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Cloning of a Tumor Antigen-related Gene in Small Cell Lung Carcinoma
Xiao-Bo Cao1, Si-De Li, Bing Wang
1Department of Immunology and Cell Biology, Shanghai Cancer Institute, Shanghai 200032, China.
Abstract:
The Small Cell lung Carcinoma cell line NCI-H128 cDNA library constructed in Lambda gtll was screened by tumor associated antigen specific monoclonal antibody 2F7. The No.4 positive clone was further investigated. The lysogen of the No.4 clone could be induced at 42 degrees to produce 165 kD fusion protein by IPTG. This fusion protein can be detected by both the monoclonal antibody 2F7 and the anti-beta-galactosidase monoclonal antibody. Antigen Competitive Assay showed that the fusion protein purified by PAGE could compete with the natural tumor associated antigen on the membrane and combine with the monoclonal antibody 2F7. PCR reaction showed the inserted DNA fragment of No.4 Clone to be about 1.3 kb. The DNA inserted fragment was sub-cloned into the plasmid vector Bluescript-sk. Northern blot showed that it was specific for H128 cell line. These results showed that the gene of the tumor associated antigen which specifically reacted with the monoclonal antibody 2F7 was cloned.
Insights
Researchers cloned the gene for a tumor-associated antigen found in Small Cell Lung Carcinoma (SCLC). This antigen specifically binds to the 2F7 monoclonal antibody, aiding in cancer research.
Area of Science:
- Molecular Biology
- Immunology
- Oncology
Background:
- Small Cell Lung Carcinoma (SCLC) is an aggressive form of lung cancer.
- Identifying specific tumor-associated antigens is crucial for developing targeted therapies.
- Monoclonal antibody 2F7 has shown specificity towards a particular antigen in SCLC.
Purpose of the Study:
- To clone and characterize the gene encoding the tumor-associated antigen recognized by monoclonal antibody 2F7.
- To confirm the specificity of the cloned antigen for SCLC cells.
Main Methods:
- Screening of a NCI-H128 SCLC cell line cDNA library using monoclonal antibody 2F7.
- Induction of a fusion protein from a positive clone and its characterization.
- Polymerase Chain Reaction (PCR) to determine the size of the inserted DNA fragment.
- Sub-cloning into a plasmid vector and Northern blot analysis for cell-specific expression.
Main Results:
- A positive clone (No.4) was identified and induced to produce a 165 kD fusion protein.
- The fusion protein reacted with both monoclonal antibody 2F7 and an anti-beta-galactosidase antibody.
- Antigen Competitive Assay confirmed the fusion protein's ability to bind monoclonal antibody 2F7, competing with the natural antigen.
- PCR analysis revealed a 1.3 kb DNA insert, and Northern blot indicated specificity for the NCI-H128 cell line.
Conclusions:
- The gene for the tumor-associated antigen specifically recognized by monoclonal antibody 2F7 has been successfully cloned.
- The cloned gene product is specifically expressed in the NCI-H128 SCLC cell line.
- This cloned gene provides a valuable tool for further research into SCLC diagnostics and therapeutics.
