小鼠富含半胱氨酸分泌蛋白-1原核表达载体的构建与表达

Construction, expression and characterization of the procaryotic expression plsmid containing mouse cysteine-rich secretory protein-1 gene

目的构建小鼠附睾富含半胱氨酸分泌蛋白-1（Crisp—1）基因的原核表达载体并在大肠杆菌中的表达。方法从小鼠附睾组织中提取总RNA，逆转录一聚合酶链反应（RT—PCR）扩增实验所需的Crisp-1基因片段，将目的基因克隆至原核表达载体pET28a（＋）上，酶切和测序鉴定后，转化大肠杆菌ER2566，经IIYFG诱导表达，表达产物经十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）和免疫印迹法鉴定。结果重组表达质粒pET28a（＋）-Crispl经双酶切后，得到2条大小分别为5369bp和660bp的片段，测序结果证实克隆的基因序列与GeneBank中的Crisp-1序列相符。重组体转化大肠杆菌ER2566后，经IPTG诱导，可表达分子质量（Mr）为30×10^3的重组蛋门，与预期值相一致。IPTG诱导4h重组蛋白的表达量最高。SDS—PAGE电泳显示表达蛋白主要以包涵体形式存在于胞质中。包涵体经纯化和复性后，Western blot实验表明所获得的重组蛋白具有较好的抗原活性。结论成功构建重组蛋白Crisp-1，为进一步研究Crisp—1的功能奠定基础。

Objective To construct a procaryotic expression plsmid of mouse cysteine-rich secretory protein-1 （Crisp-1） and express it in E. coli ER2566. Methods Total RNA was extracted from mouse epididymal tissue, and Crisp-1 gene was amplified by using reverse transcription-polymerase chain reaction （RT-PCR）. The amplified gene was inserted into procaryotic expression vector pET28a（ ＋ ） to construct a recombinant plasmid pET28a（ ＋ ）-Crisp-1. The recombinant plasmid confirmed by PCR, restriction endonuclease digestion and DNA sequence analysis was transformed into E. coli ER2566. The expression product was induced by IPTG, and its specificity and relative molecular weight weve detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （ SDS-PAGE ） and Western blotting respectively. Results The recombinant plasmid pET28a（ ＋ ）-Crispl was digested with BamH Ⅰ and HindⅢ, obtaining a band of 5369 bp and another one of about 660 bp. DNA sequence analysis also showed the cloned Crisp-1 gene sequence was completely correspondent to GeneBank data. After the recombined plasmid was transformed into E. coli ER2566 and induced by IPTC for 4 h, the SDS-PAGE analysis showed that the expressed fusion protein with a molecular weight of 30 × 10^3 existed in the inclusion body. Western blotting revealed that after purification and refolding, the protein had good antigenicity. Conclusion The eukaryotic expression vector of Crisp-1 was expressed successfully in E. coli ER2566, which provides a basis for further study on the function of this protein.