Cloning, Prokaryotic Expression Of CpsE Gene Of Streptococcus Agalactiae And Its Application On Indirect In Situ PCR

In this study, Streptococcus agalactiae (S. agalactiae)-infected tilapia that cultured in several farms (Wenchang City, Hainan) were collected. The molecular characterization、 prokaryotic expression of cpsE gene (the functional gene in S. agalactiae) and its further application to indirect in situ PCR (IS-PCR) were investigated by using molecular biology method. Objectives including:to provide comprehensive basic experimental data for studying functions of cpsE gene of S. agalactiae; to establish theoretical foundation for the development of new-style aquatic vaccine, with recombination protein encode by the functional genes of S. agalactiae as major antigen component.1. Molecular characterization of cpsE gene of S. agalactiae. Molecular characterization of cpsE gene of S. agalactiae was analyzed by using bioinformatics softwares. Results indicated that amino acid sequence deduced by cpsE was highly conserved and has revealed a surprising degree (100%) of homology among strains isolated from human and other animals. The highest content of amino acid in deduced sequence were Lys and Val, which were10.7%and10.1%, respectively; Polypeptide analyzed in this study contained a glycosyltransferases superfamily conserved domain between the amino acid residues of56and the142. Three phosphorylation sites were predicted and the hydrophilic regions were larger than hydrophobic regions. Bioinformatic software also confirmed the presence of transmembrane domain and absence of signal peptide in deduced sequence. Proportions of various structures in secondary structure were as follows:a-helice40.94%,(3-strand25.50%, β-corner5.37%and loops structure28.19%. The analysis of codon bias demonstrated that codons biased in cpsE of S. agalactiae have23including GCA et al. and that usage frequency in cpsE of S. agalactiae was distinctly different compared to that in Escherichia coli (E. coli), yeast and human, which, of significant different were20,18and24, respectively; Rare codon usage analysis showed no distinct difference between cpsE of S. agalactiae and E. coli.2. Prokaryotic expression and polyclonal antibody preparation of cpsE gene of S. agalactiae. Recombinant expression plasmid pET-32a(+)-cpsE was constructed and then was expressed into E.coli BL21(DE3) competent cells. The immunogenicity of the recombinant protein was studied by using SDS-PAGE、Western-blot and agar diffusion reaction test.The optimization of induction conditions (IPTG concentration, temperature and time) in E.coli was accomplished and let us to perform the recombinant protein induction at37℃for3h,with0.2mM IPTG in Luria Bertani (LB) medium. CpsE purification was performed by affinity chromatography. The results presented in Western-blot analysis confirm favorable antigenicity and immunogenicity of CpsE by anti-CpsE rabbit serum recognition, and the immunopotency of polyclonal antibody of rabbit anti-CpsE was1:32.3. Development and application of indirect in situ polymerase chain reaction (IS-PCR) based on cpsE gene. Mice were artificially infected by intraperitoneal injection with viable bacteria fluid of S. agalactiae isolates, viable bacteria fluid of standard S. agalactiae and sodium chloride respectively to prepare specimens of positive control, negative control and specimen for detection. cpsE gene of S. agalactiae was used as a template to design a pair of specific primers and an oligonucleotide probe. IS-PCR was developed for application to specimens of positive and negative controls and subsequently detecting the bacteria of S. agalactiae. isolates in order to study distribution pattern of S. agalactiae isolates in mouse. The positive signals sporadically appeared in the liver and the kidney of mouse after4hours since artificial injection; After8hours, the spleen and lung were detected positive signals as well; Twelve hours later, positive signals were found in stomach and intestine, too. Lots of positive spots were detected in many tissues of mouse including liver, spleen, kidney, lung, brain, stomach and intestine, whereas only little positive spots were found in cardiac muscles.