Objective ToexpresschimericantigenofHCVwithmultipleimmunodominantepitopes inE .coliforimprovingthequalityofreagentsforHCVscreening .Methods Thegenefragmentencoding HCVchimericantigenwasobtainedbymolecularcloningmethodandclonedintopQE 30 plasmidforex pressioninE .coliM 15 .TheexpressedchimericantigenwaspurifiedbyNi NTAandcoatedonELISA platestoanalyzeitssensitivityandspecificity .Results Thechimericantigenof 5 30 0 0washighlyex pressed .ELISAassayofserumsamples (including 18HCVpositiveand 17negativesera)indicatedthatthe HCVchimericantigenhadhighsensitivityandspecificity .Conclusions ChimericantigenofHCVwith typicalimmunodominantepitopescanbeusedtodevelopgoodreagentsforHCVimmunoassay .

Objective:To investigate the presence of SENV infection among patients in China,and analyze partial nucleotide sequence of SENV isolated from a patient with non A-G hepatitis.Methods:A nested polymerase chain reaction (PCR) assay with primers from ORF1 of SENV genome was established to detect SENV DNA.The PCR product was cloned and sequenced.Results:SENV DNA was positive in 2 of 7 patients with non A-G hepatitis and TTV negative.Partial gene of a SENV isolate was compared with the corresponding region of SENV isolate(AX025730)from Italy and was found that the nucleotide homology was 90%.Conclusions:The results of this study confirmed the presence of SENV infection in China.The development of a PCR assay for SENV DNA detection and the cloning,sequencing of the SENV isolate have important implication for the diagnosis and epidemiological investigation on SENV infection.

Objective:To analyze the etiological and epidemiological characteristics of hand, foot and mouth disease (HFMD) in Xi′an from 2019 to 2021, so as to provide evidence for the prevention and control of HFMD.Methods:Stool specimens and anal swabs were collected from patients with HFMD. Enteroviruses (EVs) including enterovirus 71 (EV71), coxsackievirus A16 (CVA16), CVA6 and CVA10 were detected by RT-PCR. Excel 2007 and SPSS18.0 software were used for data collection and statistical analysis, respectively. The epidemiological data of HFMD cases were analyzed by descriptive epidemiology method. The VP1 gene sequence of the representative strain of each CVA6 genotype was downloaded. Phylogenetic trees were constructed using MEGA X software and the genetic characteristics were analyzed.Results:A total of 1 531 HFMD cases were involved and 1 365 were positive for EVs with a positive rate of 89.16%. The detection rates of EV71, CVA16, CVA6, CVA10 and other EVs were 1.31% (20/1 531), 32.46% (497/1 531), 38.47% (589/1 531), 5.09% (78/1 531) and 11.23% (172/1 531), respectively. There were significant differences in the pathogen composition in HFMD cases of different clinical types (χ 2=46.14, P<0.01) and occupations (χ 2=34.65, P<0.01) as well as in different years (χ 2=462.86, P<0.01). The average age was greater in patients with CVA16 infection than in those with CVA6 or CVA10 infection ( F=6.00, P<0.01). In 2019, the HFMD cases were mainly caused by CVA16, while in 2020 and 2021, the main pathogen was CVA6. Enterovirus-positive cases showed a bimodal distribution with the main peak from May to July and the secondary peak from September to November. CVA16 was the predominant pathogen in spring and summer, and CVA6 was the predominant pathogen in autumn. CVA6 was the dominant pathogen in eight districts and counties of Xi′an; CVA16 was the dominant pathogen in six districts and counties; CVA6 and CVA16 co-circulated in one district. The CVA6 isolates belonged to two evolutionary branches of D3a subtype. Conclusions:CVA6 and CVA16 were the prevalent pathogens of HFMD and CVA6 subtype D3a circulated in Xi′an from 2019 to 2021. The pathogen composition of HFMD cases showed obvious differences in population, time and regional distribution.

Objective:To investigate the positive rates of 2019-nCoV nucleic acid in different specimens from confirmed COVID-19 cases during hospitalization and after discharge.Methods:Patients with confirmed COVID-19 were enrolled from designated hospitals. Nasal swabs, throat swabs, and specimens of stool, urine and blood were collected during hospitalization. After the patients were discharged, nasal swabs, throat swabs and stool specimens were collected during follow-up. Real-time RT-PCR was used to detect 2019-nCoV nucleic acid.Results:This study involved 25 confirmed COVID-19 cases. During hospitalization, all patients tested positive in both nasal and throat swab 2019-nCoV nucleic acid tests, and nine of them (36.00%) were positive in stool specimen test. Urine and blood specimen test results were all negative. Nasal swabs, throat swabs and stool specimens were collected from each patient 7 d and 14 d after discharge. Two patients (8.00%) tested positive for 2019-nCoV nucleic acid again in nasal and throat swab tests on 7 d, while all stool specimen tests were negative. No 2019-nCoV nucleic acid was detected in nasal swabs, throat swabs or stool samples on 14 d.Conclusions:2019-nCoV nucleic acid was detected in stool samples of confirmed COVID-19 cases during hospitalization. Nasal and throat swab nucleic acid tests turned positive again in some patients after discharge.

Objective: To investigate the function of anti-PD-1 (scFv)/IL-15/IL-15Rα-sushi fusion protein (PD-S15) to specifically bind to PD-1 in vitro and to explore its effect on NK/T cell proliferation. Methods: The human anti-PD-1 (scFv) gene sequence and human IL-15/IL-15Rα-sushi fusion gene sequence were synthesized chemically. The recombinant expression plasmid pUC57-PD-S15 was constructed by enzyme digestion and ligation of the two target genes, and then transiently transfected into HEK293T cells by lipofectamineTM 2000. The supernatants of cell culture medium were acquired, and the expression of PD-S15 fusion protein in cell culture supernatants was detected by Wb assay. PBMCs and TILs were cultured in mediums with different proportion of PD-S15/X-VIVOTM15, respectively. Then, the capacity of PD-S15 fusion protein to bind to PD-1 in vitro and its effect on the proliferation of PBMCs and the proportion of CD3+CD8+, CD3+CD4+ and CD3-CD56+ subsets were detected by flow cytometry. The effect of PD-S15 fusion protein on the proliferation of TILs was detected by cytometry. Results: The successful construction of pUC57-PD-S15 eukaryotic expression plasmid was confirmed by double enzyme digestion and sequencing, and then successfully transfected into HEK293T cells. The relative molecular weight of the target protein was approximately 55 000, and was in line with expectations. PD-S15 fusion protein could specifically combine with PD-1 in vitro (P<0.05) and stimulate NK/T cell proliferation (P<0.05). Compared with classical TILs culture method, the efficiency of activation and amplification of T cells in vitro by PD-S15 culturemethodwasbetter (P<0.01). Conclusion: PD-S15 fusion protein can specifically target PD-1 and rapidly expand NK/T cells in vitro, which lays a foundation for the selective expansion of CD8+PD-1+ antigen-specific T lymphocytes from tumor tissues and even peripheral blood.

Objective To analyze the pathogenic composition of hand, foot and mouth disease (HFMD) cases and the antibody level of enterovirus 71 (EV-A71) in healthy people in Xi'an in 2022, so as to provide evidence for the prevention and control of HFMD. Methods Anal swabs or stool specimens of HFMD cases were collected. RT-PCR was used to detect enterovirus (EV) and serotype was identified. Enzyme-linked immunosorbent assay (ELISA) was used to detect EV-A71 IgG antibody levels in healthy people. Results A total of 172 positive cases were detected from 274 HFMD clinical specimens with a total detection rate of 62.77%, including 1 case of EV-A71 (0.58%), 95 cases of CV-A16 (55.23%), 64 cases of CV-A6 (37.21%), and 1 case of CV-A10(0.58%). CV-A16 was the dominant pathogen in spring and summer, and CV-A6 was the dominant pathogen in autumn and winter（χ²= 64.376，P＜0.001）. The age of HFMD cases caused by CV-A16 was older than the cases caused by CV-A6（t = 2.709，P = 0.007）. The positive rate of EV-A71 IgG antibodies in healthy people was 36.92% (168/455). The positive rate of EV-A71 IgG antibodies in men (32.35%) was lower than that in women (43.72%), and the difference was statistically significant (χ²= 6.605 , P = 0.014). The positive rate of EV-A71 IgG antibodies in people of all ages ranged from 21.95% to 54.78%, and the difference was statistically significant (χ²= 27.623 , P<0.001). Conclusion The main pathogens of hand, foot and mouth disease in Xi'an in 2022 are CV-A16 and CV-A6 . The positive rate of EV-A71 IgG antibodies in children under 5 years old is low , and EV-A71 vaccination should be strengthened.

OBJECTIVETo explore the experimental methods that the phage peptide library technology screening human osteoblast specificity polypeptide, which will provide the basis of the experiment of the Ti surface biolization modification.

METHODSHuman calvarial osteoblasts were used as the target cells for whole-cell biopanning from a 12-mer peptide phage-display library. Cell eluent and cell lysis buffer were cultivate and count respectively after washing. Then the target cells were analyzed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence detection to authenticate the positive phage clones by human gingival fibroblast as the absorber cells. The positive phage clones were deduced by DNA sequencing.

RESULTSAfter four rounds of screening, twenty-two positive phage clones were found out from randomly selected phage monoclonals, whose single-strand DNA were extracted and sequenced. Amino acid sequence of the highest frequency peptide was MGWSWWPETWPM.

CONCLUSIONSThe specific peptide against human osteoblasts can be obtained from a phage-display peptide library for use as a new research approach and experimental basis of the biolization modification of the titanium surface.

Amino Acid Sequence ; Base Sequence ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique ; Humans ; Osteoblasts ; Peptide Library ; Peptides ; genetics ; Sequence Analysis, DNA ; Sequence Analysis, Protein ; Titanium

Objective To understand the distribution of novel coronaviruses in the external environment of confirmed COVID-19 cases. Methods Environmental surface swab specimens such as bed rails, doorknob, closestool, hand washing sink, table, locker,ward pager, mobile phone, cup, clothes, were collected from the sentinel hospital of COVID-19, and samples were collected for the nucleic acid detection by RT-PCR. Results A total of 150 environmental samples were collected from 30 confirmed COVID-19 cases, 6 samples were determined to be novel coronaviruses postive (positive rate 4.00%). The total 14 mobile phone showed 3 novel coronaviruses positive.Among the 30 confirmed COVID-19 cases, 6 cases (positive rate 20.00%)were found novel coronaviruses in the external environment. Conclusions Novel coronaviruses exists in external environment of confirmed COVID-19 cases, which indicates the potential risk of COVID-19 infection.