| Salmonella enterica serovar Typhi is a human pathogen that causes a series of disease including diarrhea, typhoid fever, lymphadenopathy, hepatosplenomegaly, encephalopathy, intestinal hemorrhage and perforation. An estimated 21,650,000 episodes of typhoid fever illness and 216 500 deaths globally occurred in 2000. In recent year, the incidence of disease caused by multi drug-resistant is increasing and this made the treatment becoming difficult. Therefore it is very important to develop novel approaches to the diagnosis, treatment and prevention of typhoid fever. All of these depending on the understanding of its pathogenesis. But humans are the only known natural hosts for S. enterica serovar Typhi infection, so the study of pathogenicity of S. enterica serovar Typhi is difficult for default of animal model. Up to now, little is known about the specific factors contributing to its ability to cause typhoid fever and its adaptation to the human host.During disease, pathogens must adapt to a series of environments, and survival in any one niche would likely need the expression of a distinct subset of virulence factors. Hence, genes that are expressed or upregulated in vivo may play an important role in disease. If the proteins encoded by these genes are antigenic , it would stimulate the body to produce antibodies. So immunogenic technique can be used to indentify these antigens. In vivo-induced antigen technology (IVIAT) is a modified immunoscreening technique that circumvents limitations of animal models, allowing direct identification of microbial proteins expressed during human infection but not during growth in standard laboratory media. This sort of proteins is called ivi-proteins and the genes is called ivi-genes. It might play an important role in the virulence of this organism. The aim of present study was to identify S. enterica serovar Typhi genes specifically expressed during human infection by using IVIAT. In this dissertation, the following experiments are conducted:Collection and adsorption of sera: Convalescent human sera were collected from typhoid patients. 3 sera with high-level antibody titer, checked by Widal's reaction, were collected ,and Equal volumes of Serum of them were pooled and adsorbed extensively against in vitro-grown serovar Typhi Ty2 organisms and against the expression host strain E. coli BL21(DE3) containing the native pET-30a expression plasmid. Both of strains were grown to late log phase in LB broth. Then pooled sera were serially adsorbed against whole cells, cell extracts and denatured cell extracts. The extracts was prepared by sonication. A 10-μl aliquot of the serum was removed after each adsorption and an indirect ELISA was performed to check the efficacy of each adsorption step. A progressive decrease in reactivity of the pooled sera with in vitro-grown S.enterica serovar Typhi was shown by ELISA, and the final adsorbed pooled convalescent-phase serum showed no significant reaction with in vitro-expressed S.enterica serovar Typhi Ty2 antigens.Construction of a genomic expression library of serovar Typhi Ty2: We use pET-30abc expression vectors, which allow the cloning of inserts in each of the three reading frames under the transcriptional control of the T7 phage promoter, to construct genomic expression library of serovar Typhi Ty2 . Three vectors can warrant the correct expresson of the insert. Vector DNA was digested with restriction enzyme BamHI, gel purified by gel extraction kit, and treated with shrimp alkaline phosphatase. Genomic DNA of S.enterica serovar Typhi Ty2 was partially digested with Sau 3AI to generate a fragment of 0.5～1.5 kb, and purified by gel extraction kit. A rational ratio of vector DNA and insert fragment was ligated and electroporated into competent E. coli DH5α. Transformants were spread on LB plates containing kanamycin. After incubation overnight at 37°C, all colonies on plates were collected and recombinant plasmid DNA was recovered and electroporated into E. coli BL21 (DE3). Finaly we constructed the genomic expression library of Ty2,which contained 6.0×104 clones, and more than ninety percent were recombinated plasmids. The library had reached the theoretic requirement.Screening for proteins expressed during the infection of S.enterica serovar Typhi Ty2: An aliquot of the expressing library , which was constructed as above in expression host BL21 (DE3), was diluted and spread on LB plates containing kanamycin to produce≈400 colonies perplate. These plates were incubated at 37°C until the clone become pinpoint big. Then a sterile nitrocellulose membrane was put onto each plate for 10 min to produce replica, and then was pick onto LB plate containing kanamycin and isopropyl-β-D-thiogalactoside (1 mM), and was incubated for 5 h at 37°C to induce expression of genes in cloned inserts. In the mean time, primary plates also incubated for 5h at 37°C and stored at 4°C as the master plates. After that, each membrane was removed, and partially lysed by exposure them to chloroform vapour for 15 min, and blocked with 10% nonfat skim milk overnight at 4°C. After washed with PBS-T, each membrane was reacted with adsorbed convalescent sera (1:200 dilution) for 1h at room temperature with mild agitation, and washed with PBS-T for 3 times. Clones reacting with antibody in adsorbed sera were detected by using peroxidase-conjugated goat anti-human Ig at a 1:20,000 dilution and an ECL chemiluminescence kit. Reactive clones were identified by their position on the master plate; each positive clone was purified and stored at -80°C as glycerol stocks. To eliminate the false positive clone farthest, a secondary screening was done by comparing those primary screen clones directly to the reactivity of a control strain, E. coli BL21(DE3) containing the pET30 vector with no insert, in a whole-colony immunoblot assay. Plasmids from individual reactive clones were purified, and the S.enterica serovar typhi DNA inserted in the vector was sequenced in both directions by using pET30-specific primers. A tertiary screening was done by cloning each of the entire predicted ORFs, generated by PCR amplification, into 5'NdeI or NcoI(if the gene has NdeI sites) and 3'NotI restriction enzyme sites in the pET30a vector and assaying the reactivity of each resultant clone compared to that of the control strain, E. coli BL21(DE3) containing the pET30a vector without insert. Seven open reading frames were obtained in this study including bcfD(t0025), grxC(t3817),sapB(t1598), t3664, t3816 , t1497 and t3689, and they were involved in pilus structure(bcfD), transport(t1497, sapB) and metabolic(grxC, t3664, t3816).RT-PCR: We analysis RNA transcription of genes identified by RT-PCR. After overnight culture of strain Ty2 in LB broth media, the total RNA was isolated by using TRIzol according to the manufacturer's instructions. RNA was then treated with RNase-Free DNaseI. Control PCRs were performed on the purified RNA preparations to ensure absence of amplification from contaminating DNA. Reverse transcription to cDNA was done by using a reverse transcription kit. Primers specific for seven identified genes and cysZ (as a control) were used in PCRs on cDNA; primers were obtained from TaKaRa Biotechnology (Dalian)Co.,Ltd.. PCR products were detected on a 1.2% agarose gel with ethidium bromide. Transcription of t3663 and t1497 were not detected after overnight growth in LB broth media. Transcription of t3689, bcfD, sapB, grxC and t3816 were detected after overnight growth in LB broth media, for they expreesed at low level and antibodies in the pooled sera reactive with these five genes were not removed during the adsorption process. The control gene, cysZ, was expressed in LB broth media as expected.In conclusion, seven in vivo induced genes of S.enterica serovar typhi Ty2 were identified by IVIAT approach this study. We marched one step on the road of exploring the key genes which help serovar typhi survive in the body and result in typhoid. And the study also provided some new molecular targets data of prophylaxis and therapy of typhoid.
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