Xu Bingfang, Copolla Michael, Herr John C, Timko Michael P
Departments of Biology, University of Virginia, Charlottesville, Virginia, 22904, USA.
Soc Reprod Fertil Suppl. 2007;63:465-77.
The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant-derived recombinant protein should provide large quantities of an inexpensive spermistatic plantibody.
鼠单克隆抗体（mAB）S19识别一种位于膜蛋白CD52上的N-连接碳水化合物抗原，称为精子凝集抗原-1（SAGA1）。这种抗原在附睾成熟过程中添加到精子表面。S19单克隆抗体与SAGA-1的结合会导致精子迅速凝集，并阻断受精前事件。先前的研究表明，S19单克隆抗体可能是一种潜在的特异性杀精剂（称为精子抑制剂），能够替代目前含有具有有害副作用的刺激性洗涤剂的杀精产品。编码S19抗体重链（H）和轻链（L）的核苷酸序列被克隆。使用编码H链和L链可变区的核苷酸序列构建了一个嵌合基因，该基因（scFv19）在转基因烟草（Nicotiana tabacum L.）中表达，以产生重组抗精子抗体（RASA）。在BY-2植物细胞悬浮培养物和再生的烟草品种Xanthi植物转化体中观察到RASA的最高表达水平，其中RASA编码序列在含有双增强子序列（2X CaMV 35S）的花椰菜花叶病毒35S启动子的控制下表达。对转基因的后续修饰，包括添加来自烟草蚀纹病毒的5'-非翻译序列（TEV前导序列）、编码区与马铃薯块茎蛋白的内质网靶向信号（pat）的N端融合以及与内质网保留信号肽KDEL的C端融合，显示RASA表达进一步增强。植物表达的RASA形成链内二硫键，主要可溶于细胞的细胞质部分。在重组RASA蛋白中引入多组氨酸（6xHIS）标签，可使用镍-氮三乙酸（Ni-NTA）色谱法快速纯化重组蛋白。扩大这种植物源重组蛋白的生产规模并优化其纯化过程，应能提供大量廉价的精子抑制植物抗体。
