| Hsps take important physiological parts in protein folding, assembling and transportation, and protecting cells from the deleterious effects of stress. They are indispensable for the survival of cells. Hsp70 is a highly conserved protein which is involved in protein transportation, folding and antigen processing and presenting. Many researches show that Hsp70 can play as a molecule carrier and enhance the antigenicity of proteins associating with it. HTNV can lead to HFRS. HTNV nucleocapsid protein (NP), a protein expressed by HTNV, has strong immunogenicity and antigenicity and can induce CTL effects. On the basis of our previous work on the colocalization and interaction of NP and Hsp70 in animals experimentally infected by HTNV, this experiment is to research the relationship between viral proteins including NP, glycoprotein G2 and several Hsps including Hsp70, Hsp27, Grp94 in infected animals. Based on the relationship, a DNA vaccine aiming at HFRS is constructed which includes both hsp70 gene and S gene. Besides, we also express Hsp70 in prokaryotic system in high level.l.The relationship between viral proteins and several Hsps in animals experimentally infected with HTNV. Newborn mice less than 72 h of age were experimentally infected with HTNV by intracerebral inoculation. The brains of mice at the 8th day post-infection were removed and prepared for tissue sections and tissue extracts. The expression of Hsp70, Hsp27, Grp94and NP, G2 were detected by immunohistochemimal staining. The possible interaction or association of viral proteins and those Hsps were analyzed by double specific antibodies-sandwich ELISA and immunoprecipitation methods. The result showed that HTNV infection led to increased expression of Hsp70, Grp94, NP and G2 but not Hsp27 at cytoplasma of neurons in brains of infected suckling mice. NP associated with Hsp70, Hsp27 and Grp94, which led to formation of NP-Hsp70-Grp94-Hsp27 complex. G2 associated with NP and Hsp27, which led to formation of G2-NP-Hsp27 complex.2.The construction and eukaryotic expression of a DNA vaccine aiming at HFRS. We amplified S gene of HTNV by PCR and mutated its termination code. Then we cloned it into eukaryotic expression vector pcDNA3.1(+), which had already been inserted with hsp70 gene, to form the S-hsp70 fusion expression vector. This recombinant plasmid was transfected into COS-7 cell by liposome and immunocytochemistry assay was used to detect the NP-Hsp70 fusion protein expression. The result showed that pcDNA3.1(+)/S2-hsp70 was successfully constructed and NP-Hsp70 fusion protein was transiently expressed in COS-7 cell.3.The expression of Hsp70 in prokaryotic system in high level. The human hsp70 gene was cloned into prokaryotic expression vector pMAL-c2X. The recombinant vector pMAL-c2X/hsp70 was transformed into E.coli DH5a. Then Hsp70 was expressed via the induction of IPTG in different temperatures and times. The expressed products were identified by SDS-PAGE and Western Blot. The levels of expression were analyzed by gel thin-layer scanning. The result showed that pMAL-c2X/hsp70 was successfully constructed and Hsp70 was successfully expressed. Under different inducing conditions, the expression levels and the proportions of soluble products were distinct. The expression level of Hsp70 was highest when induced by 0.3 mmol/L IPTG under 37 C for 5 h and the favorable condition for soluble expression was 0.3 mmol/L IPTG under 20 C for 20 h.In conclusion, our experiment proved that HTNV infection led to increased expression of a set of Hsps and the formation of Hsps-viral proteins complex, which may demonstrate the chaperone roles of Hsps in the synthesis, transportation and maturity of viral proteins during viral replication in the host cells. We also constructed a plasmid expressing Hsp70-NP complex in eukaryotic system and efficiently expressed Hsp70 in prokaryotic system, which provided a basis for vaccines to prevent and treat HFRS.
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