Screening Lung Cancer Serum Markers Aptamers By SELEX Technique

Lung cancer is one of the fastest morbidity and the highest mortality of malignant tumors,which has become a serious threat to the people's health and life nowadays.Serum tumor markers can reflect tumorigenesis and lesion to a certain extent.At present,the clinical application of combined detection of serum tumor markers in lung cancer has improved the accuracy of lung cancer diagnosis and has strong persuasion.It has become one of the main methods of early diagnosis of lung cancer.SELEX(Systematic evolution of ligands by exponential enrichment)is a new combinatorial chemistry technology which is developed in the early 90 s,and it is a kind of technology which can be used to screen the oligonucleotide with high specificity and high affinity.The technology combined with PCR amplification techniques in vitro,which makes it possible to screen out ssDNA or RNA molecules that specifically bind to the target molecule in a randomized single-stranded library constructed in vitro.The method is simple and the screening period is short.The oligonucleotide molecules of target molecules are called aptamer.Aptamers have not only the recognition properties of antibody molecules but also the binding ability of antibodies.Almost all of the areas involving the field of antibody diagnosis can be replaced by aptamers.At the same time,aptamers have many advantages,such as simple preparation,low cost,good stability,wide range of target molecules,easy modification,low immunogenicity,reusability and so on.Therefore,the detection of serum tumor markers in lung cancer can be used as an important way of early diagnosis for lung cancer patients.In this paper,we applied the subtractive SELEX technique to screen the serum specific aptamers from lung cancer patients and did the clinical verification for its specificity.Specific research contents and results are as follows:1.The removal of high abundance proteins in serum: the mainly experimental material of this study is the serum of patients with lung cancer.Screening the aptamer that specifically binds to a tumor protein marker from a complex lung cancer serum,it is first necessary to consider isolating proteins that are highly abundant in serum but do not have special biological information,as much as possible to retain may act as a tumor marker of low abundance proteins,lay the foundation for the success of screening the lung cancer patient serum specific aptamer.In the experiment,the high abundance protein was removed by acetonitrile precipitation method,and the ratio of acetonitrile to serum was optimized.The results of SDS-PAGE electrophoresis showed that the serum: water: acetonitrile =1:2:0.5,the effect is good,basically reached the purpose of high abundance protein removal.2.The preparation of Streptavidin magnetic beads: utilize the characteristics binding between avidin and biotin,used on the magnetic beads in the surface layer coated streptavidin,and add primer bio-P11 in PCR system,The biotin-labeled dsDNA was amplified.Then,dsDNA was combined with streptavidin beads to prepare the ssDNA secondary library.The binding efficiency of magnetic beads with different concentrations of streptavidin was different.To this end,we optimized the optimal concentration of streptavidin and 0.05 ml magnetic beads.The results showed that when streptavidin concentration was 0.01mg/ml,streptavidin and 0.05 ml magnetic beads were close to saturation and the coupling rate was the highest.3.Screening of aptamers: utilize subtractive SELEX technology to screen aptamer,lung cancer serum is the positive screening target(positive),normal human serum is the screening target for anti(negative),Detection of specific ssDNA in the screening of aptamers by RT-qPCR.After nine rounds of screening,the positive and negative RT-qPCR curves were obviously separated,and the number of cycles between the negative and positive curves almost reached 4 to 6 cycles.The aptamers were sent to Shanghai Sangon for sequencing,the sequences of the serum markers of lung cancer were obtained,and two high abundance of aptamer 1 and aptamer 2 were selected,sent to Shanghai Sangon synthesis.4.Clinical validation of aptamers and lung cancer serum binding specificity: selected 50 cases of lung cancer clinical patients serum was positive,normal human serum was negative.To detect the specificity of the binding of the serum of patients with lung cancer with aptamer 1 and aptamer 2.The detection of RT-qPCR results showed that the RT-qPCR detection curve of the serum of 50 cases of lung cancer patients with normal human serum were separated,that aptamers 1 and 2 aptamers can be combined with the high specificity of lung cancer patient serums.In this paper,we took carboxylated agar beads as screening medium and lung cancer patients serum as a target for the first time.Meanwhile,we screened the serum specific aptamers by subtracting SELEX technique,detected 50 cases of clinical cases of lung cancer serum samples and verified the specificity of the combination of two nucleic acid aptamers and lung cancer serum.The paper provided a scientific basis for establishing new methods of lung cancer based on nucleic acid aptamer during the early diagnosis and treatment.