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Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.
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Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.
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Objective:To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli ( E. coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods:Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results:Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass ( Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×10 3, 37.1×10 3, 31.9×10 3, 30.8×10 3, 40×10 3 and 54.4×10 3, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions:The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines.
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Objective To express and purify two domains GcⅠand GcⅡof Xinjiang hemorrhagic fever virus (XHFV) glycoprotein, and to study its immunogenicity and the effects on immune response in mice. Methods The prokaryotic expression plasmids of pET28a-GcⅠand pET32a-GcⅡwere constructed and transformed into E. coli BL21, respectively. The expression and purification conditions of rGcⅠand rGcⅡproteins were optimized. The antigenicity of the fusion protein was detected by Western Blot and enzyme-linked immunosorbent assay (ELISA). BALB/c mice were immunized by protein immunization and DNA priming-protein boosting. The mice were randomly divided into 5 groups, including pVAX1-GcⅠ+rGcⅠgroup, pVAX1-GcⅡ+rGcⅡgroup, rGcⅠgroup, rGcⅡgroup and saline group (control group) with 7 mice in each group. The serum antibody titer of mice was detected by indirect ELISA, and the immune effect was evaluated by spleen T lymphocyte proliferation assay and cytokine content determination. Results The fusion proteins rGcⅠand rGcⅡwere purified and obtained, which could react with positive serum of sheep and had good antigenicity. After three immunizations, the IgG levels in the serum of each experimental group were significantly higher than those in the control group (all P<0.001). The serum antibody titers of the experimental groups were reached above 1:12800. Among them, the concentration of Th2 type cytokine interleukin-4 (IL-4) in the spleen cell culture supernatant of rGcⅡ[(79.97±7.47) ng/L] and pVAX1-GcⅡ+rGcⅡgroup [(61.43±9.27) ng/L] was significantly higher than (24.29±3.81) ng/L of the control group, respectively (all P<0.01). The highest mass concentration [(42.46 ±2.60) ng/L] of Th1 type cytokine interferon-γ(IFN-γ) was observed in the pVAX1-GcⅡ+rGcⅡ group, which was significantly higher than (20.33±1.67) ng/L of the control group, and the difference was statistically significant (P<0.001). That showed a significant antigen-specific splenic T lymphocyte proliferation (P<0.001). Conclusions The purified recombinant proteins rGcⅠand rGcⅡhave good immunogenicity, which can make the immune system T lymphocytes tend to Th2 response, and pVAX1-GcⅡ combined with recombinant protein GcⅡ can induce better antigen-specific immune effect. And pVAX1-GcⅡ combined with recombinant protein GcⅡis expected to be used as vaccine candidates for the prevention and control of XHFV.
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The disease caused by Rickettsia is an important zoonotic disease. Due to the complexity of the ecological environment of Xinjiang, the vectors, host and species of Rickettsia are complex and diverse; its natural foci are widely distributed, so they have brought great harm to local human health and livestock production. With global warming, rapid development of social economy and promotion of foreign economic and trade exchanges in Xinjiang, these may become potential factors for the diversity of Rickettsia vectors and hosts. In recent years, some rickettsial disease such as the onset of Q fever, ehrlichiosis and anaplasmosis, are increased at some areas of Xinjiang, thus, the Rickettsia and rickettsial diseases have been widely concerned. At present, a variety of new Rickettsia species have been detected and isolated from several areas of Xinjiang, and their public health significance have also been paid more and more attention. In this paper, we briefly summarized the research status about reported Rickettsia species identification, media host and popular distribution in the Xinjiang region over the past 20 years, to providing scientific basis for a systematic study and diagnosis, prevention and treatment of Rickettsia and rickettsial disease.
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OBJECTIVE To investigate the antitumor effect of cadmium (Ⅱ) complex of pyrazolone derivatives 1-phenyl-3-methyl-4-propionyl-5-pyrazolone salicyloyl hydrazide-cadmium (Ⅱ) (Cd-PMPP-SAL) on the murine melanoma B16 cells in vitro and in vivo and its mechanisms. METHODS B16 cells were incubated with Cd-PMPP-SAL at 1.0, 1.5, 3.0, 5.0 and 10.0 mg·L-1 for 24, 48 or 72 h. The prolifera? tion rate of B16 cel s was evaluated by MTT assay. B16 cel s were incubated with Cd-PMPP-SAL at 6.25, 12.50 and 25.00 mg·L-1 for 24 h, while cell morphology was observed by Hoechst33258 staining. Apop?tosis of B16 cells was detected by Annexin Ⅴ-FITC/PI staining. The activity of caspases in B16 cells was detected by caspase activity assay. C57BL/6J mice were inoculated subcutaneously with B16 cells to establish a tumor-bearing model. Five days later, Cd-PMPP-SAL at 6.25, 12.50 and 25.00 mg·kg-1 was injected into tumors of C57BL/6J mice once a day for 12 d. The body mass was recorded daily. One day after the last administration, all the mice were killed and the tumor was harvested. Tumor volume and mass were measured, and the tumor inhibitory rates were calculated. Pathological changes of the tumor, liver and lung were observed under a microscope. The expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) in tumor tissues were detected by immuno?histochemistry. The apoptotic cells in transplanted tumor tissues were detected by TUNEL. RESULTS Cd-PMPP-SAL inhibited the proliferation of B16 cells. The IC50 was 4.946 mg · L-1, and 95% confidence interval was 4.24-5.65 mg · L-1. The apoptosis rates(12.8 ± 1.4)% and (18.4 ± 0.4)% of Cd-PMPP-SAL 12.50 and 25.00 mg · L-1 groups were significantly higher than those of control group (1.7 ± 0.1)% (P<0.01). The activity of caspase 3 and 9 of Cd-PMPP-SAL 25.00 mg · L-1 group was significantly higher than that of control group (P<0.01), but there was no significant difference in caspases 3/7. The relative tumor volumes of Cd-PMPP-SAL 6.25, 12.50 and 25.00 mg · kg-1 treated groups from the 8th day of treatment were significantly decreased compared with the model group (P<0.01). The result of paraffin sections showed that the transplanted tumor tissues in Cd-PMPP-SAL 12.50 and 25.00 mg · kg- 1 groups exhibited different degrees of necrosis, but there was no significant pathological damage to the liver or lung tissues of mice. Compared with model group, expressions of VEGF and FGF2 in Cd-PMPP-SAL 12.50 and 25.00 mg · kg-1 treated groups were significantly inhibited (P<0.05), and apoptotic cell rates were significantly higher (P<0.05). CONCLUSION Cd-PMPP-SAL can inhibit growth of B16 cells in vivo and in vitro, which may be associated with induction of tumor cell apoptosis and inhibition of tumor angiogenesis.
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Objective To construct two DNA vaccines based on two glycoprotein antigen segments of Xinjiang hemorrhagic fever virus (XHFV) and to evaluate the immune responses in BALB/c mice following vaccination.Methods Two recombinant expression plasmids pVAX1-GcⅠand pVAX1-Gc Ⅱ were constructed by inserting XHFV YL04057 strain Gc Ⅰ (1 229-1 349 aa) and Gc Ⅱ (1 443-1 566 aa) fragments into the eukaryotic expression vector pVAX1 and then were identify by restriction enzyme digestion and sequencing analysis.The recombinant expression plasmids were transfected into mice by hydrodynamics-based transfection.Immune responses induced in mice were evaluated by testing the proliferation of T cells with MTT,measuring serum antibody level with ELISA and detecting cytokines in the supernatant of spleen cell culture with ELISA kit.Results The recombinant expression plasmids were successfully constructed as indicated by the results of restriction enzyme digestion and sequencing analysis.Expression of Gc Ⅰ and Gc Ⅱ genes in mice liver tissues was detected.Antibody titers in mice immunized with pVAX1-GcⅠor pVAX1-Gc Ⅱ were higher than those in mice immunized with pVAX1.Compared with pVAX1,pVAX1-Gc Ⅱ significantly enhanced the proliferation of splenic T lymphocytes and the expression of IFN-γ (P<0.01).Conclusion The constructed two DNA vaccines for XHFV can induce specific humoral and cellular immune responses in mice.pVAX1-Gc Ⅱ is better than pVAX1-GcⅠin immunogenicity and protective efficacy,suggesting that it can be used as a promising candidate for the development of DNA vaccine for XHFV.
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The Crimean-congo hemorrhagic fever virus(CCHFV)is a geographically widespread fatal pathogen.Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections.In this study,expression vectors carrying series truncated fragments of the NP(nueleoeapsid protein)gene from the S fragment of CCHFV strain YL04057 were constructed.The recombinant proteins were expressed in E.coli and purified for detection.The antigenic of the truncated fragments of NP was detected with a polyclonai serum(rabbit)and 2 monoclonal(mAbs)(14B7 and 43E5)against CCHFV by Western-blot analyses.The results showed that the three expressed constructs,which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum.The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.
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The genomic DNA were extracted from the leaves of Polygonatum roseum (Liliaceae) in Xinjiang and the primers were designed according to conservative sequences of Polygonatum lectins gene. The complete ORF of Polygonatum roseum agglutinin (PRA) gene was amplified as a fragment of 550 bp, which was identical with predicted size. Like most of the plant lectin genes, there was no intron in the PRA gene. The ORF of the gene encoded 159 amino acid residues, in which included a signal sequence of 28 amino acid residues at its N-terminus. The cDNA sequence had 92% identities compared with the published sequence. The amino acid sequence and SWISS-MODEL analysis indicated that the three-dimensional structure of PRA strongly resembled with that of monocot mannose-binding lectins, which comprised with three antiparallel four-stranded beta-sheets arranged as a 12-stranded beta-barrel. The recombinant pGEX4T-1-PRA and pMAL-p2x-PRA prokaryotic expression vectors were constructed to produce GST-PRA and MBP-PRA fusion proteins in E. coli, respectively. SDS-PAGE of the fusion protein demonstrated that the PRA lectin protein migrated at a size of 14 kD. The immunization was performed by intra-muscular injection of pcDNA3-PRA, and the antiserum was detected by ELISA. Western blotting analysis showed the antiserum specifically bound the lectin protein. The establishment of such an expression system might provide materials for further investigation of the properties and functions of PRA proteins. It also laid the basis for plant genetic engineering on its defensive functions to pests and diseases.