摘要
血小板血栓形成是血管闭塞性疾病的主要成因。其表面的糖蛋
白Ⅱb/Ⅲa是血小板聚集最终共同途径的关键要素。阻断GPⅡb/Ⅲa
与纤维蛋白原的结合，能高效地抑制血小板的聚集，进而使血小板血
栓不能形成。SZ-21是我室制备的一株针对GPⅢa的单克隆抗体，经
家兔颈动—静脉血栓模型证实能显著降低血栓形成，显示了在抗血栓
治疗中的潜在价值。为降低鼠源性单抗免疫原性（HAMA）及全抗体Fc
段对血小板的破坏，本研究将SZ-21制备成小分子抗体，并对这一抗
体片段进行了纯化、复性和体外性质的研究。
一 SZ-21单克隆抗体可变区基因的克隆和序列分析
采用反转录-聚合酶链式反应（RT-PCR）技术，从分泌抗血小板膜
糖蛋白Ⅲa单克隆抗体SZ-21杂交瘤细胞中克隆了该抗体的重、轻链
可变区（V区）基因，并测序分析。SZ-21重链（V_H）基因全长为342 bp，
轻链（V_L）基因全长为306 bp，分别编码114和102个氨基酸。基因序
列登录号为VH：AF354053，VL：AF354054。
二 SZ-21单链抗体（SZ-21ScFv）基因的筛选和表达
为最大限度地保留单链抗体的血小板结合活性，利用噬菌体展示
技术进行了高亲和力筛选。将SZ-21 V_H和V_L基因分别与含有
（Gly_4Ser）_3多肽接头的载体pSW1-ScFv连接，构建成单链抗体基因
pSW1-SZ21ScFv，克隆到噬菌体分泌型表达载体pHEN1中。同时亲
和层析纯化血小板膜糖蛋白Ⅲa，用糖蛋白Ⅲa作为抗原进行高亲和力
筛选。经过四轮“吸附—洗脱—扩增”的富集过程，phage-ELISA
得到一个具有与血小板高亲和力的克隆，转化E.coli HB2151，表达
了可溶性ScFv融合蛋白。ELISA和Western blot证实该融合蛋白保
留了亲本抗体的血小板结合特性。
三 SZ-21单链抗体高效表达载体的构建及其在大肠杆菌中的融合表达
将上述筛选得到的单链抗体基因亚克隆到大肠杆菌融合型表达载
体pET20b中，得到pET20b-21ScFv。通过IPTG诱导在大肠杆菌E coli
抗血小板膜糖蛋白基因工程抗体的制备和性质研究 摘 要
BLZI（DE3）PlysS中表达了功能分子。SDS－PAGE和 Western blot显
示表达产物的分子量为27000。该融合蛋白除少量为分泌型表达外，
主要以包涵体形式存在，表达量占菌体总蛋白的 21％，约 20 m叭。同
时探索了该抗体片段的最佳复性条件。把包涵体形式表达的 SZ上 单
链抗体片段用尿素溶解，经过蛋白质的体外重折叠复性过程，成功地
得到具有活性的单链抗体片段。ELISA和Western印迹检测证实，该
鱼链抗体保留了与血小板特异结合的能力。
四SZ毛 的纯化和体外性质研究
通过NINTA树脂亲和吸附纯化，获得具有活性的高纯度单链抗
体片段，纯度达95％以上。流式细胞仪检测证实该单链抗体能与人脐
静脉内皮细胞和血小板反应。体外实验表明，SZ2 单链抗体能够抑
制 ADP诱导的血小板聚集，其抑制能力呈剂量依赖性，其 IC刃为 12
ug／mL，在浓度为20 ug／mL时达到最大抑制率。对花生四稀酸诱导的
血小板聚集亦有相似的抑制作用。此外，SZ上 单链抗体同样能够抑
制纤维蛋白原与血小板的结合，显示了其抗血栓作用。
以上结果表明，制备的基因工程抗体具有原鼠单克隆抗体的抗
血栓形成能力，减少了全抗体的人抗鼠抗体反应（HAMAh 同时该小
分子抗体片段去除了抗体的FC段，避免鼠源性单克隆抗体引起血小
板严重下降的副作用。该单链抗体有望用于抗血栓治疗。
五抗血小板膜糟蛋白单抗SZ毛 嵌合Fab片段基因构建和表达研究
利用基因克隆技术，从杂交瘤细胞系 SZ上 中克隆出轻、重链可
变区基因，然后亚克隆到含有人恒区基因C＊和C。的质粒pSWIFab
中，构建成人一鼠嵌合Fab抗体片段质粒pSZ2，在大肠杆菌中表
达了可溶性＊b抗体片段。SDS－PAGE显示在分子量（Mr）45 000处
有一诱导条带，与该嵌合抗体的理论计算值相符。表达量约为 800 fig／L，
ELISA和Western检测证实表达产物具有与血小板结合的活性。
Platelet thrombus formation is a major contributor to various
cardiovascular diseases caused by vascular occlusion. Glycoprotein
GPIIb/IIIa on the surface of platelets plays a key role in the final common
pathway of platelet aggregation. Blocking the binding of GPIIb/IIIa to
fibrinogen receptor could inhibit platelet aggregation potently and
effectively, hence inhibit the formation of thrombi.
SZ-2 1 is a murine monoclonal antibody reactive with the GPIIIa. Its
anti-thrombotic effects were confirmed in rabbit carotid-vein thrombus
formation models. It suggested that SZ-2 1 is a potential candidate for
treating cardiovascular diseases. To reduce the human anti-mouse
antibody response (HAMA) and the potential complement梞ediated lysis
of platelets caused by Fe region of the Ab, we produced and purified the
fragments of SZ-2 1 antibody. The anti-thrombotic effects of the fusion
protein was characterized in vitro.
1 Cloning and sequences of the variable regions genes of SZ-21
monoclonal antibody
The genes encoding the light- and heavy-chain variable regions(V~
and VL) have been cloned by RT-PCR from the SZ-2 1 hybridoma of the
anti-platelets GPIIIa murine monoclonal antibody and sequenced. VH
gene is 342 bp and VL gene is 306 bp, the genes consist of 114 and 102
amino acid residues respectively. Accession numbers of the VH and VL in
genebank are AF3 54053 and AF3 54054.
2.. Screening and expression of a single chain antibody fragment
(ScFv)
III Cf
The DNA encoding the variable regions were attached to the
oligonucleotide of the linker peptide by means of recombinant DNA
technique and single chain antibody fragment (ScFv) was obtained. Then
we ligated the ScFv into phage display vector pHENI and constructed the
phagemid pHJENI-2 1 ScFv. The high affinity phage display technology
was used to retain the SZ-2 1 ScFv binding activity to platelets in great
effort. Purified GPIIIa was coated on the tissue culture flask, affinity
panning was used to select antigen-positive recombinant phage antibody.
Clone of antibody fragment with good reactivity to platelets was isolated
after 4 round of enrichment and soluble ScFv was produced in the E. coil
HB2 151. The ScFv fragment with the similar binding activity to platelets
as the mAb SZ-2 1 was confirmed by ELISA and Western blot.
3. Construction and high expression of single chain antibody fragment
of mAb SZ-21
The screening ScFv gene was ligated into highly expressed vector
pET2Ob, and the fusion protein was expressed in Eschrichia coli
BL21(DE3)PlysS by IPTG induced. The induced band was in the MW
27000 distance. The recombinant ScFv fragment was mostly produced in
the form of inclusion bodies and its yield was up to 21 % of the total
amount of bacteria protein. A series steps, including cell breakage,
inclusion body dissolution, and protein refolding were used to obtain
fusion protein in an active form. The ScFv fragment remaining binding
activity to platelet was confirmed by ELISA and Western blot.
4.. Purification and characterization of the SZ-2lScFv fragment
The SZ-2 1 ScFv purity was over 95% through affinity chromatography.
It was shown that the ScFv fragment reacted with endothelial cells and
platelets by flow cytometry. It inhibited ADP-induced platelets
aggregation in a dose-dependent manner and t
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