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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Zheng, W. Y. Yan, F. Wang, Y. Zhang, Z. C. |
| Description | Country affiliation: China Author Affiliation: Zheng WY ( Department of Infectious Disease, The 253th Hospital of P.L.A., Hohhot, China zhengwydid@126.com.); Wang Y ( Department of Pharmacy, Affiliated Hospital of Inner Mongolia Medical University, Hohhot, China.); Zhang ZC ( Department of Infectious Disease, The 253th Hospital of P.L.A., Hohhot, China.); Yan F ( Department of Infectious Disease, The 253th Hospital of P.L.A., Hohhot, China.) |
| Abstract | We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity. |
| e-ISSN | 16765680 |
| Journal | Genetics and Molecular Research |
| Issue Number | 4 |
| Volume Number | 14 |
| Language | English |
| Publisher | Fundação de Pesquisas Científicas de Ribeirão Preto |
| Publisher Date | 2015-10-05 |
| Publisher Place | Brazil |
| Access Restriction | Open |
| Subject Keyword | Antibodies, Bacterial Immunology Antibodies, Monoclonal Bacterial Outer Membrane Proteins Brucella Animals Antibody Specificity Cell Line, Tumor Mice Mice, Inbred Balb C Rabbits Research Support, Non-u.s. Gov't Discipline Genetics Discipline Molecular Biology Discipline Bioinformatics |
| Content Type | Text |
| Resource Type | Article |
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