Screening And Evaluation Of DNA Aptamer That Recognizes Swine Anaphylatoxin C5a

The anaphylatoxin C5 a is synthesized via the cleavage of the amino end of the a chain of the fifth component of complement C5 during complement activation, and released into the bloodstream. Studies confirms that C5 a is a multiple-effect molecule that help to accelerate the destruction of pathogens. Anaphylatoxin C5 a is a chemoattractant for inflammatory cells and mediator of the ‘cytokine storm’, such as interleukin(IL)-8, tumour necrosis factor(TNF)-a and IL-6.Moreover, it also works on the endothelial cell lining of blood vessels to induce expression of adhesion molecules for leukocytes, which in concert direct the migration of inflammatory cells toward sites of antigen deposition where they ingest patho genic material, and in turn alter vascular permeability, induce smooth muscle contraction and promote inflammatory cell migration. Therefore C5 a is an important risk factor in various inflammatory diseases, it has become a new target of inflammatory diseases treatment, the study of inflammatory mediators C5 a has a very important significance not only to reveal the inflammatory reaction in the molecular mechanism, but also to develop effective anti- inflammatory drugs.Aptamers are ss DNA or RN A molecules that fold into unique tertiary structures that allow them to bind molecular targets with high affinity and specificity. Although action principle is similar to antibodies, simpler, more easier to synthesise, nonimmunogenic classes of C5 a inhibitors could be derived from short, single-stranded nucleic acid oligomers known as aptamers in clinical treatment. Therefore aptamers are gaining increasing attention for their potential as pharmaceutical lead compounds or diagnostic agents. In order to develop the treatment of inflammatory diseases of swines, it is very necessary to screen aptamers of C5 a.In this study, we constructed a 36-nucleotide variable region DN A library to select looped DNA aptamers against recombinant swine anaphylatoxin C5 a by combining C E-SELEX; And antagonistic activity of aptamers were investigated in vitro. The main contents include:Swine C5 a protein was expressed with Prokaryotic expression system. Test content:(1)The prokaryotic expression plasmid(p ET32 a and p ET28a) containing the full- length C5 a clone and encoding a soluble 82-amino acid, His-tagged swine C5 a recombinant protein was obtained from Sangon Biotech.The protein was expressed in Escherichia coli BL21(DE3) strain.(2) SDS-PAGE, western blotting and other methods to identify whether successful expression of recombinant C5 a.(3)Cells were induced with IPTG.(4)Cells were resuspended in prechilled NTA-0 buffer and sonicated. The sonicated sample was centrifuged, collected the supernatant and Soluble C5 a was purified by nickel affinity chromatography under nondenaturing conditions.(5)Cell chemotaxis and induction was performed with swine neutrophils to identificate the biological activity of protein C5 a.Screening and evaluation of the pig C5 a single-stranded DNA aptamers.(1) The recombinant C5 a protein as a target, use CE-based efficient separation capability of CE-SELEX technology, after six rounds of screening and, ultimately, can have the protein higher binding DNA aptamer group force;(2) A high-throughput sequencing and bioinformatics technology to do structural analysis of candidate aptamers;(3) Then by measuring the EMSA and SPR technology aptamers and C5 a protein affinity and specificity;(4) Finally, in vitro chemotaxis assays and flow cytometry to detect swine aptamers S1 antagonize C5 a ability biological activity.Through the above test, the following results were obtained:We constructed two strains recombinant plasmid of p ET32a-C5 a and pET28a-C5 a and optimized their induction conditions after transfecting into E. coli BL21 successfully.SDS-PAGE and Western-blotting analysis showed that the recombinant protein C5 a wad about 10,000 by expression; The protein was purified by N i-NTA affinity chromatography. Then the purified proteins identified by mass spectrometry, its primary structure is correct and more than 90% at the concentration of 2.5 g / L. C5 a had the activity to chemotactic and induce neutrophil.This study revealed that swine C5 a protein involved in the inflammatory process mechanism and laid the foundation for the preparation of C5 a antagonists.Single-stranded DNA aptamers screening of swine C5 a showed that capillary electrophoresis peak can be seen clearly in the target protein and nucleic acid complex peak pattern by combining CE-SELEX and amplification 6 rounds. We obtained 55,000 raw sequences after high-throughput sequencing. Six representatives were selected to measure affinity with C5 a after sequence statistics, alignments and analysis. S1 binded specifically to swine C5 a with 4.81 μM dissociation constant as measured by surface plasmon resonance(SPR). Moreover, In vitro, S1 inhibited C5 a induced chemotaxis of neutrophils. Flow test results showed that the aptamer S1 can inhibit the binding of C5 a with neutrophil surface C5 a R, thereby inhibiting its activity.The following conclusions were obtained from the above tests:(1) Successfully Constructed two strains recombinant plasmid of pET32a-C5 a and p ET28a-C5 a. Swine C5 a protein obtained in prokaryotic expression was identificated by MS, its primary structure is correct. C5 a had the activity to chemotactic and induce neutrophil.This study revealed that swine C5 a protein laid the foundation for the preparation of C5 a antagonists.(2) Highly efficient filtering criteria of CE-SELEX technology combined with high-throughput sequencing was established to screen C5 a single-stranded DNA aptamer fastly and efficiently.(3) The aptamer analysis process for HTS data was initial established, not only to improve the screening efficiency, and to avoid the disadvantages of LTS. It is more objective selection of candidate aptamers by sequence analysis software.(4) In the chemotaxis experiments, aptamer S1 and S2 can effectively inhibit C5a-induced PMN chemotaxis, but compared the ability to antagonize, S1 was stronger than S2, and more therapeutic potential for inflammatory diseases in swine.