中国医师杂志
中國醫師雜誌
중국의사잡지
JOURNAL OF CHINESE PHYSICIAN
2014年
5期
577-580
,共4页
脑炎病毒,日本%抗体,单克隆%克隆,分子
腦炎病毒,日本%抗體,單剋隆%剋隆,分子
뇌염병독,일본%항체,단극륭%극륭,분자
Encephalitis virus,Japanese%Antibodies,monoclonal%Cloning,molecular
目的 克隆抗日本脑炎病毒单克隆抗体2F2重链可变区(VH)基因.方法 从分泌抗日本脑炎病毒单克隆抗体2F2的杂交瘤细胞中提取总RNA,通过RT-PCR扩增重链可变区基因.凝胶回收纯化后与pMD-18T载体连接,重组载体转化于宿主菌DH5α,挑取菌落PCR鉴定后测序并进行分析.测序正确的质粒经EcoRI和XhoI酶切、纯化后的产物和原核表达载体pRSET A在SolutionI作用下连接,转化BL21(DE3)对重组质粒进行表达.经Tricine-十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重链可变区表达产物的表达水平,用ELISA检测表达产物能否与日本脑炎病毒结合.结果 确定了2F2VH基因序列,长度为354 bp,编码118个氨基酸,第22位和第96位残基是维持抗体结构的半胱氨酸,含有特异性CDR1、CDR2和CDR3区域,符合鼠源性免疫球蛋白基因的特征.ELISA检测结果显示原核表达的2F2重链可变区产物可与日本脑炎病毒特异性结合.结论 获得了抗日本脑炎病毒单克隆抗体2F2重链可变区基因.
目的 剋隆抗日本腦炎病毒單剋隆抗體2F2重鏈可變區(VH)基因.方法 從分泌抗日本腦炎病毒單剋隆抗體2F2的雜交瘤細胞中提取總RNA,通過RT-PCR擴增重鏈可變區基因.凝膠迴收純化後與pMD-18T載體連接,重組載體轉化于宿主菌DH5α,挑取菌落PCR鑒定後測序併進行分析.測序正確的質粒經EcoRI和XhoI酶切、純化後的產物和原覈錶達載體pRSET A在SolutionI作用下連接,轉化BL21(DE3)對重組質粒進行錶達.經Tricine-十二烷基磺痠鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)檢測重鏈可變區錶達產物的錶達水平,用ELISA檢測錶達產物能否與日本腦炎病毒結閤.結果 確定瞭2F2VH基因序列,長度為354 bp,編碼118箇氨基痠,第22位和第96位殘基是維持抗體結構的半胱氨痠,含有特異性CDR1、CDR2和CDR3區域,符閤鼠源性免疫毬蛋白基因的特徵.ELISA檢測結果顯示原覈錶達的2F2重鏈可變區產物可與日本腦炎病毒特異性結閤.結論 穫得瞭抗日本腦炎病毒單剋隆抗體2F2重鏈可變區基因.
목적 극륭항일본뇌염병독단극륭항체2F2중련가변구(VH)기인.방법 종분비항일본뇌염병독단극륭항체2F2적잡교류세포중제취총RNA,통과RT-PCR확증중련가변구기인.응효회수순화후여pMD-18T재체련접,중조재체전화우숙주균DH5α,도취균락PCR감정후측서병진행분석.측서정학적질립경EcoRI화XhoI매절、순화후적산물화원핵표체재체pRSET A재SolutionI작용하련접,전화BL21(DE3)대중조질립진행표체.경Tricine-십이완기광산납-취병희선알응효전영(SDS-PAGE)검측중련가변구표체산물적표체수평,용ELISA검측표체산물능부여일본뇌염병독결합.결과 학정료2F2VH기인서렬,장도위354 bp,편마118개안기산,제22위화제96위잔기시유지항체결구적반광안산,함유특이성CDR1、CDR2화CDR3구역,부합서원성면역구단백기인적특정.ELISA검측결과현시원핵표체적2F2중련가변구산물가여일본뇌염병독특이성결합.결론 획득료항일본뇌염병독단극륭항체2F2중련가변구기인.
Objective To obtain the heavy chain variable region (VH) gene of monoclonal antibody 2F2 against Japanese encephalitis virus (JEV).Methods Total RNA was isolated with Trizol from hybridoma 2F2 cells,and cDNA of VH was amplified with reverse transcription polymerase chain reaction (RT-PCR) and sequenced.The putative VH gene was expressed in E.coli,and the expressed products was detected with enzyme linked immunosorbent assay (ELISA) to determine the activity to bind JEV.Results The VH gene was 354 bp in length which encodes 118 amino acids.Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the VH gene was successfully expressed and purified from inclusion bodies.ELISA result also demonstrated that VH gene expressed products bind purified JEV.Conclusions The VH gene of monoclonal antibody 2F2 against JEV had been cloned.
