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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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Aim: The aim of this study was to investigate the effect of microRNA-1224-5p (miR-1224-5p) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). Subjects and Methods: Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000. TECs were divided into three groups, namely a control (CON) group without transfection, a negative control (NC) group transfected with negative control oligonucleotides and green fluorescent protein (GFP), and a micro-up (MU) group transfected with miR-1224-5p mimic and GFP. The expression of miR-1224-5p was quantified via quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The proliferation of TECs was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the optical density value at 490 nm was measured after every 24 h. Apoptosis was detected via flow cytometry using a 7-aminoactinomycin/APC Annexin V kit. The migration and invasion of TECs were detected using transwell assay. The tube formation ability was evaluated using the tube formation assay. Results: Oligonucleotides were successfully transduced into TECs, and the expression of miR-1224-5p was specifically upregulated. The results of qRT-PCR analysis showed that the expression of miR-1224-5p was significantly upregulated in the MU group (2–ΔΔCt = 3.27 ± 0.15) than in the CON group (2–ΔΔCt = 1) and NC group (2–ΔΔCt = 1.08 ± 0.11) (P < 0.01). The results of MTT assay showed that the cell proliferation was significantly inhibited in the MU group at four time points than in the CON and NC groups (P < 0.01). Flow cytometry analysis revealed the significant increase in apoptosis of cells from the MU group (19.29% ± 0.95%) than those from the CON (8.73% ± 0.64%) and NC (9.51% ± 0.56%) (P < 0.01) groups. The migration ability was significantly inhibited in MU group (51.0 ± 3.6) as compared with CON (77.7 ± 2.5) and NC (79.2 ± 3.5) groups (P < 0.01). The invasion ability of TECs was significantly inhibited in MU group (9.8 ± 1.3) than in CON (15.8 ± 0.8) and NC (15.4 ± 0.9) groups (P < 0.01). The ability of tube formation of TECs was completely inhibited in MU group but remained unaffected in CON and NC groups. Conclusions: miR-1224-5p may serve as a potential tumor suppressor in HCC. Upregulation in miR-1224-5p expression may decrease proliferation, induce apoptosis, inhibit migration and invasion, and suppress tube formation in TECs of human HCC
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Objective In order to lay the foundation for establishment of a clean grade Meriones meridianus popu-lation, and provide reference for the formulation of Meriones meridianus parasite detection standard.Methods A survey of parasite infection in Meriones meridianus was carried out, and three kinds of antiparasitic drugs, tetramethrin/per-methrin, praziquantel, and tinidazole were used in high-, medium-and low-doses for eradication of the target parasites. Results Three types of parasites were detected among the animals.The infection rate of surface parasite Myobia musculi was 82.1%(23/28), that of intestinal parasite Hymenolepis nana was 78.1% (25/32) and Tritrichomonas muris was 90.6%(29/32).Significant results of parasite eradication were achieved with the antiparasitic drugs.Medicated bath u-sing 1%tetramethrin/permethrin for 30 s every 4 days for 5 times and 1.5%tetramethrin/permethrin for 30 s every 4 days for 3 times completely eradicated the surface Myobia musculi infection.Praziquantel in a dose of 31 mg/kg by daily gastric gavage for consecutive 7 days was the optimal dosage to completely eradicate the intestinal Hymenolepis nana.Tinidazole in a dose of 42 mg/kg administered by gastric gavage daily for consecutive 7 days was the optimal dosage for eradication of in-testinal Tritrichomonas muris infection.Conclusions Three types of parasites are detected in Meriones meridianus in our laboratory, i.e.surface infection with Myobia musculi, and intestinal infection with Tritrichomonas muris and Hymenolepis nana.Tetramethrin/permethrin, praziquantel and tinidazole can be used to effectively eradicate these three types of para-sites, respectively.
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Objective The purpose of this study was to investigate the reproductive characteristics of indoor-housed Meriones meridianus.Methods According to the data of Meriones meridianus from 1996 to 2002 in the Center for Laboratory Animal research of Xinjiang, the sexual maturity period of male and female animals, the gestation period of female animals, the litter size, weaning rate, survival rate, sex ratio per month and different fetal times were statistically analyzed.Results Sexual maturity period of male and female animals was 109.3 ±21.0 days and 106.3 ±21.7 days, and gestation period of female animals was 21.3 ±1.4 days.The litter size, weaning rate of different fetal times showed no significant difference compared between those of the first to seventh litter ( P>0.05) , and the survival rate of fourth and sixth litter were lower than that of the average offspring per litter, but the difference was no significant (P>0.05).The sex ratio of from first to sixth litter indicated no significant difference ( P>0.05 ) , and the average proportion of male and female was 1.4:1.0.During a year, the animals almost stopped to reproduce from September to November, however, the differences of litter size between the other months were statistically not significant ( P >0.05 ) , the weaning rate and survival rate per month suggested a significant difference ( P <0.05 ) between some of the months.Conclusions Compared with the background data of wild Meriones meridianus, the laboratory reproduction of Meriones meridianus show some differences, mainly, the season of breeding is shifted to December, and the number of reproduction increased by one or two litters.Our results provide useful reference for laboratory animalization of Meriones meridianus.
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Objective To conduct morphological observation and gene identification of the strain of flagellate iso -lated from Cricetulus migratorius in the Xinjiang Research Center for Experimental Animals .Methods The ileocecal con-tents of C.migratorius were microscopically examined on direct smear with Wright-Giemsa staining , and the total RNA iso-lated from Xinjiang C.migratorius was extracted and 16S rRNA was amplified by PCR , and then sequenced .Furthermore the homology was compared and the phylogenetic tree was developed using MEGA 5.22 software.Results Morphological observation indicated that the isolated flagellate was Tritrichomonas muris.The 16S rRNA gene sequence of the Xinjiang C. migratorius isolate shared highly homology with that of other Tritrichomonas.Phylogenetic tree analysis indicated that the 16S rRNA gene of Xinjiang C.migratorius isolate was classified into a subgroup with T.muris 16S rRNA (U85966.1), but was relatively distant relative from other related tritrichomonas.Conclusions The flagellate isolated from Xinjiang C. migratorius is identified to be T.muris by both morphological observation and 16S rRNA gene analysis.