| With the development of recombinant DNA technology, there has been substantialsuccess made in developing microbial cell surface display technology. Surface display is apowerful technique that uses natural microbial functional components to express aheterologous protein or peptide on the surface of host cells through linkage with agenetically-encoded anchor protein. Cell surface display has been used in many filed,including whole cell biocatalysts, identifying protein-protein interaction, selection antibodyand live oral vaccine construction. A variety of new systems have been reported for yeast, andbacteria except phage.Yeast surface display was divided into agglutinin system and flocculation systemaccording the different anchor protein. Agglutinin system is the most popular one and alsocommercially available. With many great successes using this system, it also has a limitationfor host strains and the host should be replaced the native promoter of AGA1with a GAL1-10promoter by genetic modification. The novel and simple yeast surface display system with asingle expression vector should be constructed.With the development of drug resistance and more regulations and bans on the use ofanticoccidial drugs，vaccination is urgently needed to control the chicken coccidiosis. In viewof the complex life cycle of coccidia and protein expression with stage characteristics, thevaccine is less effective. The EtMic2, a microneme protein of E. tenella, is secreted from themicroneme and involved in host-cell invasion. Previous studies suggested that the EtMic2protein showed partial protection of the chicken from E. tenella infections.S. cerevisiae is generally regarded as safe and benign organisms and the yeast cell wallcomponents would modulate immunity of hosts. The use of S. cerevisiae as a vaccine vehiclehas been used to protect hosts against bacteria and viral diseases. Yeast’s post-translational processing is eukaryotic, and thus common to higher eukaryotic cells. So, it expressed theeukaryotic protein (such as E. tenella protein) is better than bacteria. S. cerevisiae has beenused in poultry industry to against coccidiosis; however, the use of S. cerevisiae as a livevaccine vehicle in parasitology has never been reported.Stimulate and improve the body immune system is the active ingredient from the yeastcell wall. In order to evaluate the impact of EtMic2, the differences of the cell wall proteingroup between the EtMic2displayed strain and the wild-type yeast strain were detected usingtwo-dimensional electrophoresis.1. Establishment of novel surface display system of S. cerevisiaeIn this study, we constructed a universal vector pYSD using a bidirectional expressionvector pSP-G2as basic vector, which carrying the AGA1gene driven by the PGK1promoterin one direction and the AGA2-expression cassette driven by the TEF1promoter in the reversedirection, and uses the geneticin, a G418resistant gene as the selection marker fortransformants. EGFP and EtMIC2were cloned on the plasmid pYSD and transformed inwild-type (CICC1224and HAO) as well as laboratory strains (EBY100and W303-1A) of S.cerevisiae. The new system has been confirmed by displaying EGFP and EtMic2. To test ifthe native Aga2protein has any effect on the heterologous protein display, the AGA2genewas deleted from the strain EBY100. The results indicate that the native expressed Aga2protein has no effect on the display efficiency of heterologous proteins. In order to detect ifthe expression of heterologous proteins does have any effect on the yeast growth, an analysisof the growth rates and doubling times among the different strains (HAO/pYSD,HAO/pYSD–EtMIC2, HAO/pYSD-GFP and HAO) was performed, the result suggested thatthe expression of the EGFP and EtMic2proteins on the surface of different S. cerevisiaestrains has a nil effect on the growth rate. Because all the display elements are put into oneexpression vector, the new system is much simpler to use, and there is no need for any geneticmodification of the host strains; therefore, the new system can be used in wild-type as well aslaboratory strains of S. cerevisiae.2. Construction and evaluation of immune protective effect of EtMic2live oralvaccine The Western Blot analysis was employed to further demonstrate that the EtMic2wasdisplayed on the yeast cell surface. Because the Aga2-EtMic2fusion protein is linked to thecell wall anchor protein Aga1p through two S-S bonds, the Aga2p-EtMic2fusion protein canbe released by DTT (100mM) treatment. The DTT extracted Aga2p-EtMic2fusion proteinwas detected using anti-EtMic2monoclonal antibody. Freshly harvested yeast cells wereadministered using oral gavage to chickens, at21days post immunization, all groups wereorally challenged with3000freshly sporulated E. tenella oocysts except the unchallengedcontrol. Serum antibody and cytokines were detected using ELISA and cell immune level wasmeasured by MTT. The results showed that the yeast oral vaccine could stimulate humoral aswell as cell mediate immune responses. In addition, the live oral vaccine can improve weightgains (80.9%), reduced cecal pathology (lesion score is2.3±0.46) and reduced oocyst fecalshedding (76.56%) compared with non immunized controls. These results suggested thatEtMic2displayed on the cell surface of S. cerevisiae could be used as potential live vaccineagainst chicken coccidiosis.3．The influence of EtMic2to yeast cell wall proteomicStimulate and improve the body immune system is the active ingredient from the yeastcell wall. The comparative cell wall proteomic between vaccine strains (HAO/pYSD-EtMIC2)and wild-type yeast (HAO) were analyzed with two-dimensional gel electrophoresis (2-DE)followed by MS protein identification.2-DE gel analysis showed210protein spots from eachstrain at the same processing conditions and20protein spots were differentially expressed(silver staining intensity greater than or equal to2), including14up-regulated expressedprotein spots and4down-regulated expressed protein spots. Mass spectrometry successfullyidentified16protein spots. The cellular function analysis of differential expressed proteinsmainly involved in cell mediated control of cellular organization, cellular catalytic,metabolism and protein fate and synthesis. The main metabolic pathways involved in theglycolytic pathway, protein secretory pathway, oxidative stress, and amino acid metabolicpathways. |
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