The Experiment And Application Research On A New-typed Brucella Allergen

Brucellosis is a kind of infected-metamorphism disease existed in human beings and animals. The characteristics of delayed-type hypersensitivity reaction for Brucellosis provide precondition for its specific skin trial diagnosis. The combination of skin trial and serology has great value for the diagnosis of urgent and tardy brucellosis, and the skin trial can distinguish the artificial positive reaction caused by Yersin 09 in serology test with brucellosis.Though brucellin includes many kinds, it can be divided into two species, such as rough brucellin and purified brucellin, they all can be intituled allergen. Rough brucellin includes strong-poisoned-brucellin, feeble-poison brucellin, dog-strained brucellin, strypped-down brucellin, and no outer-fountained-protein brucellin. In the course of pick-up for the skin diagnosis products, such as brucellin, the different strain, bacteria proteins, and lipopolysacchride, can all affect the quality of brucellin. The Brucella bacteria proteins, lipopolysacchride and nucleic acid had been extracted and analyzed by the use of the methods such as ultra-speed centrifugation and chemical extraction methods. The trichloroacetic acid (TCA) and ammonium sulfate (AS) synthesis chemical reaction had been filtrated to purify Brucella enduring-heat protein, namingly purified rotein allergen of Brucella (Br-PPA), then it had benn thoroughly studied and ideal results were obtained.Materials and objects:Materials: Brucella suis 2,Brucella vaccine strain 104M,Purified brucellin from France,Brucellin saled at marketplace in our courtry,The KunMing series mouse. Observed objects: Brucellosis patientsjnfected crowds of Brucella, The irnmuned crowds, Tuberculoses,other infected patients and healthy crowds. Methods:The preparation of Br-PPA: The Brucella suis 2 which had been examined eligible were inoculated on murphy-glucose-arar culture medium, and were cultured in biochemistry box at 37 degree for 24 hours. The representative bacterium falls were picked out and inoculated on live agar slant culture medium for 48 hours, the lawn was washed out with sterilized physiological brine and executed strong bacteria loquid, the concentration was 5 multiply 109 numbers per milliliter. The strong bacteria liquid was sterilized at 15 pounds for 20 minutes. The sterilized liquid was centrifuged at 4000g per minute, the upper liquid was extracted and added equal quantity saturated ammonium sulfate, and was salted out at 4 degree for 12 hours. The deposited protein was centrifuged at speed lOOOOg per minute, the centrifuged deposited protein was dissolved with PBS, and the dissolved protein was deposited with trichloroacetic acid. The deposited protein was dissolved with PBS again, and the purity of sample liquid was determined. The deposition with trichloroacetic acid was repeated, and the deposited sample was dialysed with distilled water and PBS. The purified allergen after dialysis was filtered and frozen in vaccum. After the product was examined up to grade, it was diluted and packed out.The allergen was determined its protein contents by the use of Lowry method, its amylose contens were determined used by anthracene-ketone colorimetry method, its nucleic acid contents were determined by the use of purple-spectrophotometer. The safety trial was completed by the means of observing the alive-keeping situation for KunMing series mouses weighing 18-20g. The causing-sensitive animals were prepared by using guinea-pigs weighing 300-400g which had been immune with Brucella vaccine 104M. The effect-value was determined by the use of Br-PPA and French referenct standard products 4IU. The stability test was completed by themeans that the diluent products were made skin trial, and were used to the determination of effect-value, and were compared with reference standard products after the diluent products being put at 37 degree for 45 days and 4 degree for half a year.The observation of security effect for Br-PPA: The occurrance rate of fast-type reaction was compared among brecellosis and crowd...