文档介绍：Cloning and Prokaryocytic Expression of Omp25 Gene of Brucella
Abortus and Studies of Antigen Specificity
Abstract
Brucella is a Gram-negative facultative intracellular parasitic bacteria .It is a zoonotic
disease that was caused by brucella SPP and severes affectting humans health and the
development of animal husbandry. Accurate diagnosis is very important for effective
control and eradication of the brucellosis and researching a suitable brucella diagnostic
antigen is the aim of the people,Therefore, the Brucella outer membrane protein which is
to immunogenicity and protective antigens is ing researches focus.
The gene of Brucella outer membrane protein was downloaded from
gene was selected by analysis that showed high gene homologous of the Brucella species at
present .The experiment select the Omp25 of A544 for the Preliminary study. In
the studies, a gene recoding ouetr membrane portein 25kDa was amplified from the
genomic DNA template of by Polymerase chain reaction (PCR) technique. A
fragment of about 642bp was obtained by PCR from the A544 genomic
amplified fragments were digested with the restriction endonuclease EcoR I,
Xho I ,then linked to expression vector pGEX-6p-1 and formed a pGEX-Omp25
Identification by PCR , double enzyme shearing and sequencing exerted respectively to
binated vehicle bacterium liquid and binated plasmid showed the correct
inserting position of the interesting gene in the vector. The binant fusion protein
(pGEX-Omp25) was expressed in the form of inclusion bodies at high level in BL21
induced by mmol/L IPTG for 4h. The Molecular weight of binant fusion protein
was about 49kDa.
The purifieation of pGEX-Omp25 was done by electrodialysis
western-blotting analysises the reactionogenicity of the binant fusion
protein(pGEX-Omp25), which found the protein with Virginia's adjuvants emulsification
immune rabb