02249naa a2200349 a 450000100080000000500110000800800410001902400380006010000160009824501180011426000090023252012480024165000160148965000200150565000130152565000150153865000300155365000320158365000190161565000180163465000230165265000120167565000210168765000100170865300180171865300130173670000180174970000140176770000170178170000160179877300850181415168132024-02-01       1995    bl uuuu     u00u1 u    #d7 a10.1016/0165-2427(94)05305-c2DOI1 aSEOW, H. F.  aExpression, biological activity and kinetcis of production of recombinant ovine TNF-alpha.h[electronic resource]  c1995  aAbstract: Ovine tumour necrosis factor-alpha (OvTNF-α) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-α cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNFα (rOvTNF-α) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30°C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-α from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-α achieved in E. coli JM109 and AM207 were approximately 1 mg L−1. Both rOvTNF-α and recombinant human TNF-α (rhTNF-α) exerted cytotoxicity on L929 cells. However, rOvTNF-α but not rhTNF-α stimulated proliferation of ovine thymocytes. Maximum levels of TNF-α mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.  aDNA primers  aGene expression  aGenetics  aImmunology  aPolymerase chain reaction  aRecombinant fusion proteins  aSheep diseases  aT-lymphocytes  aBiologia Molecular  aDoença  aEscherichia Coli  aOvino  aBase Sequence  aClonagen1 aROTHEL, J. S.1 aPEPIN, M.1 aDAVID, M. J.1 aWOOD, P. R.  tVeterinary Immunology and Immunopathologygv. 44, n. 3/4, p. 279-291, Jan. 1995.