ABSTRACT
<p><b>OBJECTIVE</b>To develop a targeting protein for Xenopus kinesin-like protein 2 (TPX2) C' terminal SBP-3 x Flag-tagged HCT 116 cell model.</p><p><b>METHODS</b>Homologous arms were amplified by polymerase chain reaction (PCR), and then the adeno-associated virus (AAV) -targeting vector of TPX2 was constructed. HCT 116 cells were targeted after the viruses were packaged. Positive cell clones with neomycin resistance gene were obtained by G418 and PCR screening. Finally, the neomycin gene cassette was excised after the targeted clones were infected with adenovirus expressing Cre-recombinase, and the TPX2 C' terminal SBP and 3 x Flag endogenous double-tagged HCT 116 cells were obtained by PCR screening.</p><p><b>RESULTS</b>Two positive cell clones with neomycin resistance gene were obtained by PCR screening. The positive clones with neomycin resistance gene excised were obtained by Cre adenovirus infection, and the knock-in of SBP-3 x Flag gene was verified by Western blot analysis.</p><p><b>CONCLUSION</b>The TPX2 C' terminal SBP-3 x Flag tagged HCT 116 cell model was successfully established.</p>
Subject(s)
Humans , Cell Cycle Proteins , Genetics , Colorectal Neoplasms , Genetics , Pathology , Dependovirus , Genetics , Gene Targeting , Genetic Vectors , HCT116 Cells , Microtubule-Associated Proteins , Genetics , Nuclear Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection.</p><p><b>METHODS</b>By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined.</p><p><b>RESULTS</b>After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions to cellular receptors and the fusion activity of the virus.</p><p><b>CONCLUSION</b>The SARS-CoV spike protein and membrane proteins are able to elicite efficient neutralizing antibodies in SARS patients. The neutralizing antibodies we generated in this study may be more promising candidates for prophylaxis and treatment of SARS infection.</p>