大菱鲆(Scophthalmus maximus)蛋白质二硫键异构酶SmPDIA3的表达分析和功能验证

EXPRESSION ANALYSIS AND FUNCTIONAL VERIFICATION OF PROTEIN DISULFIDE ISOMERASE SMPDIA3 IN TURBOT SCOPHTHALMUS MAXIMUS

本研究利用SMART-RACE技术克隆了大菱鲆SmPDIA3基因。SmPDIA3基因cDNA序列全长2083bp,包括1479bp的开放阅读框,编码492个氨基酸, GenBank登录号:MG765516。组织表达分析发现:SmPDIA3基因在肠、鳃、肝脏等组织中均有表达,其中在肝脏中表达量最高,脑中最低。进一步研究了温度胁迫后SmPDIA3基因在肠和肝脏组织中的表达变化规律,发现随着胁迫温度的升高,SmPDIA3基因的相对表达量总体上升,并在28°C时达到最大。利用原核表达技术,构建了pET-28a-PDIA3原核表达载体,IPTG诱导后融合蛋白主要在上清中表达。将上清中的蛋白分离纯化后,利用Westernblot技术验证了目的蛋白的准确性。将纯化后的目的蛋白浓缩后,利用BCA蛋白浓度测定试剂盒测得蛋白浓度为243.18μg/mL。最后设计变性溶菌酶的复性实验,验证了SmPDIA3蛋白具有分子伴侣功能,能够辅助变性蛋白的正确折叠。

We cloned the SmPDIA3 gene of turbot Scophthalmus maximus using the SMART-RACE technique. The cDNA sequence of SmPDIA3 is 2083bp in length and includes a 1479bp open reading frame encoding a 492 amino acid signal peptide containing 17 amino acids. The predicted molecular weight is 54.92kDa, the theoretical isoelectric point is 5.40, and GenBank accession number is MG765516. Tissue expression analysis showed that SmPDIA3 gene was expressed in intestinal, iliac, liver, and other tissues, with the highest expression in the liver and the lowest in the brain. In addition, we studied the expression of SmPDIA3 gene in the intestine and liver tissues after a thermal stress. We found that the relative expression of SmPDIA3 gene increased with the increase of temperature, and reached the maximum at 28°C. Using prokaryotic expression technology, the pET-28a-PDIA3 prokaryotic expression vector was constructed and the target protein was successfully expressed in the supernatant. Finally, the refolding experiment of denatured lysozyme was designed to verify the function of the SmPDIA3 protein. We found that SmPDIA3 protein can assist the correct folding of denatured proteins, and has obvious molecular chaperone function.