Comparative analysis of structural characteristics and epitopes in S proteins between SARS-CoV-2 and SARS-CoV / 浙江大学学报·医学版

OBJECTIVE@#To provide data support for the study of pathogenic mechanism of SARS-CoV-2 at the molecular level, and provide suitable candidate targets for vaccine, antibody and drug research and development through comparative analysis for structural characteristics and epitopes of S protein of SARS-CoV-2 and SARS-CoV.@*METHODS@#Based on the reference sequences of S protein, physical and chemical properties, hydrophobicity, signal peptide, transmembrane region, domain, secondary structure, tertiary structure analysis and antigenic epitopes prediction were carried out. Meanwhile, the tissue expression, related pathways and reactome pathways of angiotensis Ⅰ converting enzyme 2 (ACE2) and C-type lectin domain family 4 member M (CLEC4M) receptors were analyzed.@*RESULTS@#The amino acid sequence of S protein of SARS-CoV-2 and SARS-CoV has a 75.80% consistency. The structural characteristics of the two coronaviruses are highly consistent, but the secondary structure and tertiary structure of SARS-CoV-2 is not as obvious as SARS-CoV. ACE2 and CLEC4M are expressed in alimentary system, heart, kidney, lung and placenta. The main related the pathways of renin-angiotensin system, protein digestion and absorption pathway, and the reactome pathways of metabolism of angiotensinogen to angiotensins, GPCR ligand binding, are related to typical symptoms of coronavirus disease 2019 induced by SARS-CoV-2. Three pairs of highly or completely homologous epitopes of S protein were obtained. The 600-605, 695-703 and 888-896 amino acid residues in SARS-CoV-2 were highly homologous with 586-591, 677-685 and 870-878 amino acid residues in SARS-CoV, respectively.@*CONCLUSIONS@#The similarity of S protein of SARS-CoV-2 and SARS-CoV determines that they have similar infection patterns and clinical manifestations. The candidate epitopes with high reliability can provide reference for virus diagnosis and vaccine development.

Expression and significance of Sirt1 in colorectal cancer / 中国医师进修杂志

Objective:Sirtuins family is involved in the regulation of many biological events in the cells of the body. As one of the important members, SIRT1 may participate in the formation and development of colorectal cancer. We detected the expression of SIRT1 in colorectal cancer and adjacent normal mucosa to explore its role and significance in the pathogenesis of colorectal cancer.Methods:One hundred and twenty surgical specimens of patients from Xinhua Hospital Affiliated to Dalian University who were hospitalized for colorectal cancer from January 2018 to July 2018 were selected as experimental group. The normal mucosa tissues more than 10 cm away from the tumor focus were taken as the control group. The expression of SIRT1 in colorectal cancer and normal mucosa was detected by immunohistochemistry SP method and Western blot. The different expression of SIRT1 in different organs of digestive tract and parts of human system was compared with Expression Atlas Database. The relationship between SIRT1 expression and clinical pathological data was analyzed to explore the role and significance of SIRT1 in colorectal cancer.Results:SIRT1 protein was mainly expressed in the tumor cell nucleus. The positive staining was brownish yellow, and it was highly expressed in rectal cancer; Sirt1 expression was positively correlated with the depth of tumor invasion, differentiation and tumor size, and the difference was statistically significant ( P<0.05); SIRT1 was highly expressed in human digestive tract, but there was no significant difference in the expression of SIRT1 in various organs of digestive tract; Sirt1 may function through the PI3K-AKT signaling pathway in colorectal cancer. Conclusions:SIRT1 plays the role of oncogene in the development of colorectal cancer, and increasing expression of SIRT1 promotes the development of colorectal cancer. SIRT1 may be a marker of early diagnosis of colorectal cancer, which is of great significance.

Identification of differentially expressed genes in peripheral blood mononuclear cells of patients with hepatocellular carcinoma and its regulatory network analysis / 浙江大学学报·医学版

OBJECTIVE@#To identify the differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) and to analyze their regulatory network.@*METHODS@#The DEGs in PBMCs of HCC patients were screened based on GEO database. The functional enrichment analysis and interaction analysis were carried out for DEGs. MCODE algorithm was used to screen core genes of DEGs, and the mirDIP and starBase online tools were used to predict upstream miRNAs and lncRNAs of the core genes.@*RESULTS@#A total of 265 DEGs with a high credibility were identified, which were mainly enriched in the biological activity, such as regulation of cell proliferation, metabolic regulation, cell communication and signaling, and inflammatory diseases according to Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and the two analyses were correlated. Four diagnostic candidate genes were identified, including FUS RNA binding protein, C-X-C motif chemokine ligand 8, cullin 1 and RNA polymerase Ⅱ subunit H. Subsequently, 10 miRNAs, 1 lncRNAs and 38 circRNAs were predicted, and finally a lncRNA/circRNA-miRNA-mRNA-pathway regulatory networks was constructed.@*CONCLUSIONS@#The diagnostic candidate genes and its regulatory network in HCC PBMC have been identified based on data mining, which could provide potential tumor biomarkers for early diagnosis and treatment of HCC.

Inhibitory effects of recombinant human Hespintor on proliferation, migration and invasion of hepatocellular carcinoma cells / 重庆医学

Objective To perfect the purification method of recombinant fusion protein of Hespintor (rHespintor) for increasing the protein extraction efficiency,and to investigate its effects on the proliferation,migration and invasion of hepatoblastoma cell line HepG2.Methods In the recombinant protein extraction,the inclusion body washing process was added and the protein purification buffer system was changed.BAPNA was used as the substrate.The inhibitory effect tof purified rHespintor on trypsin hydrolysis was detected.The blank group served as the control group.The MTT test,cell scratch wound healing test and tumor cell invasion test were performed to detect the effect of rHespintor on growth of hepatoblastoma HepG2 cells and its effect.Results The urea gradient washing on the inclusion body protein could effectively remove the vast majority of impure proteins from the targeted protein.After one-step purification,the target protein rHespintor exhibited a high inhibition effect of trypsin hydrolysis,and the inhibitory effect was exhibited a dose-dependent manner.After acting on hepatoblastoma HepG2 cells with rHespintor,the cell proliferation ability was inhibited,the migration ability was reduced and the number of invaded cells were significantly decreased.Conclusion rHespintor can significantly inhibit the proliferation,migration and invasion of hepatoblastoma cell line HepG2 cells in vitro.

CDC42-interacting protein 4 gene is down trans-regulated by HBV DNA polymerase trans activated protein 1

Hepatitis B Virus [HBV] DNA polymerase transactivated protein 1 [HBVDNAPTP1] is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique [GenBank accession no: AY450389]. The biological function of HBVDNAPTP1 was investigated in this study. We constructed a vector pcDNA3.1 [-]/myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization [SSH]. The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels. HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1

Prokaryotic expression, purification and activity analysis of recombinant human serine protease inhibitor Hespintor Kazal Domain / 生物工程学报

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.

Cloning and Sequencing of the Gene Encoding Variant-Specific Surface Antigen from Giardia lamblia / 中国寄生虫学与寄生虫病杂志

Objective To clone and sequence variant-specific surface antigen gene from Giardia lamblia isolate SUCH/89/BTMRI/2(C2) derived from human in China. Methods Total genomic DNA of G.lamblia was extracted and a full-length variant-specific surface antigen gene fragment was amplified by pelymerase chain reaction (PCR). The PCR product was cloned into pMD19-T simple-vector, transformed into an Escherichia coli JM109 strain and then sequenced. The sequence analysis for cloned fragment was finished by Vector NTI 9.0 software for the homology of Giardia variantspecific surface antigen gene to that of sequences publishend in GenBank. Results The full-length variant-specific surface antigen gone fragment from G. lamblia was found to be 2 142 bp, encoding a 713 amino acid polypeptide and contained a single open reading frame (ORF). The deduced polypeptide sequence was rich in cysteine (11.8 mol%), most of which occurred with in 29 copies of the 4-amino acid CXXC motif, one GGCY-tetrapeptide motifs and three NXS consensus N-linked glycosylation sites. This polypeptide was also rich in threonine (10.2 mol%), glycine (12.1 mol%) and alanine (10.1 mol%). Like other previously identified VSPs, it contained a highly conserved hydropbebic Cterminal region. The homology of G. lamblia SUCH/89/BTMRI/2(C2) variant-specific surface antigen gene to that of sequence (TSA417) published in GenBank was 99% both at the nueleotide and the amino acid levels. Conclusion The full length variant-specific surface antigen gene from the isolate of G. lamblia has the common characteristics with other previously identified VSPs.

ADHESION OF HEAT-KILLED BIFIDOBACTERIUM ADOLESCENTIS TO HUMAN COLORECTAL CARCINOMA CELL / 微生物学通报

The assay investigated mainly the adhesion phenomenon of heat killed Bifidoba cterium adolescentis DM8504 to human colorectal carcinoma cell line CCL 22 9 Moreover, the assay discussed the adhesion mechanism of Bifidobacterium Resu lts found heat killed bifidobacterium would be the same ability to adhere and c o lonize to human intestinal epithelial cells in vitro as live bifidobacterium, an d their adhesion had to depend on its spent culture supernatant(SCS) The adh esin of Bifidobacterium adolescentis was possibly lipoteichoic acid(LTA)w h ich existed on the cell wall of bacteria and was secreted into the SCS LTA bou nd to the heat resistant proteins of cell surface of bacteria, and extended out from the cell surface Moreover, the bifidobacterial adhesin rece ptor of intestinal epithelial cells was possibly saccharides or glycopolymers

Cloning, prokaryotic expression and tissue expression profile of human novel gene HBVDNAPTP1 transactivated by hepatitis B virus DNA polymerase / 解放军医学杂志

Objective To clone the human target gene HBVDNAPTP1 transactivated by hepatitis B virus DNA polymerase obtained by screening with suppression subtractive hybridization (SSH) and bioinformatics techniques. To construct prokaryotic expressive vector of HBVDNAPTP1 gene, induce the expression of recombinant protein in E. coli, and analyze the expression level of HBVDNAPTP1 gene in human tissues. Methods The DNA fragment of HBVDNAPTP1 was amplified by reverse transcription polymerase chain reaction (RT-PCR) taking mRNA from HepG2 cells as the template, and the correct DNA fragment was then inserted into inducible prokaryotic expressive vector pET-32a (+). The competent BL21 (DE3) E. coli was transformed, and then cultured and induced with IPTG. The expressed HBVDNAPTP1 was confirmed with Western blot. UniGene database was used to analyze the chromosome mapping and tissue expression profile of HBVDNAPTP1 gene. Results The DNA fragment of HBVDNAPTP1 was amplified by RT-PCR. HBVDNAPTP1 expressive vector was constructed. After transformation with pET-32a(+)-HBVDNAPTP1 and induction with IPTG, recombinant HBVDNAPTP1 was expressed and confirmed by Western blot. The expression of genomic location of HBVDNAPTP1 gene was low in multiple-tissues with the exception of pituitary gland, tonsil, tongue, thymus, trachea and umbilical cord. Conclusion The recombinant HBVDNAPTP1 gene could be expressed in prokaryotic expression system of E. coli. The chromosome mapping and tissue expression level of HBVDNAPTP1 gene is tentatively conceived.