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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 prokaryotic express mouse fertilized antigen-1 (FA-1) gene. Methods: Primers were designed and the coding sequence of FA-1 gene was amplified by RT-PCR from mouse testis total RNA. The amplified product was cloned into the GST fusion expression vector pGEX-2T. Induction of GST fusion FA-1 expression by IPTG. Results: Gel electrophoresis showed that the molecular weight of PCR amplification products was consistent with the expected 508bp. The recombinant plasmid was digested with BamH I / EcoR I to generate a fragment of about 500 bp. Sequence analysis of the recombinant plasmid showed that the sequence of the insert corresponded to the published FA-1 gene coding sequence. Under the induction of IPTG, recombinant BL21 efficiently expressed a product with the similar molecular mass as 43 kDa (expected to be 44.2 kDa). This product can be purified by the GST fusion protein purification kit. Conclusion: The mouse FA-1 coding sequence has been successfully cloned into the GST fusion expression vector pGEX-2T.