1.Detection and analysis of protein gene product 9.5 positive nerve fibers in liver of patients with alveolar echinococcosis
ZHANG Rui ; LIU Hui ; DOU Jingrui ; ZHANG Kainan ; Yakufu Tuoheti4 ; LYU Guodong
China Tropical Medicine 2023;23(10):1017-
Abstract: Objective To explore the distribution characteristics of protein gene product 9.5 (PGP9.5) in the proximal and distal lesions of liver tissue in patients with alveolar echinococcosis (AE), and to clarify the relationship between the positive nerve fiber density of PGP9.5 in the proximal lesion of liver tissue of patients with AE and clinical pathological features and biochemical indexes. Methods From July 2019 to July 2022, 59 patients with AE who were hospitalized in the Department of Hepatobiliary Surgery of the First Affiliated Hospital of Xinjiang Medical University were selected, and their liver tissues at the proximal and distal ends of the lesion were collected, and their clinicopathological data and biochemical index information were collected at the same time. Immunohistochemical method was used to detect the density of PGP9.5 positive nerve fibers in the proximal and distal liver tissues of 59 patients with AE, and to analyze the difference between the density of PGP9.5 positive nerve fibers in the proximal and distal liver tissues of patients with AE, and to further analyze the relationship between the density of PGP9.5 positive nerve fibers in the proximal liver tissues of patients with AE and clinicopathological features and biochemical indexes. Results The nerves in the proximal lesion of the liver tissue in patients with AE increased, mainly distributed in the outer layer of the fibrous capsule enclosing the lesion, and no obvious abnormalities were observed in the distal nerves. The density of PGP9.5 positive nerve fibers in the liver tissue of patients with AE was significantly higher than that in the distal part of the lesion, with statistical significance (Z=-4.237, P<0.05). The density of PGP9.5 positive nerve fibers in the liver tissue of patients with AE was correlated with the increase of liver volume (Z=-2.632, P<0.05). Conclusions The area of PGP9.5 positive nerve fibers in the proximal liver tissue of patients with alveolar echinococcosis increases, suggesting that PGP9.5 positive nerve is involved in the pathogenesis of AE, and its specific role needs further study.
2.Genomic epitopes of Xinjiang hemorrhagic fever virus and the evaluation of its laboratory testing value
Huahua ZHANG ; Guoyu ZHAO ; Yang LI ; Shen SHI ; Ran AN ; Jiangling YUAN ; Dongliang LIU ; Jingrui DOU ; Tao LUO ; Surong SUN ; Yujiang ZHANG
Chinese Journal of Laboratory Medicine 2023;46(2):127-136
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.