CAJ | 학술논문

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、6xHis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 overlapextension,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31 000.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2FgVH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.
목적 재진핵세포중표체Zhejiang Children's Hospital(ZCH)-7-2F9(간칭2F9)단련항체(scFv2F9)이획득고활성적목적 단백,위2F9기타류형적기인공정항체급기면역독소적연구전정기출.방법 근거pSectag2A다극륭위점、(G4S)3급scFv2F9기인서렬,설계함유SfiI、EcoRI매절위점、6xHis이급종지밀마TGA적인물,채용고보진적Taq매,통과중첩연신병접법(splicing by overlapextension,SOE)확증극륭도scFv2F9기인,장확증도적목적 기인편단극륭도pGEM T-easy재체,측서핵실후재극륭도pSectag2A진핵분비형표체재체중.양성중조질립pSectag2A/scFv2F9전염중화창서란소(CHO)세포진행목적 단백적순시표체,장배양상청농축후,류식세포술관찰기대친본단항적조체효과.결과 진핵분비형표체재체pSectag2A/scFv2F9재CHO세포중획득성공표체,기상대분자질량위31 000.친본직표단항2F9-FITC피진핵세포표체적scFv2F9조체후기양성세포백분수、평균형광강도화최고봉치분별하강료90.02%、63.30%화63.38%.결론 극륭도적2FgVH화2F9VL기인급구건적진핵분비형표체재체pSectag2A/scFv2F9시정학적,scFv2F9재CHO세포중획득성공적표체,차유교고적식별화결합인CD14항원적공능.
Objective Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant hpopolysaccharide (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 x 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) cells forexpression.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 geue was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supornatant of transformed CHO cells with an Mr of 31 000.The blocking test showed that the positive cell 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 scFv2~,while those were 4.55%,10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.Conclusions The scFv2F9 against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity,which may be useful for the further studies on its b.manized antibodies.