CAJ | 학술논문

B7-2蛋白为免疫球蛋白超家族(IGSF)成员之一.采用PCR法从分泌鼠抗人B7-2抗体的杂交瘤细胞株克隆出该单克隆抗体的重链(VH)和轻链(VL)可变区基因,通过重叠延伸PCR(SOE-PCR)方法,在VH和VL可变区基因间引入连接肽(Gly4Ser)3编码序列,体外构建抗人B7-2的鼠源单链抗体基因B7-2-ScFv.利用新型杂交病毒HyNPV的Bac-to-Bac表达系统,将B7-2-ScFv基因克隆到杆状病毒转移载体pFastBacHTa中,获得重组转移载体pFastBacHTa-B7-2-ScFv,转化大肠杆菌DH10Bac,获得重组杆粒Bacmid-B7-2-ScFv.将此重组杆粒的DNA转染Sf9细胞,获得重组病毒HyNPV-ScFv.感染该重组病毒的BmN细胞经SDS-PAGE和Western blotting分析表明,至感染24 h时目的蛋白已有表达,96 h达到最高表达水平,表达产物的分子质量约31 kD.用该重组病毒感染家蚕5龄起蚕,感染后96 h蚕体表现出明显的发病症状.RT-PCR结果表明,5龄起蚕感染后48 h,在脂肪体内已有目的基因转录,一直持续到感染后120 h;SDS-PAGE和Western blotting分析表明,5龄起蚕感染96 h后方可在脂肪体中检测到目的蛋白表达.构建并在家蚕中成功表达鼠抗人B7-2单链抗体,为深入研究和开发该抗体奠定了基础.
B7-2단백위면역구단백초가족(IGSF)성원지일.채용PCR법종분비서항인B7-2항체적잡교류세포주극륭출해단극륭항체적중련(VH)화경련(VL)가변구기인,통과중첩연신PCR(SOE-PCR)방법,재VH화VL가변구기인간인입련접태(Gly4Ser)3편마서렬,체외구건항인B7-2적서원단련항체기인B7-2-ScFv.이용신형잡교병독HyNPV적Bac-to-Bac표체계통,장B7-2-ScFv기인극륭도간상병독전이재체pFastBacHTa중,획득중조전이재체pFastBacHTa-B7-2-ScFv,전화대장간균DH10Bac,획득중조간립Bacmid-B7-2-ScFv.장차중조간립적DNA전염Sf9세포,획득중조병독HyNPV-ScFv.감염해중조병독적BmN세포경SDS-PAGE화Western blotting분석표명,지감염24 h시목적단백이유표체,96 h체도최고표체수평,표체산물적분자질량약31 kD.용해중조병독감염가잠5령기잠,감염후96 h잠체표현출명현적발병증상.RT-PCR결과표명,5령기잠감염후48 h,재지방체내이유목적기인전록,일직지속도감염후120 h;SDS-PAGE화Western blotting분석표명,5령기잠감염96 h후방가재지방체중검측도목적단백표체.구건병재가잠중성공표체서항인B7-2단련항체,위심입연구화개발해항체전정료기출.
B7-2 protein iS one member of the immunoglobulin superfamily.The variable domain genes encoding heavy (VH)and light(VL)chains of B7-2 antibody were cloned from a murine anti.human B7-2 hybridoma cell line.Using spli-cing by overlapping extension PCR(SOE-PCR)approach,coding sequence of a short linker peptide(Gly4Ser)3 was in-serted between the VH and VL gene fragments to construct the anti-human B7-2 single-chain antibody gene B7-2-ScFv of murine origin in vitro.The mouse B7-2.ScFv gene was subcloned into a baculovirus transfer plasmid pFastBacHTa by using the Bac-to-Bac expression system of the new hybrid virus HyNPV to obtain the recombinant transfer vector pFast-BacHTa-B7-2.ScFv which was subsequently transformed into E coil DH10Bac to generate the recombinant Bacmid-B7-2.ScFv Sf9 cells were transfected with the Bacmid-B7-2-ScvF,DNA and the recombinant baculovirus Bac-B7-2-ScFv was obtained.SDS-PAGE and Western bIot analysis of BmN cells infected by the recombinant baculovirus showed that the expressed product could be detected at 24 hpi(hours post infection)and reached the maximum level at 96 hpi,with a molecular weight of about 31 kD.Symptoms on the infected 5th instar newly exuviated Bombyx mori Iarvae were obvious at 96 hpi.RT-PCR results re-vealed that B7-2.ScFv gene transcription was detected from 48 to 120 hpi in fat body of the 5th instar newly exuviated Bombyx mori larvae.SDS-PAGE and Western blot analysis indicated that the target protein was detected in fat body until 96 hpi.Construction and successful expression of murine anti-human single-strand antibody in Bombyx moil lays a good foundation to study and develop this antibody further.