung tumor expression library was prepared as described in Example 2. Sera were obtained from SCID mice containing post-passage human squamous epithelial and adenocarcinoma tumors. These sera were pooled and injected into normal mice to produce anti-tumor serum. Approximately 200,000 PFU were screened from the unamplified library using this antiserum. Using goat anti-mouse IgG-A-M (H+L) alkaline phosphatase secondary antibody, NBT / BCIP (BRL Labs) was used for color development, and about 40 positive phage plaques were identified. The plaques were purified, and the phagemids were cut into 9 clones, which were inserted into the pBK-CMV vector for expression in prokaryotic cells or eukaryotic cells.
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