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目的:克隆人异质性胞核核糖核蛋白I(hnRNP I)基因并构建原核表达载体,用纯化的重组蛋白进行系统性硬化症(SSc)体外诊断应用研究。方法:从体外培养的HeLa细胞中提取总RNA,用逆转录-聚合酶链反应(RT-PCR)扩增hnRNP I基因,构建原核表达载体pET-30a-hnRNP I,在原核表达系统E.coliBL21(DE3)中表达重组蛋白。重组蛋白纯化后作为抗原对包括系统性硬化症(SSc)、系统性红斑狼疮(SLE)、干燥综合征(SS)、混合型结缔组织病(MCTD)、未分化型结缔组织病(UCTD)、类风湿性关节炎(RA)、正常对照(control)在内的血清相应抗体进行ELISA检测。结果:成功构建了原核表达载体pET-30a-hnRNPI,在IPTG诱导下在E.coli BL21(DE3)中高效表达相对分子质量(Mr)为59600的重组hnRNP I蛋白,重组蛋白可以可溶蛋白和包涵体蛋白两种形式存在。hnRNP I蛋白抗体在SSc中阳性率(48.72%)明显高于其他各组(P<0.05)。结论:利用构建的原核表达载体成功表达出hnRNP I蛋白,此重组蛋白可用于SSc的临床诊断检测。
OBJECTIVE: To clone the human heterogeneous nucleocapsid protein I (hnRNP I) gene and construct a prokaryotic expression vector. The purified recombinant protein was used to study the in vitro diagnosis of systemic sclerosis (SSc). Methods: Total RNA was extracted from HeLa cells cultured in vitro. The hnRNP I gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-30a-hnRNP I was constructed and expressed in prokaryotic expression system E.coli BL21 (DE3) to express the recombinant protein. Recombinant proteins are purified and used as antigens to treat systemic sclerosis (SSc), systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), mixed connective tissue disease (MCTD), undifferentiated connective tissue disease (UCTD) Rheumatoid arthritis (RA), normal control (control), including serum corresponding antibody ELISA test. Results: The prokaryotic expression vector pET-30a-hnRNPI was successfully constructed and the recombinant hnRNP I protein with high molecular weight (Mr) of 59600 was efficiently expressed in E. coli BL21 (DE3) induced by IPTG. The recombinant protein could express soluble protein and Inclusion body protein exists in two forms. The positive rate of hnRNP I protein in SSc (48.72%) was significantly higher than that in other groups (P <0.05). Conclusion: The hnRNP I protein was successfully expressed by using the constructed prokaryotic expression vector. The recombinant protein can be used in the clinical diagnosis of SSc.