CAJ | 학술논문

为尽快研制出国产HTLV抗体诊断试剂，首先从福建HTLV流行区1名HTLV感染者外周血细胞中克隆出HTLV-Ⅰ的全长膜基因（env），继而结合文献报道、PSA软件的亲疏水性分析和EPI软件的B细胞表位分析数据，选择了gp46中段开始延伸至gp21N端212个氨基酸（aa185～aa396）的基因，并在3′端通过（GlySer）2与人工合成的HTLV-Ⅱ型的型特异性表位区基因嵌合，插入原核表达载体pRSET，在E．coli中得到了高效表达，目的蛋白产量约占菌体总蛋白的30%。通过Triton-X100洗涤，低浓度尿素逐步变性处理，8mol/L尿素溶解后纯度在75%左右，经电泳洗脱纯化，最终纯度可达95%左右，纯蛋白得率约40%。经Westernblotting检测，该蛋白对4份HTLV-Ⅰ型和2份HTLV-Ⅱ型血清均有较强反应，而对4份阴性血清无反应，从而有可能用于研制HTLV抗体诊断试剂盒。
위진쾌연제출국산HTLV항체진단시제，수선종복건HTLV류행구1명HTLV감염자외주혈세포중극륭출HTLV-Ⅰ적전장막기인（env），계이결합문헌보도、PSA연건적친소수성분석화EPI연건적B세포표위분석수거，선택료gp46중단개시연신지gp21N단212개안기산（aa185～aa396）적기인，병재3′단통과（GlySer）2여인공합성적HTLV-Ⅱ형적형특이성표위구기인감합，삽입원핵표체재체pRSET，재E．coli중득도료고효표체，목적단백산량약점균체총단백적30%。통과Triton-X100세조，저농도뇨소축보변성처리，8mol/L뇨소용해후순도재75%좌우，경전영세탈순화，최종순도가체95%좌우，순단백득솔약40%。경Westernblotting검측，해단백대4빈HTLV-Ⅰ형화2빈HTLV-Ⅱ형혈청균유교강반응，이대4빈음성혈청무반응，종이유가능용우연제HTLV항체진단시제합。
For development of an antibody detection reagent for humanT-lymphotropic virus (HTLV), a full-length envelope(env) gene of HTLV type Ⅰ (HTLV-I) was cloned from peripheral blood cells of a HTLV-Ⅰ infected individual in a HTLV endemic region of Fujian, and then a gene fragment of 212 amino acid length from the midpiece of gp46 extending to the N-terminal of gp21 was selected as well as a type specific epitope region of HTLV type Ⅱ env gene to construct a HTLV Ⅰ+Ⅱ chimeric gene. This chimeric fragment was inserted into prokaryotic expression vector pRSET and expressed in E. coli. The yield of the target recombinant protein was about 30%. The final purity is about 95% after electrophoresis elution purification. Detected by Western blotting, this recombinant protein showed strong reaction with four HTLV-Ⅰ positive sera as well as two HTLV-Ⅱ positive sera, and had no response to four HTLV negative sera. Thus, this recombinant antigen can be used to develop a diagnostic reagent for HTLV antibody detection.