Study On Gene Sequence Characteristics And Expression Of CCL20in Mauremys Mutica

Mauremys mutica SMART full-length cDNA library constructed in our lab, andscreening of macrophage inflammatory protein-3α (MaMIP-3α or MaCCL20for)full-length cDNA sequence, Maccl20, full-length cDNA of2318bp in the5’non-coding District (Untranslated region, UTR) of length1549bp,3’UTR length475bp open reading frame ORF)(Open reading frame of261bp, and encodes86aminoacids of the polypeptide chain, the molecular weight of9.739kDa., theoreticalisoelectric point is9.004. Integrated hydrophilic surface antigen index three indicators,forecasting the formation of the amino acid region of the epitope7. The homologyanalysis showed that the yellow pond water turtle and discoloration of the lizard(Anolis carolinensis), red jungle fowl (Gallus gallus) and macrophage inflammatoryprotein-3α closest relationship. The PCR results showed that: MaMIP-3α expression inthe liver, kidney, heart, spleen, the spleen, the highest expression level.Mauremys mutica macrophage inflammatory protein-3α (Mauremys muticaMacrophage inflammatory protein,3α, MaMIP-3α or MaCCL20of) design offull-length cDNA-specific primers and PCR cloning techniques from the theMauremys mutica liver cloned tissue MaMIP-3α gene sequence of the mature peptide.The mature peptide sequence will be inserted into PMD18-T cloning vector,transformed into E. coli E. coli DH5α and the plasmid was obtained by PCR andsequencing identified the purpose of double digestion fragments inserted into theexpression vector pET-32a to build into the prokaryotic expression of the recombinantplasmid pET-32a-of CCL20and the expression plasmid was transformed into E. coliBL21pET-32a-of CCL20fusion protein was successfully expressed after IPTGinduction. Polyclonal antibodies to the fusion protein antigen. After sequencinganalysis showed that mature too MaCCL20the gene sequence inserted into theexpression vector of the Pet-32a. After IPTG induction and SDS-PAGE gelelectrophoresis and Western bolt analysis showed that expression of the fusion proteinof relative molecular mass of about30KD, consistent with the target protein size and react specifically with the His tag monoclonal antibody. His Bind nickel columnpurification after SDS-PAGE gel electrophoresis a single band. Experimental resultsshow that the success of the Mauremys mutica MaCCL20, polyclonal antibody. Toestablish the detection of Mauremys mutica of endogenous MaCCL20ofimmunoassays, enzyme-linked immunosorbent assay, radioimmunoassay method laidthe foundation for subsequent exploration of macrophage inflammatory protein-3α inthe Mauremys mutica of non-specific role in the immune response to provide someimportant information.