Rearrangement of light chain gene of anti-tac antibody

Abstract

This study is concerned with the gene rearrangement of light chain gene of anti-Tac antibody which reacts specifically with human activated T cell. The main part of this study is in the following three area: first, large scale isolation of mCh28-$JK_1B_3$ 18 DNA which is a modified Charon 28 DNA containing the light chain DNA for the purpose of obtaining $JK_1B_3$ 18 DNA (light chain DNA). Second, $JK_1B_3$ 18 DNA were subcloned into pBR 322 and large amount of light chain DNA was electroeluted after BamHI digestion for the further study. Third, the electroeluted $JK_1B_3$ 18 fragment was mapped by the various restriction endonuclease by comparing with the restriction enzyme map of mouse embrionic light chain DNAs reported. Phage DNA mCh28-$JK_1B_3$ 18 was transfected to E. coli LE 392 in the presence of $CaCl_2$. The resulting phage plaques were hybridized nick translated $J_K$ probe to identify presence of $JK_1B_3$ 18 DNA. Many plaques showed positive result. To obtain $JK_1B_3$ DNA, phage DNA was digested with BamHI restriction enzyme and electroeluted the 7.2Kb fragment. The fragment was ligated with pBR 322 and transformed to E. coli HB101. The transformant were identified by the methods of colony hybridization, restriction digestion, and southern blotting hybridization. Transformation efficiency was about $6\times10^6$ transformant per ug pBR 322 DNA. Transformant with recombinant plasmid (pBR322-$JK_1B_3$) was mass-cultured and plasmid DNA was isolated in pure form by the CsCl-EtBr equilibrium density gradient. Finally $JK_1B_3$ 18 light chain DNAs were isolated from hybrid plasmid by electroelution method after BamHI digestion. Anti-Tac antibody light chain gene were digested with various restriction enzymes and mapped comparing with mouse embrionic light chain gene. Light chain gene are well rearranged by the results of this study. $J_1-J_2$ cluster was deleted and C region are linked to $J_3-J_4-J_5$ cluster. In the case of this deletion 2 HindIII, 1...