Vaccines from infectious agents
A vaccine composition and selected technology, which can be used in anti-infective drugs, anti-infective drugs, genetic engineering and other directions, and can solve problems such as undetermined properties of peptide fragments
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Embodiment 1
[0031] Example 1: Preparation of heat-induced HSPs
[0032] The cells of the extracellular pathogen Mycobacterium bovis (BCG) were cultured at 37°C to stationary phase with conventional medium, heat-shocked at 42°C for 0.5 hours, or at 39°C for 5 hours, and cultured overnight to induce heat-shock-containing cells. Formation of products of protein and antigenic peptide fragments. Pathogen cells are then washed in phosphate-buffered saline (PBS) and resuspended in a homogenization buffer, especially a hypotonic buffer, such as containing 2 mM MgCl 2 10mM phosphate, pH7.4. The cells are then disrupted by any suitable technique, e.g. with a cell homogenizer (e.g. French press, Ultraturrax or Waring mixer), lysed with detergents such as Tween or Triton, complement lysed at 37°C, or re-frozen, e.g. in liquid nitrogen melt. Cell lysates are then processed by centrifugation, typically at 3-5000g for 5 minutes to remove nuclei and cell debris, followed by high-speed centrifugation a...
Embodiment 2
[0036] Example 2: Immunization with induced HSPs; immunity in vaccine recipients
[0037] Vaccine compositions containing HSP complexes were prepared as described in Example 1. Mice and rabbits were vaccinated by injecting 1-10 μg of stress protein complexes in phosphate buffered saline, with the same dose one month after the initial injection. vaccine booster. Immunity induced by pathogens was analyzed by Western blot using total M. bovis proteins. Antibody titers of 1:1-10000 are usually obtained, and cytotoxic T cell activity against pathogen-infected cells can also be detected in immunized mice. Challenge with fixed M. bovis at 6, 12 and 18 months after primary immunization resulted in good antibody responses with titers ranging from 1:1 to 10,000, indicating a good memory response in immunized animals.
Embodiment 3
[0038] Example 3 Comparison of Peptides Binded in Constitutive and Induced HSP Complexes
[0039] M. tuberculosis was grown to saturation in Sauton's medium at 37°C for 3 days. A 4 ml aliquot of the stationary culture was used to inoculate 500 ml of Sauton's medium in 2 liter Erlenmeyer flasks, and the culture was grown overnight at 30°C. The temperature of the log phase culture was then raised to 40°C and grown for an additional 4 hours before the bacteria were harvested by centrifugation at 10000 rpm for 10 minutes. Non-induced (constitutive) HSPs were isolated from initial cell cultures by centrifugation at 37°C.
[0040] Cell pellets from centrifuged samples were resuspended in lysate containing 0.5% Tween and HSPs prepared from induced and non-induced cells by ammonium sulfate precipitation as described in Example 1. Purified HSPs were resuspended in 10% acetic acid and boiled for 15 minutes to elute HSP-bound peptides. Denatured HSPs were precipitated in a Beckman air...
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