抗人CD14新克隆ZCH-7-2F9单链抗体的真核表达和初步鉴定  

Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14

作　　者：宁铂涛[1] 汤永民[1] 曹江[2] 沈红强[1] 钱柏芹[1] 

机构地区：[1]浙江大学附属儿童医院血液肿瘤科,杭州310003 [2]浙江大学附属邵逸夫医院临床实验室

出　　处：《中华儿科杂志》2008年第8期605-609,共5页Chinese Journal of Pediatrics

基　　金：国家自然科学基金资助项目（30170391）;浙江省自然科学基金资助项目（Y206009）

摘　　要：目的在真核细胞中表达Zhejiang Children’s Hospital（ZCH）-7-2F9（简称2F9）单链抗体（scFv2F9）以获得高活性的目的蛋白，为2F9其他类型的基因工程抗体及其免疫毒素的研究奠定基础。方法根据pSectag2A多克隆位点、（G4S）3及scFv2F9基因序列，设计含有SfiI、EcoRI酶切位点、6×His以及终止密码TGA的引物，采用高保真的Taq酶，通过重叠延伸拼接法（splicing by overlap extension，SOE）扩增克隆到scFv2F9基因，将扩增到的目的基因片段克隆到pGEM T—easy载体，测序核实后再克隆到pSectag2A真核分泌型表达载体中。阳性重组质粒pSectag2A／scFv2F9转染中华仓鼠卵巢（CHO）细胞进行目的蛋白的瞬时表达，将培养上清浓缩后，流式细胞术观察其对亲本单抗的阻滞效果。结果真核分泌型表达载体pSectag2A／scFv2F9在CHO细胞中获得成功表达，其相对分子质量为31000。亲本直标单抗2F9-FITC被真核细胞表达的scFv2F9阻滞后其阳性细胞百分数、平均荧光强度和最高峰值分别下降了90．02％、63．30％和63．38％。结论克隆到的2F9V。和2F9VL基因及构建的真核分泌型表达载体pSectag2A／scFv2F9是正确的，scFv2F9在CHO细胞中获得成功的表达，且有较高的识别和结合人CD14抗原的功能。Objective Acute monocytic leukemia （AML）-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide （LPS） receptor （designated as CD14） molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of MS, since they can be recognized and bound by mouse-anti- human CD14 monoclonal antibody （McAb）. However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus ＂ humanized antibody＂ is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody （scFv） with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv （ scFv2F9 ） in eukaryotic cells and obtain the scFv2F9 protein with a high biological activity. Methods Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 × His and stop code TGA sequences, scFv2F9 gene was amplified through splicing by overlap extension （SOE） using the high fidelity Taq polymerase. Positive recombinants （pSectag2A/scFv2F9） were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary （CHO） ceils for expression. Western-Blot and flow cytometry （FCM） were carried out to determine the relative molecular mass （Mr） and binding activity of scFv2F9. Results The cloned scFv2F9 gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO calls with an Mr of 31 000. The blocking test showed that the positive ceil percentages, the mean fluorescence intensity （MFI） and the peak of channel （ peak Ch） were reduced by 90. 02%, 63. 30% and 63.38%, respectively after blocking with scFv2F9, while those were 4. 55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells

关 键 词：单链抗体 中华仓鼠卵巢(CHO)细胞 CD14 真核 表达 

分 类 号：R563[医药卫生—呼吸系统] R392.11[医药卫生—内科学]