Construction Of Hantaan Virus Pseudovirion And Preliminary Study On Effect Of The Envelope Glycoprotein Glycosylation On Biological Characteristics

Due to there generally was existed glycosylation, the viral envelope proteins which all kinds of protein molecules embedded in the lipid bilayer, was customary called the viral envelope glycoprotein (GP). GP is one of the most important viral structural protein. So far, research has shown that Glycan structure, glycosylation manner and the degree of glycosylation of surface on the viral envelope glycoprotein played a crucial role in the virus pathogenicity, viral particle assembly, mature, release, viral immune evasion and antiviral immunity etc. In recent years, research on glycosylation of the viral envelope glycoprotein has increasingly become a focus of current molecular virology research. In this paper, from the following two aspects, preliminary study on N-glycosylation site mutation on GP of Hantaan virus (HTNV) affect humoral and cellular immune activity.1、Construction and Identification of Recombinant pseudovirus Containing multi-N Glycosyla tion sites mutant of Hantaan envelope glycoproteinBased on the previous research, five N-linked glycosylation site mutants of Hantaan virus envelope glycoprotein, which were designed as N134, N235, N347, N399 and N928, was firstly constructed using multisite-directed mutagenesis.Then, a recombinant pseudovirus particle was constructed by co-transfecting the Lentiviral expression vector carrying five N-linked glycosylation site mutants and packaging vectors into the HEK293 cells. The titers of pseudoviral particles were 2.0×107TU/ml (rLV-M carrying unmuted M gene),4.0×107TU/ml (rLV-M’ carrying muted M gene), and 1.0×108TU/ml (rLV carrying Empty vector), respectively.2、Research on multi N-glycosylation site mutation to affect the immunological charac- teristics of hantaan viral envelope glycoprotein using recombinant pseudoviral techniqueC57BL/6 mouse was respectively intramuscular immunized with three recombinant recombinant pseudovirus stains, rLV-M, rLV-M’ and rLV. Inactivated vaccine immunized mice were set as positive controls and rLV, rLV+ajuvant, ajuvant、NS were as negative controls.ELISA and Cell microculture neutralization test (CMNT) showed that the recombinant recombinant pseudovirus stains constructed by us can induce C57BL/6 mice to generate specific anti-HTNV humoral response. Geometric mean titers (GMT) of the specific anti-HTNV antibody of rLV-M and rLV-M’ immunized groups was 1:1386 and 1:587, respectively and GMT of neutralizing antibody of rLV-M immunized groups and rLV-M’ immunized groups was 1:160 and 1:113, respectively. It was significantly higher than those vaccinated group (1:269 and 1:28) (p<0.01)。 There also are significant differences between rLV-M immunized group and rLV-M’immunized group (p<0.05)Secretion of INF-y by spleen cells of immunized mice was measured using ELISPOT method and specific killing activity against target cells of spleen cells of immunized mice using CTL assay. The results indicated that secretion of INF-y of Inactivated vaccine immunized mice groups was significant higher comparing to rLV-M and rLV-M’ immunized mice groups (p<0.01), and secretion of INF-y of rLV-M immunized mice groups were higher those of rLV-M’ immunized mice groups (p<0.05).Animal protection test results showed that recombinat recombinant pseudovirus carrying M gene (whether or not the mutation) could provide specific immune protection for mice immunized following challenged with high lethal dose of Hantaanvirus. But the strength of this specific immune protective activity is much lower than the inactivated vaccine immunized mice groups (p<0.01) and those of rLV-M immunized mice groups were higher rLV-M’ immunized mice groups (p<0.05).Through this research experiments, the following conclusions can be drawn that there was composite and superimposed effect on anti-HTNV GP humoral and cellular immunity induced by HTNV Gn, Gc carrying five N-glycosylation sites mutations compared with the single N-glycosylation sites mutant of HTNV M gene.Compared with the single mutant gene HTNV M N-glycosylation sites, five N-glycosylation sites while mutations HTNV Gn, Gc induce anti-HTNV GP humoral and cellular immunity in mice immune responses of the composite and having a degree of stacking effect. Mainly for inducing immunity in mice against HTNV GP-specific antibodies and neutralizing antibody titers decreased, but far better than expected, obviously, but the level of cellular immune responses are induced to produce enhanced. The main trend is the titer of against HTNV GP specific antibody and neutralizing antibody partly decreased, but far from expected. However the cellular mediated immune response has for some degree enhanced.