摘要
目的:克隆和原核表达小鼠受精抗原-1(FA-1)基因。方法:自行设计引物,用RT-PCR从小鼠睾丸组织总RNA扩增FA-1基因的编码序列。将扩增产物克隆至 GST 融合表达载体pGEX-2T。以IPTG诱导GST融合FA-1的表达。结果:凝胶电泳显示PCR扩增产物的分子量与预期的508bp一致。重组质粒经BamH I/EcoR I双酶切后产生一个约500bp的片段。对重组质粒的序列测定表明,插入片段的序列与发表的FA-1基因编码序列相符。在IPTG的诱导下,BL21重组菌高效表达出一个分子量与43kDa相近的产物(预期44.2kDa)。该产物可通过GST融合蛋白纯化试剂盒得到纯化。结论:小鼠FA-1编码序列已被成功地克隆至GST融合表达载体pGEX-2T。
Objective:To clone and prokaryotically express mouse FA-1 antigen.Methods:The encoding sequence of FA-1 genewas amplified from mouse testis total RNA by RT-PCR.The PCR product was cloned into the GST fusion expression vector pGEX-2T.and GST fusion FA-1 was induced by IPTG.Results:ln gel electrophoresis the molecular weight of the PCR product agreed with the ex-pected 508bp.A fragment of some 500bp was produced from the BamH I/EcoR Ⅰ digestion of the recombinant plasmid.The sequence ofthe insert corresponded with published encoding-region sequence of FA-1 gene.A product with a molecular weight close to 43kDa wasinduced by IPTG,which could be purified by a commercial GST fusion protein purification kit.Conclusion:The encoding sequence ofmouse FA-1 gene was successfully cloned into the GST fusion expression vector pGEX-2T.
出处
《福州总医院学报》
2000年第3期20-22,共3页
Journal of Fuzhou General Hospital
