摘要
应用氯化铯－超离心法从抗人纤维蛋白单克隆抗体ＳＺ一６３杂交瘤细胞抽取总ＲＮＡ，经Ｏｌｉｇｏ（ｄＴ）纤维素柱层析分离出ｍＲＮＡ。采用ＲＴ－ＰＣＲ技术，扩增出了ＳＺ－６３重链和轻链可变区基因。应用基因重组技术将ＳＺ－６３可变区基因片段与人免疫球蛋白γ１重链ＣＨ１和轻链恒区基因进行拼接，构建噬菌体ＳＺ－６３／ＨｕＦａｂ表达载体，并在大肠杆菌中表达。表达的嵌合抗体Ｆａｂ片段为可溶性，ＥＬＩＳＡ及Ｗｅｓｔｅｒｎｂｌｏｔ检测证实能特异地与人纤维蛋白呈结合反应，表达量约为２１０μｇ／Ｌ。
RNA was selected by oligo (dT)-cellulose columnchromatography from the total RNA isolated frommurine antifibrin monoclonal antibody SZ-63 hybrido-ma cells. cDNA coding for heavy and light variable re-gions were amplified by reverse transcription-poly-merase chain reaction. The amplified fragments werethen cloned and sequenced. The nucleotides of SZ-63VH and Vk were 354 and 321 respectively. The aminoacid sequence of the heavy and light chain of SZ-63were also deduced. The variable region genes of SZ-63were linked with human immunoglobulin γ1 CH andkCL genes by means of recombinant DNA technique.The phagemid SZ-63 Fab/Hu expression vector wasconstructed and introduced into E. coli HB2151 for ex-pression of soluble chinieric Fab fragment. The resultsof western blot and enzyme-linked immunosorbent as-say (ELISA) showed that the expressed products re-tained the capability of binding to crosslinked fibrin.The concentration of the expressed products in the cul-ture supernatant was about 210μg/L.
出处
《中华血液学杂志》
CSCD
北大核心
1995年第9期462-464,共3页
Chinese Journal of Hematology
基金
核工业科学基金
江苏省卫生厅资助
