The Selection Of Aptamers To Anti-CEA

Background and objectives:Lung cancer is the highest incidence and mortality of maligant tumors around the world. The overall 5-year survival is very low. The key to improve the prognosis is early diagnosis and early treatment. Currently, the detection of serum tumor markers(TMs) is one of the principal diagnostic methods for early diagnosis of lung cancer. There were some TMs have been used in lung cancer,including neuron-specific enolase(NSE) ? carcinoembryonic antigen(CEA) ?pro-gastrin-releasing peptide(Pro-GRP) ? cytokeratin 19 fragments(CYFRA21-1) ?squamous cell carcinoma associated antigen(SCC-Ag) etc, but they have low sensitivity and specificity. It will improve diagnostic positive rate of lung cancer with combined detection of the serum level of TMs. Thus, the selection of aptamers to known and unknown serum TMs of lung cancer through SELEX technique is the hotspot in lung cancer research. Aptamers are a group of artificial oligonucleotides which are selected from random single-stranded oligonucleotide libraries. They can specifically bind target molecules with high affinity. Aptamers have many merits as follows. They are easy to be prepared and modified. They are stable, and there is wide range of target molecules, low immunogenicity. The function of aptamers is similar to antibodys but better than antibodies in some characteristics. Thus, the diagnostic methods which based on aptamers found a new method for early diagnosis of lung cancer.Methods: Using Anti-CEA for the screening target, and carboxyl magnetic microspheres(CMM) as medium, through subtractive SELEX technique to screen aptamers to Anti-CEA. After selection, we had a preliminary-results, which were transformed in the competent cell of Ecoli.DH5 ?, Then we separated different oligonucleotides, using interlaced PCR technique to identify positive clones.Results:(1)We applied of enzyme-linked immunosorbent assay(ELISA) to compare the ability of fixing HBs Ag between magnetic bead and resin. We selected CMM as medium for SELEX technique.(2) Compared asymmetric PCR andCMM-SA separation to prepare oligonucleotide ss DNA, we choosed CMM-SA separation to prepare ssDNA. Besides, we optimized the method further, and improved the screening efficiency.(3)After 10 rounds subtractive SELEX screening to Anti-CEA, we obtained oligonucleotide sub-library enrichment for Anti-CEA.(4)The enriched library were transformed in the competent cell of Ecoli.DH5 ?,and we separated 14 clones. Using interlaced PCR technique to identify thoes clones and select the clones with high specificity and high affinity. Finally, we obtained No.2aptamer, which has a high specificity to Anti-CEA. Meanwhile, the No.2 aptamer was sequenced.Conclusions: To screen the aptamers to Anti-CEA plays an important role in early diagnosis and treatment of lung cancer, and aiming to lay the experimental foundation for screening aptamers to other serum TMs of lung cancer.