Intracellular Antibodies: Development and Applications by Antonino Cattaneo, Silvia Biocca

By Antonino Cattaneo, Silvia Biocca

Recent advances within the box of recombinant antibodies have accepted the manipulation of genes encoding particular antibodies, therefore permitting their ectopic expression in a wide selection of non-lymphoid cells. This quantity describes how the ectopic expression of antibodies, as secreted or as intracellularly retargeted molecules, could be exploited to dam organic services or to confer new phenotypic features (e.g. resistance to a virus). this can be the 1st publication describing this rising expertise, that is receiving expanding consciousness for software in lots of diverse fields and organic platforms - from human gene remedy to plant biotechnology.

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LH~I=C~H=2:::::1=C=H=3==1=============2nd lst cDNA strand .. etc. Clone into approriate bacterial or mammalian expression vector Fig. 3·3· Using RACE to clone V regions. When V region prirners are unable to amplify the correct V regions, RACE may be performed using general prirners independent of the V region sequence being amplified. After the first cDNA strand has been synthesized, it is tagged using either terminal transferase to add a tail of As or RNA ligase to add a specific oligonucleotide (both indicated by the bold arrow in the figure).

B) Polding and assembly begin cotranslationally. The N-terminal domains begin to fold while the nascent H chain is still on the polysome. When at least the VH and CH1 have folded and the intradomain disulfides formed, nascent H may assemble with preexisting L chains. Some L chains form L1 homodimers. In B cells, Hm and Hs homodimerization might also involve chains synthetized on the same polysome. Reprinted with permission from ref. 82. 44 Intracellular Antibodies: Development and Applications assembly of Ig chains also begins on nascent H chains (Fig.

30. Persie L, Roberts A, Wilton J et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 1997; 187:9-18. 31. Heinrichs A, Milstein C, Gherardi E. Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-side PCR. J. Immunol. Methods 1995; 178:241-251. 32. Nissim A, Hoogenboom HR, Tomlinson IM et al. Antibody fragments from a 'single pot' phage display library as immunochemical reagents.

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