鼠抗人B7-2分子单链抗体基因的构建及其在家蚕中的表达  

Construction of Murine Anti-human B7-2 Molecular Single Chain Antibody Gene and its Expression in Bombyx mori

作　　者：胡静平[1] 董海辉[1] 邱玉华[1,2] 陈永井[1,2] 王文兵[3] 黄光镁[1] 吴小锋[4] 朱江[1] 

机构地区：[1]苏州大学基础医学与生物科学学院,江苏苏州215123 [2]苏州大学医学生物技术研究所,江苏苏州215007 [3]江苏大学生命科学研究院,江苏镇江212013 [4]浙江大学动物科学学院,杭州310029

出　　处：《蚕业科学》2010年第2期274-281,共8页ACTA SERICOLOGICA SINICA

基　　金：国家科技重大专项课题(No.2009ZX09103-705);江苏省自然科学基金项目(No.BK2004203)

摘　　要：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24h时目的蛋白已有表达,96h达到最高表达水平,表达产物的分子质量约31kD。用该重组病毒感染家蚕5龄起蚕,感染后96h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48h,在脂肪体内已有目的基因转录,一直持续到感染后120h;SDS-PAGE和Western blotting分析表明,5龄起蚕感染96h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。B7-2 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 splicing by overlapping extension PCR （SOE-PCR） approach, coding sequence of a short linker peptide （Gly4Ser） 3 was inserted between the VH and VL gene fragments to construct the anti-human B7-2 single-chain antibody gene BT-2-ScFv of murine origin in vitro. The mouse BT-2-ScFv gene was subctoned 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. coli DHIOBac to generate the recombinant Bacmid-B7-2-ScFv, Sf9 cells were transfected with the Bacmid-B7-2-ScFv DNA and the recombinant baculovirus Bac-B7-2-ScFv was obtained. SDS-PAGE and Western blot 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 larvae were obvious at 96 hpi. RT-PCR results revealed 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.

关 键 词：B7-2蛋白 单链抗体 杂交病毒HyNPV 家蚕 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]