Objective:To screen antigen of Helicobacter pylori outer membrane proteins by murine infection model.Methods:Parallel two-dimensional gel electrophoresis (2D) of outer membrane proteins extracted from Helicobacter pylori strain SS1 was performed.Western blot of a duplicate 2D gel hybridized with serum from H.pylori-infected murine was employed.Immunogenic H.pylori proteins identified in this way were digested in gel by trypsin and the mass of generated peptides were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS).The data obtained from peptide mass finger-printing (PMF) were searched using the internet available database.Results:32 proteins were identified and they are in good agreement with typical protective antigens which reacted with serum from H.pylori-infected patients.Conclusion:The results suggest that murine model of H.pylori may be valid to screen antigens for human vaccination and the proteins identified in this paper are valuable for the selection of H.pylori protective antigens as well.

Thrombin-like enzymes (TLEs) are studied widely because of their therapeutic potential in myocardial infarction and thrombotic diseases. We synthesized the DNA fragment encoding thrombin-like enzyme calobin from Agkistrodon caliginosus (Korean Viper) venom by fusion PCR and expressed it in Pichia pastoris. After induction by 0.5% methanol for 48 h, the expression level of recombinant calobin reached 3.5 g/L in medium. The recombinant calobin was purified by Q-Sepharose Fast Flow ion-exchange chromatography and Sephacryl-S-100 gel filtration chromatography. Purified sample had a molecular weight of 32 kD shown in SDS-PAGE. It hydrolyzed fibrinogen and formed a light white hydrolysis circle in fibrinogen plate. SDS-PAGE analysis showed that recombinant calobin cleaved Aalpha-chain of fibrinogen specifically, and produced an appropriately 40 kD new band. However, we failed to find its fibrin-clot formation activity.
Agkistrodon ; Animals ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Endopeptidases ; biosynthesis ; genetics ; Thrombin ; biosynthesis ; genetics ; Viper Venoms ; enzymology

Anthrax is a zoonosis caused by Bacillus anthracis, which seriously affects human health. In recent years, a special phenomenon is found that the metabolic active of a bacterium remains after it is killed. To development of a KBMA (killed but metabolically active) Bacillus anthracis vaccine candidate strain, a plasmid pMAD and a recombinase system Cre-loxP were used to knockout the uvrAB gene of B. anthracis AP422 which lacks both of two plasmids pXO1 and pXO2. The results of PCR and RT-PCR shows that uvrAB genes were deleted from B. anthracis AP422 chromosome successfully. The constructed B. anthracis AP422deltauvrAB was inactivated by photochemical treatment (PCT) including an exposure in a long-wave-length ultraviolet (UVA) light and a treatment of 8-Methoxypsoralen (8-MOP), then the metabolic activity were detected by the method of MTS. The results showed that the killed B. anthracis AP422deltauvrAB maintained a highly metabolic activity for at least 4 hours, showing a state of KBMA. The KBMA strain of B. anthracis AP422deltauvrAB provides the prospective vaccine candidate strain for anthrax.
Anthrax ; immunology ; microbiology ; prevention & control ; Anthrax Vaccines ; genetics ; immunology ; radiation effects ; Bacillus anthracis ; genetics ; immunology ; Gene Knockout Techniques ; Methoxsalen ; pharmacology ; Ultraviolet Rays ; Vaccines, Inactivated ; genetics ; immunology