Screening Of Molecular Markers For The Early Diagnosis Of Lung Cancer By T7 Bacteriphage Display

Background Effective biological markers for early diagnosis and prognosis prediction of lung cancer are currently lacking. Single biomarker lacks the specificity and sensitivity according to the diverse types of lung cancer, so tne union screening of many molecular markers is the research direction for the early screening. Identifing and cloning the specific or related gene will provide valuable biomarkers for clinical diagnosis.Objective To establish a lung cancer T7 phage-peptide dispiay library and use it as a valuable source in screening biomarkers for early dignosis.Methods (1)Construction of T7 phage display library drived from lung cancer tissue Total RNA was extracted separately from 30 lung cancer tissue samples according to a standard Trizol protocol. These samples contain all sorts of lung cancer. Equal amounts of total RNA from the 30 tissues were pooled together after the integrity of total RNA being confirmed. Next, mRNA was isolated from total RNA and the agarose gel electrophoresis showed the range of mRNA size. Then reverse transcription, end modification, ligation to Directional EcoR I/Hind III Linkers, EcoR I/Hind III digesting , eliminating excess linkers and small fragments which were less than 300bp were performed. After the cDNA was ligated with T7Select10-3b EcoR I/Hind III Vector Arms ,the lung cancer phage display cDNA library was constructed by package reaction in vitro and plate proliferation. (2)Evaluate the primary library and amplify it The serial 1:100 dilutions were prepared by adding 10μl sample to 990μl medium. Added 100μl different dilution sample,250μl BLT5403 host cells and 3 ml prewarmed(55℃) top agarose into sterile tubes,then poured the admixture onto prewarmed(37℃) LB/Amp plate. Allowed the plate to sit undisturbed for several minutes until the top agarose hardened ,then inverted and incubated for 4h at 37℃. Counted the plaques on every plate and determined the complexity of primary library. Chose 20 plaques stochastically and put every plaque into a tube containing 100μl of 100mM EDTA(PH 7.5). Vertex the tube briefly ,then heat at 65℃for 10min. Cool to toom temperature and centrifuge at 14000rpm for 3min to get the supernatant which was amplified by PCR. Analyzed the recombination rate via the result of 1.5% agarose gel electrophoresis. Used the same way to calculate the phage titer of amplified phage library .The phage titer was the number which described as plaque forming units×10×dilution. (3)Enriching IgG from the sera of lung cancer group and control group 10μl protein-A/G agarose beads were placed into 1.5 ml Eppendorf tubes to get IgG. Then the agarose beads were incubated with 15μl sera. After validating IgG we had enriched, 20 tubes of IgG from lung cancer sera and 20 tubes of IgG from control sera were combined to make IgG pools of control and lung cancer respectively. (4)Enrichment for lung cancer specific phage-peptide clones To enrich for phage clones that bind to IgGs specifically associated with lung cancer, we performed five rounds of positive and negative selection. 20μl control IgGs were incubated with 30μl amplified phage library pool diluted at 1:40 with 10% BSA at4℃for 2 hours. The beads with non-specifically bound phage particles were discard, and the supernatant was collected to be incubated with 20μl lung cancer IgG pool. Infected and propagated specifically bound phage particles. This is one biopanning cycle. Five rounds of affinity selection were carried out for enrichment of lung cancer-specific phage clones. Determed the recombination ratio and titer of the phage-peptide library after each cycle of affinity selection. (5)Markers screening by ELISA To find the clear lung cancer specific markers, we chose 3000 phage clones randomly after five rounds of affinity selection and explored two further cycles of ELISA. Read the result at 450nm using an microplate reader. The phages with the ratio of not less than 2.0(lung cancer v.s. control) were selected as the potential markers for lung cancer diagnosis. (6)DNA sequencing and protein function prediction The insert of positive phages selected by ELISA were sequenced. The gene alignment was blasted at NCBI to find the exact or most similar known gene. Then the relation between these gene and cancer was predicted.Results (1)The complexity of primary phage-peptide library was 5.4×10~6 pfu/ml and PCR identification of 20 randomly picked clones showed that recombination ratio was 60%. The average titre of amplified library is 9.6×10~8pfu/ml .(2)The recombination ratio and titer of the phage-peptide library had a large increasing after five rounds of affinity selection,which reflected that the phages with some feature had been enriched. (3)13 positive phage clones were selected by ELISA and sequenced. One of them is novel gene. Other gene have been reported that they have relation with cancer. Furthermore there are three groups of same gene.Conclusion (1)The cDNA library constructed by T7 phage could well display little abudance peptide. (2)The phages which displayed lung cancer specific peptide were enriched after five cycles of affinity selection. (3)These gene selected by ELISA may be molecular markers for detection of lung cancer.