Immunoproteomics of membrane proteins of Shigella flexneri 2a 2457T

AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.

Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics

Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated ani- mals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, FOFI ATP synthase subunit beta, and OMPI. These provide many candidate immunoreactive proteins for vaccine development.

Immunoproteomic Assay of Antigenic Surface Proteins in Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus （S. zooepidemicus） is a zoonotic pathogen with worldwide distribution. Lacking suitable vaccine and virulent maker is still bottleneck to control this infection. An immunoproteomic approach has been used to screen the membrane-associated and cell wall-associated proteins of S. zooepidemicus isolate in China CY to discover vaccine candidate antigens and therapeutic agents. Finally, 11 membrane-associated proteins, and 13 cell wall-associated proteins were successfully identified. BLAST （www.sanger.ac.uk） results also indicated that nucleotide sequences of majority identified proteins shared high homology （60%） with S. zooepidemicus, except for AC1-3, AC5, AC8, and AC13. Moreover, genes for 7 of the identified proteins were detected from CY; compared with ST171, 3 of them （AM1, AM8 and AC11） were only found in virulent strains （CY）. All of the proteins identified in this study remain not to be reported in S. zooepidemicus. Some of the proteins serve a vital role in the immune system and reproduction of host species according to available data, while the functions of the rest were seldom researched.

Identification of Antigens Common to Streptococcus suis Serotypes 2 and 9 by Immunoproteomic Analysis

Streptococcus suis is a Gram-positive pathogen that causes serious diseases in pigs. In addition to S. suis serotype 2 （SS2）, S. suis serotype 9 （SS9） is another prevalent serotype, which is frequently isolated from the organs of diseased pigs in China. An immunoproteomic-based approach was developed to identify antigens common to SS2 and SS9 for vaccine development. Cell wall proteins extracted from SS2 strain HA9801 were screened by two-dimensional Western blot using anti-SS2 sera, anti-SS9 sera, or pre-immune sera pooled from specific pathogen free （SPF） mice. Protein spots on preparative gels were excised and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which led to the identification of four shared immunogenic proteins （arginine deiminase, translation elongation factor-Ts, o-acetylserine lyase, and 1-phosphofructokinase）. The genes encoding these four proteins from SS9 strain GZ0565 were cloned and their proteins were overexpressed in Escherichia coli BL21. Western blot analysis of these recombinant proteins using the convalescent serum of an SPF mini-pig inoculated with the SS2 strain, anti-SS2 sera, and anti-SS9 sera pooled from SPF mice further confirmed the immunogenicity of these proteins. These immunogenic proteins, which are encoded by genes that are reasonably conserved among SS2 and SS9 strains, could be developed as vaccine candidates.

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

Overexpression of Annexin A2 Is Associated with Abnormal Ubiquitination in Breast Cancer

Abnormal expression of annexin A2 contributes to metastasis and infiltration of cancer cells. To elucidate the cause of abnormal expres- sion of annexin A2, Western blotting, immunoproteomics and immunohistochemical staining were performed to analyze differentially ubiquitinated proteins between fresh breast cancer tissue and its adjacent normal breast tissue from five female volunteers. We detected an ubiquitinated protein that was up-regulated in the cancer tissue, which was further identified as annexin A2 by mass spectrometry. These results suggest that abnormal ubiquitination and/or degradation of annexin A2 may lead to presence of annexin A2 at high level, which may further promote metastasis and infiltration of the breast cancer cells.

Study roadmap for high-throughput development of easy to use and affordable biomarkers as diagnostics for tropical diseases:a focus on malaria and schistosomiasis

Background:Interventions are currently being used against‘infectious diseases of poverty’,which remain highly debilitating and deadly in most endemic countries,especially malaria,schistosomiasis,echinococcosis and African sleeping sickness.However,major limitations of current‘traditional’methods for diagnosis are neither simple nor convenient for population surveillance,and showed low sensitivity and specificity.Access to novel technologies for the development of adequate and reliable tools are expressly needed.A collaborative project between African Network for Drugs and Diagnostics Innovation and partner institutions in Africa and China aims to screen suitable serological biomarkers for diagnostic pipelines against these‘diseases of the poor’.Methods:Parasite-specific exposed versus unexposed individuals were screened and sera or urine/stools were collected through case-control studies in China and African countries.Target genes/open reading frames were selected,then will be cloned and cell-free expressed,quantified and immuno-detected.Target antigens/epitopes will be probed and screened with sera from exposed or unexposed individuals using a high-throughput antigen screening platform as the study progresses.The specificity and sensitivity of highly immunoreactive biomarkers will be evaluated as well,using enzyme-linked immunosorbent assays or dipsticks.Discussion:This roadmap explicitly unfolds the integrated operating procedures with focus on malaria and schistosomiasis,for the identification of suitable biomarkers that will aid the prioritization of diagnostics for population use.However,there is need to further validate any new diagnostic through comparison with standard methods in field deployable tests for each region.Our expectations for the future are to seek regulatory approval and promote the use of diagnostics in endemic areas.