Serex Method Of Screening And Identification Of Lung Squamous Cell Carcinoma-associated Antigen

Background:Currently, only a limited numbers of tumor markers for lung cancer diagnosis. It's seldom detected at an early stage and 80% patients had been advanced. In malignant transformation, mutant gene products and dysregulated proteins can become tumor associated antigens(TAAs) and activate immunoreactions, and the autoantibodies for TAAs might occurrence before clinical symptom. Therefore, the autoantibodies exist in sera of cancer patients will become new dependable biomarkers, and offer the methods for TAAs screening. SEREX(Serological analysis of recombinant expression libraries)using autoantibodies in sera provides a powerful approach to identify TAAs, and combined together with phage display to increase the efficiency of screening.Objective:For more new biomarker, such as serum autoantibodies may improve the early detection of lung cancer. However, microheterogeneity of majority tumor, one of gene mutants, chromosome aberration and changes of protein, would not existence in all phenotypes. Our study select the most common lung cancer—human lung squamous cell carcinoma for screening , and objective is construction human lung squamous cell carcinoma T7 phage display cDNA library from the tissues of NSCLC patients. By using of cancer sera contain antibodies which react with autologous cellular antigens (tumor-associated antigens TAAs), the main purpose of this study is to screening and identify non small cell lung cancer associated antigen which can be used in NSCLC early detection or target treatment.Currently only a limited number of diagnostic tumor marker for non-small cell lung cancer (NSCLC).Methods:mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 lung squamous cell carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library. Then, the lung squamous cell carcinomas T7 phage display cDNA libraries were panning against the sera from 24 patients with NSCLC. Then the enriched libraries were screened with the pool of sera from NSCLC patients, the positive clones encoding antigenic genes were obtained after screening , and the nucleotide sequences of positive were identified and analyzed with BLST software in GenBank . Some clones selected for phage peptide array with independent serum.Results:Lung squamous cell carcinomas T7 phage display cDNA library was established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the phage titer of the amplified library were 3.2×1010 pfu/ml. PCR amplification of random plaque show insert ratio were 95% (23/24) in human lung squamas cell carcinoma library. Insert range from 300 bp to 1 500bp. After immunoscreen the T7 phage display cDNA libraries with serum from patients with lung squamas cell carcinoma, fifty positive clones were obtained and sequence results show they are derive from 15 genes, 12 of 15 genes were homologous to the genes known in GenBank. The other three genes expressed sequence tags ( ESTs) were not found in GenBank.Conclusion:The lung squamas cell carcinoma T7Select phage display cDNA library from NSCLC were constructed.15 NSCLC associated antigen genes were obtained by SEREX from T7 phage display cDNA libraries.