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目的 探讨HBsAg与结核杆菌热休克蛋白70（MtHSP70）融合表达质粒在真核细胞中的表达。方法 用真核表达质粒pCI-neo作为载体构建HBsAg及其与MtHSP70的融合表达质粒（PCI-S,PCI-S-HSP70）;用脂质体介导的转染法将重组质粒转染 HepG2细胞,48h后,采用RT-PCR、免疫细胞化学和ELISA检测重组质粒在HepG2细胞中的表达。结果 质粒pCI-S和pCI-S-HSP70转染的HepG2细胞总RNA用RT-PCR可检测到目的基因mRNA的表达;pCI-S和pCI-S-HSP70转染的HepG2细胞用免疫细胞化学检测,结果显示在胞浆及核周有大量的阳性颗粒;ELISA检测pCI-S转染的细胞培养上清液HBSAg为阳性,而pCI-S-HSP70转染的细胞培养上清液HBSAg为阴性;在细胞裂解液中,二者转染的细胞均为阳性。结论 HBsAg与Mt.HSP70的融合表达质粒可在HepG2细胞中表达,但表达的融合蛋白不分泌出细胞外。
Objective To investigate the expression of fusion protein of HBsAg and Mycobacterium tuberculosis heat shock protein 70 (MtHSP70) in eukaryotic cells. Methods The recombinant plasmids were transfected into HepG2 cells by lipofectin-mediated transfection method using the eukaryotic expression plasmid pCI-neo as a vector to construct HBsAg and its fusion expression plasmid with MtHSP70 (PCI-S, After 48h, the expression of recombinant plasmid was detected by RT-PCR, immunocytochemistry and ELISA in HepG2 cells. Results The mRNA expression of target gene was detected by RT-PCR in total RNA of HepG2 cells transfected with plasmid pCI-S and pCI-S-HSP70. The expression of pCI-S and pCI-S-HSP70 transfected HepG2 cells was detected by immunocytochemistry , The results showed a large number of positive particles in the cytoplasm and perinuclear; HBSAg positive for pCI-S transfected cell culture supernatant was detected by ELISA, and HBSAg was negative for pCI-S-HSP70 transfected cell culture supernatant; In the cell lysate, both transfected cells were positive. Conclusion The fusion expression plasmid of HBsAg and Mt. HSP70 can be expressed in HepG2 cells, but the expressed fusion protein is not secreted extracellularly.