The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9...The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.展开更多
Ralstonia solanacearum infecting Davana （Artemisia pallens Wall.） from commercial nurseries in India was isolated on modified semi selective media （SMSA）. Here, we report a new host for Ralstonia solanacearum i.e....Ralstonia solanacearum infecting Davana （Artemisia pallens Wall.） from commercial nurseries in India was isolated on modified semi selective media （SMSA）. Here, we report a new host for Ralstonia solanacearum i.e. davana. It has huge demand in medicinal and aromatic industries. Isolate was confirmed as race-l, biovar-3 by morphological, physiological, biochemical and pathogenicity studies. Two sets of primers （OLI 1 ＆ Y2 and Y 1 ＆ Y2） were used in this study. Further, the identity of the isolate was confirmed by serological diagnostic kit obtained from International Potato Research Center, Lima, Peru and single chain variable fragment antibody specific to Ralstonia solanacearum used to confirm the casual organism.展开更多
In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and mai...In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment.Additionally,it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system.As an alternative,this study modifies the hyperstable single-chain fragment variable(scFv)antibody to eliminate cancer using its linear shape.The scFv(F8)antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site.Furthermore,a cell-penetrating peptide(CPP)was attached to the scFv(Ras)antibody model to allow entrance to the cell,creating CPP-scFv(Ras).Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blotting,and the binding assay were performed to prove its effectiveness.As a result,CPP-scFv(Ras)was successfully engineered and bound to the antigen,HRas(G12V).展开更多
文摘The single chain variable fragments of antibodies(scFvs) against cTnI were screened from the phage display antibody library by using cTnI as the target antigen. After four rounds of panning, four clones(H2, G5, A9, B9) from the phage display antibody library were verified to show higher binding affinity for cTnI by ELISA and to contain the variable region genes of the light and heavy chains of scFvs by sequencing. The variable region genes of scFvs H2 and G5 were successfully amplified by polymerase chain reactions(PCR) and cloned into expression vector pPELB and expressed as a soluble protein in E.coli Rosetta, whose expression yield was about 2% of total proteins. The expressed proteins were purified by nickel(Ni) affinity chromatography and a single band is shown in the position of 28 kDa on SDS-PAGE. The western blot analysis result verifies that the expressed scFv proteins are capable of binding with monoclonal antibodies against hexa-histidine, indicating that they are hexa-histidin-tagged aim proteins. The immunoassay demonstrates that the expressed scFv proteins are able to specifically react with cTnI molecules. The association constant(K_A) values range from 1.2×10 4 to 1.7 ×10 5 L/mol that are correspondent to the affinities of polyclonal antibodies against cTnI from rabbits. These antibodies can be valuable reagents for the immunoassay of cTnI.
文摘Ralstonia solanacearum infecting Davana （Artemisia pallens Wall.） from commercial nurseries in India was isolated on modified semi selective media （SMSA）. Here, we report a new host for Ralstonia solanacearum i.e. davana. It has huge demand in medicinal and aromatic industries. Isolate was confirmed as race-l, biovar-3 by morphological, physiological, biochemical and pathogenicity studies. Two sets of primers （OLI 1 ＆ Y2 and Y 1 ＆ Y2） were used in this study. Further, the identity of the isolate was confirmed by serological diagnostic kit obtained from International Potato Research Center, Lima, Peru and single chain variable fragment antibody specific to Ralstonia solanacearum used to confirm the casual organism.
文摘In the modern pharmaceutical industry,monoclonal antibodies are often used as therapeutic agents.However,they are restricted to cell surface antigens due to their inability to penetrate the outer cell membrane and maintain normal function in the reducing environment.Additionally,it can lead to cytotoxicity since it attacks cancerous cells by mimicking the human immune system.As an alternative,this study modifies the hyperstable single-chain fragment variable(scFv)antibody to eliminate cancer using its linear shape.The scFv(F8)antibody model was modified to recognize human Ras protein by altering residues in the antigen-binding site.Furthermore,a cell-penetrating peptide(CPP)was attached to the scFv(Ras)antibody model to allow entrance to the cell,creating CPP-scFv(Ras).Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE),western blotting,and the binding assay were performed to prove its effectiveness.As a result,CPP-scFv(Ras)was successfully engineered and bound to the antigen,HRas(G12V).
