罗非鱼HSP70(tHSP70)蛋白的生物信息学分析、真核表达及质谱鉴定

运用生物信息学技术分析罗非鱼热休克蛋白70(tilapia heat shock protein,tHSP70)的理化特性、糖基化位点、跨膜区域、蛋白的细胞定位及信号肽与虹鳟等其他动物HSP70的相似性,以人工合成cDNA为模板,通过聚合酶链式反应扩增其完整CDS,并将此DNA片段与真核表达载体pGAPZa-A连接,构建pGAPZa-HSP70表达质粒,在毕赤酵母GS115中表达tHSP70蛋白,对表达上清液进行SDS-PAGE及Western-blotting分析后,采用Ni²⁺IDA层析脱盐纯化目的蛋白,并对所表达的蛋白进行糖基化PAS染色鉴定。对表达的蛋白进行SDS-PAGE后,将所获得的非预定大小(约100 ku)条带进行电离飞行时间质谱鉴定,并确定其蛋白种类。结果表明:tHSP70所含氨基酸数量为640,分子量为70 274.5 u,等电点为5.49。在哺乳动物网织红细胞(体外)的半衰期为30 h,在酵母内半衰期大于20 h,而在大肠杆菌内的半衰期也大于10 h,不稳定指数为36.64,脂肪族指数为85.58。可能分别含有6个N-和O-糖基化位点,所克隆tHSP70基因与目的基因完整编码区序列完全一致,所构建的pGAPZa-HSP70质粒能在GS115中成功表达,诱导表达上清液经SDS-PAGE及Western-blotting分析后发现,除70 ku目的蛋白外,还出现一条100 ku条带。经糖基化PAS染色证明,此100 ku蛋白可能是HSP70糖基化的结果,且能被人的兔抗HSP70抗体识别,质谱鉴定证明该条带即是tHSP70蛋白。本研究所表达tHSP70蛋白为后续罗非鱼细胞学及抗原提呈等免疫学研究奠定了基础。

Bioinformatics analysis,eukaryotic expression and mass spectrum identification of the tilapia HSP70 protein

The physical and chemical property, glycosylation site, transmembrance domain, cellular localization, signal peptide of tilapia HSP70(tHSP70)and percent identity to the HSP70 derived from rainbow trout and other species(tHSP70)were analyzed by bioinformatics methods, and the full CDS of tHSP70 was amplified and cloned by using and the artificially synthesized cDNA. The expression plasmid of pGAPZa-HSP70 was constructed by the insertion of tHSP70 CDS into the pGAPZa-A vector, and the tHSP70 protein was then expressed in host strain of GS115. The supernatant was used to conduct the SDS-PAGE and Western-blotting, followed by the purification of tHSP70 by Ni₂⁺IDA affinity chromatography. Then the PAS staining was used to identify if the glycosylations occurred in tHSP70 protein after the expression from GS115. Then the non-expected band(about 100 bp)in the SDS-PAGE was identified by standard MALDI-TOF-MS procedure. Bioinformatic analysis showed tHSP70 contains 640 amino acids, the molecular weight is 70 274.5 mol/L and the isoelectric point is 5. 49. The half-life of tHSP70 is 30 h in mammalian reticulocyte(in vitro)and greater than 20 h in yeast, while it is more than 10 h in Escherichia coli. Additionally, the instability index and aliphatic index of tHSP70 is 36. 64 and 85. 58, respectively. Moreover, tHSP70 might totally contain 6 O-linked and 6 N-linked glycosylation sites. The cloned tHSP70 sequence was 100% identity to the sequence in NCBI database. The HSP70 protein could be efficiently expressed by methane induction of pGAPZa-HSP70 plasmid in GS115 strain. Except the 70 ku band, one 100 ku was observed on the SDS-PAGE and Western blotting gels. The PAS staining specific to the glycosyl radicals was performed and the positive signals indicated that this 100 ku band might be the consequence of glycosylations after expression in GS115. Western blotting showed the protein currently expressed was recognized by rabbit anti-human HSP70 antibody, and the MALDI-TOF-MS result demonstrated that the 100 ku protein was exactly the tilapia HSP70, 100% identity to the tilapia HSP70 in NCBI database. The present study provided experimental materials for the future cellular and immunological researches in tilapia.