Bioinformatics Analysis And Recombinant Expression In Vitro Of Fasciola Giganticatwo CD59 Like Proteins

The surface of the Fasciola is rich in glycoprotein.These proteins are directly related to host tissues and body fluids.They are the biochemical reactions of parasites to host,the position of the body's immune response and physiological response,and are important for the host immune mechanism and immune escape of Fasciola.It is important to study the body-to-cover protein of Fasciola on the immunization,treatament and vaccine of Fasciola.In this experiment,the CD59-1 and CD59-2 protein gene sequences of large tracts were obtained by PCR amplification and sequence sequencing.The size of FgCD59-1 protein is 122 amino acid residues,and the size of FgCD59-2 protein is 127 amino acid residues.The two proteins have a signal peptide protein at the N end,and a GPI anchorage protein at the C end,and there is a conservative domain "CCXXXXCN" in the sequence of LY6/CD59.Through sequence alignment analysis showed that FgCD59-1,FgCD59-2,fasciolagigantica protein sequence of FhCD59-1,FhCD59-2 and F.hepatica protein sequence similarity is very high.Sequencing and analysis of 6 clones of CD59-1 protein from F.gigantica and 3 clones sequencing analysis of CD59-2 protein of F.gigantica sinensis confirmed that FgCD59-1 and FgCD59-2 protein polymorphism was not high.The FgCD59-1 and FgCD59-2 protein sequence and FhCD59-1,FhCD59-2 protein,as well as the other 43 protein phylogenetic tree,FgCD59-1 and F.hepatica CD59 like protein FhCD59-1,FhCD59-4,FhCD59-5,FhCD59-6,FhCD59-7 and FhCD59-8 belong to the same group.FgCD59-2 and FhCD59-2 belong to the same group.The software of biological information through the application of SWISS-MODEL,modeller and a series of FgCD59-1,FgCD59-2 protein and primary structure,secondary structure and two three level structure,transmembrane domain,subcellular localization was predicted and analyzed,and the prediction model was constructed by homology more accurate FgCD59-1 and FgCD59-2 protein.According to the sequence of FgCD59-1 and FgCD59-2 gene,two pairs of specific primers with enzyme cut sites were designed,and RNA from Fasciola sinensis was extracted,and the target fragments were amplified by RT-PCR.Cloning and sequencing proved that the clones.were cloned and cloned.After identifying the correct sequence,two enzymes cut out the sticky end and connect it to the expression vector pET-32a,and successfully constructed the expression plasmid pET-32a-FgCD59-1 and pET-32a-FgCD59-2 with Trx tag and 6His tag.The correct recombinant plasmid was transformed into the expressed bacterial BL21,and the protein expression was induced by IPTG.The expression conditions such as expressing bacteria,expressing temperature and inducing IPTG expression were optimized,and then the inclusion body of fusion protein was obtained.After refolding of inclusion body denaturation,the soluble protein of the protein sequence was obtained,and the expression products were identified by SDS-PAGE and Western blot.The results show that FgCD59-1 fusion protein and FgCD59-2 fusion protein have been obtained.In this study,two protein genes of FgCD59-1 and FgCD59-2 were obtained and analyzed.The protein structure was predicted and analyzed,and two homologous models were constructed.The expression vector of 2 protein genes was successfully constructed and 2 fusion proteins were obtained.It lays the foundation for further study of the structural properties of the two proteins and their role in the immune process of large Fasciola.