Screening Of Infection-related Factors From Salmonella Enteritidis By In Vivo Induced Antigen Technology

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent serovars with respect to human Salmonella infections worldwide, and also one of the common human foodborne pathogens and frequently associated with the consumption of contaminated meat (mainly poultry) and eggs. Currently, despite the pathogenesis of poultry S. Enteritidis infection has gained some progress, there are still many unknown areas. Although the commercial vaccines to protect poultry against S. Enteritidis are much more, most of them are still not fully effective or safe. Therefore, further study on the pathogenesis, new drug targets and vaccine candidates of S. Enteritidis is particularly important.In vivo induced antigen technology (IVIAT) is an immunologic screening technique using sera from host infected pathogen to identify pathogen antigens that are immunogenic and expressed in vivo during host infection, and it also integrates both principles and methods of immunology and genomics. Genes and gene pathways identified by IVIAT may play a role in virulence or pathogenesis during host infection. They would be very useful on the development of novel therapeutic methods, vaccines or diagnostic technigues.1. Construction of S. Enteritidis genomic expression libraryGenomic DNA was extracted from S. Enteritidis strain CMCC50041, and digested incompletely with Sau3A Ⅰ under optimized conditions to yield fragments of0.4to2.7kb which were then gel purified and extracted. At the same time, pET30-a/b/c(+) expression vectors, were prepared by digestion with BamH Ⅰ and treated with shrimp alkaline phosphatase (SAP). Each of the three vectors was separately ligated with genomic DNA fragments, and then the ligated products were transformed into E.coli DH5α. The frequency and sizes of the insertions were assessed by PCR amplification of a random sample. After overnight incubation, colonies were collected from plates by scraping, subsequently plasmid DNA was harvested and stored frozen at-70℃. The results indicate more than90%of transformants contained inserts of sizes ranging from0.4to2.7kb randomly, and the genomic library consisted of approximately1.68×105clones and covered the genome for nearly5times.2. Screening of infection-related factors from S. Enteritidis by IVIATEqual volumes of chicken antisera prepared for higher titers were pooled, and extensively adsorbed with in vitro-derived CMCC50041antigens and E.coli BL21(DE3) antigens (including whole cells, cell lysates and secreted proteins) respectively. The efficacy of each adsorption step was analyzed with an indirect-ELISA against both CMCC50041antigens of whole cells, cell lysates and lipopolysaccharide and E.coli BL21(DE3) antigens (such as whole cells and cell lysates). Furthermore, positive clones were selected with dot-immunobinding blot through primary screening to secondary screening and SDS-PAGE, subsequently sequenced and analyzed by NCBI blast. The results indicate that there are57reactive clones and which encoded43different proteins. The putative ivi proteins were grouped into7categories:molecule synthesis/degradation, transport system, regulation, virulence, metabolism, others and those with unknown functions, and they were involved in various aspects of pathogenicity and basic survival of S. Enteritidis.3. Differential expression level of infection-related factors from S. Enteritidis detected in vivo and in vitro by real-time fluorescent quantitative PCROf the43putative ivi genes,10(bigA, metQ, SEN3752, fadJ, rlpA, glpA, SEN2804, SEN3610, nadR and ssaD) were selected for further analysis of gene expression by real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) which was performed with SYBR green and analyzed with2-ΔΔCT.These genes were selected based on their putative functions in order to maximize the representativeness of genes chosen for further analysis. The expression of10putative ivi genes was tested under in vivo conditions after challenge with CMCC50041via intravenous inoculation measured at12,24,36and48hr pi. The results indicate that expression of the mostly analyzed genes were upregulated in vivo compared to their expression in vitro except for fadJ. Of the upregulated genes in vivo condition, the expression level of five genes (metQ, bigA, glpA, SEN2804and SEN3610) were volatility, two genes (SEN3752and ssaD) were continuous upregulation and remaining two genes (rlpA and nadR) were continuous downregulation. By assessing the expression of10putative ivi genes during the infection process, it could be seen that genes identified by ⅣAT were expressed in vivo. At the same time, it also initially revealed that the expression pattern of different function genes presented timing, diversity and other characteristics, and there is a complex mechanism between bacteria and host interaction during the infection process.