Production and characterization of scFV Monoclonal antibody against Cry2B and scFv Polyclonal antibody against Cry1Ac through phage display technology
KrishiKosh
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Title |
Production and characterization of scFV Monoclonal antibody against Cry2B and scFv Polyclonal antibody against Cry1Ac through phage display technology
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Creator |
Chidanand.A.Rabinal
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Contributor |
Narayana.Moger
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Subject |
Plant Biotechnology
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Description |
Cry2B and Cry1Ac protein were used to produce single chain fragment variable (scFv) monoclonal antibody for Cry2B and scFv polyclonal antibody for Cry1Ac. The Tomlinson I library was amplified and the scFv phages were made displayed on their surface. They were incubated with immobilized antigen in immunotube for further amplification. Such four rounds of biopanning process were carried out for selection of scFv polyclonal antibody against both the proteins. The ELISA test results indicated that the fourth biopan phage particles had higher binding affinity to the target antigens. In case of Cry2B, forty-five clones were selected from fourth biopan and the same were subsequently used to produce monoclones. In case of Cry1Ac, because of their poor affinity with antigen during affinity selection process only polyclonal antibody was produced. The two clones viz., pscFvCry2B19 and pscFvCry2B43 of Cry2B were selected based on monoclonal phage ELISA and further confirmed by the cross specificity test with random mutant Cry2B proteins. The selected clones were sequenced with LMB3 forward and pHEN reverse primer and characterized. The scFv fragment of pscFvCry2B19 and pscFvCry2B43 is 746 bp and 747 bp long and they shared homology to each other at DNA and amino acid level, except at C? terminal end. According to the sequence analysis the clone pscFvCry2B19 consist of 55.09% G+C and 44.91% A+T. Similarly pscFvCry2B43 has 54.89% of G+C and 45.91% of A+T. Both the clones have similar conserved domains in Ig super family between 47 to 119 positions. The homology search with BLASTn algorithm indicated 96% identity to Homo sapiens partial scFv genes. These observations indicated that both the scFv antibodies bind to a single epitope. |
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Date |
2016-11-07T18:25:40Z
2016-11-07T18:25:40Z 2010 |
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Type |
Thesis
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Identifier |
http://krishikosh.egranth.ac.in/handle/1/84296
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Format |
application/pdf
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Publisher |
UAS, Dharwad
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