Objective:To develop a camelid single-domain antibody (SdAb) recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH), and to apply it in Western blot and ELISA. Methods:Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones. Then those clones were confirmed by Western blot, and their variable domain of heavy chain of heavy chain antibody(VHH) nucleotide sequence were determined. The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a. The SdAb was then expressed and purified, and its ability to detect CD-GDH protein in multiple assays was further explored.Results:Six positive clones were obtained, among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification. The purified GA4 binded well with CD-GDH with a Kd value of 3 nmol/L. In Western blot and ELISA, GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH. Conclusions:A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed, which provides a very useful tool for detecting CD-GDH.

To further investigate the molecular genetic characteristics of enzomatic nasal tumor vi-rus of goats(ENTV-2)in Yunnan Province,this study measured and analyzed the entire genome of ENTV-2 in Yunnan Province.The results showed that the complete genome sequence of the EN-TV-2 YN2023 strain(GenBank accession number:PP682590.1)was successfully obtained.The YN2023 strain has a total length of 7 307 bp and a typical structure of 5'-M5-gag-pro-pol-env-M3-3'.Whole genome sequence homology analysis showed that the nucleotide homology between YN2023 strain and 41 reference strains ranges from 85.3％to 95.5％.The whole genome evolution-ary tree indicates that the YN2023 strain is closely related to the prevalent strains in China,with certain genetic diversity and geographical clustering.The analysis of the gag gene evolutionary tree shows that the gag gene cluster of YN2023 strain is on a branch of the ENTV-2 gag gene,and YN2023 is clustered on the same small branch as enENTV-FJ1 and GDQY2017 strains,with the closest genetic relationship.The env gene evolutionary tree shows that YN2023 is on the same branch as GDQY2017,GDZJ2022,ENTV-2CHN1-6,ENTV-FJ1,and ENTV-FJ3,and is also on the same branch as GDQY2017,indicating a close genetic relationship.Recombination analysis showed that the YN2023 strain underwent a potential recombination event between breakpoint positions 6378-7478 bp,with the Chinese Chongqing strain enENTV-CQ1(OR669623.1)as the primary parent and the Chinese Sichuan strain BH(MT254062.1)as the secondary parent.This study enriches the genomic information of the ENTV-2 strain in Yunnan Province and provides data sup-port for the genetic variation of ENTV-2 in Yunnan Province.

To further investigate the molecular genetic characteristics of enzomatic nasal tumor vi-rus of goats(ENTV-2)in Yunnan Province,this study measured and analyzed the entire genome of ENTV-2 in Yunnan Province.The results showed that the complete genome sequence of the EN-TV-2 YN2023 strain(GenBank accession number:PP682590.1)was successfully obtained.The YN2023 strain has a total length of 7 307 bp and a typical structure of 5'-M5-gag-pro-pol-env-M3-3'.Whole genome sequence homology analysis showed that the nucleotide homology between YN2023 strain and 41 reference strains ranges from 85.3％to 95.5％.The whole genome evolution-ary tree indicates that the YN2023 strain is closely related to the prevalent strains in China,with certain genetic diversity and geographical clustering.The analysis of the gag gene evolutionary tree shows that the gag gene cluster of YN2023 strain is on a branch of the ENTV-2 gag gene,and YN2023 is clustered on the same small branch as enENTV-FJ1 and GDQY2017 strains,with the closest genetic relationship.The env gene evolutionary tree shows that YN2023 is on the same branch as GDQY2017,GDZJ2022,ENTV-2CHN1-6,ENTV-FJ1,and ENTV-FJ3,and is also on the same branch as GDQY2017,indicating a close genetic relationship.Recombination analysis showed that the YN2023 strain underwent a potential recombination event between breakpoint positions 6378-7478 bp,with the Chinese Chongqing strain enENTV-CQ1(OR669623.1)as the primary parent and the Chinese Sichuan strain BH(MT254062.1)as the secondary parent.This study enriches the genomic information of the ENTV-2 strain in Yunnan Province and provides data sup-port for the genetic variation of ENTV-2 in Yunnan Province.

Objective:To develop a camelid single-domain antibody (SdAb) recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH), and to apply it in Western blot and ELISA. Methods:Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones. Then those clones were confirmed by Western blot, and their variable domain of heavy chain of heavy chain antibody(VHH) nucleotide sequence were determined. The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a. The SdAb was then expressed and purified, and its ability to detect CD-GDH protein in multiple assays was further explored.Results:Six positive clones were obtained, among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification. The purified GA4 binded well with CD-GDH with a Kd value of 3 nmol/L. In Western blot and ELISA, GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH. Conclusions:A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed, which provides a very useful tool for detecting CD-GDH.

OBJECTIVE To study the immunotherapeutic effect of chimeric virus-like particles(VLPs) based on hepatitis E virus(HEV) against human papillomavirus type 16(HPV 16) tumor. METHODS HPV16 E7 was inserted into the p239 protein of HEV to form the recombinant chimeric protein p239-HPV16 E7. The constructed recombinant protein was expressed by Escherichia coli, purified, and then refolded, and the protein was detected by electron microscopy and dynamic light scattering to confirm size and shape. Then, the C57B/L mice were immunized with the protein grain, and the lymphocyte differentiation of mouse spleen was detected by flow cytometry and enzyme-linked immune spot immunoassay; in addition, TC-1 tumor cells were used to construct tumor models in C57B/L mice to evaluate the anti-tumor immune effect of protein particles in mice. RESULTS After refold in vitro, the structure of chimeric protein was observed under electron microscopy, and the size of particle was 22.80 nm. The obtained protein particles induced favorable specific cellular immune response in C57B/L mice. Compared with the control group, the proportions of CD3+/CD4+ and CD3+/CD8+ in spleen lymphocytes of experimental groups were significantly different(P＜0.05), and effector T cells secreting IFN-γ interferon were also increased remarkably. At the same time, the obtained protein particles could effectively inhibit the growth of tumor cells in TC-1 tumor-bearing mice, and the mice did not die during the experimental period, while the tumors in the control mice grew rapidly and all died after 6 weeks. CONCLUSION Chimeric protein p239-HPV16E7 which was expressed in prokaryotes can form virus-like particles and effectively induce anti-tumor immunity against HPV16.

Objective: To investigate the clinical outcomes of skin and soft tissue expansion in the repairment of neck scar. Methods: From March 2009 to May 2018, 15 patients with postburn scar contractures on neck, were admitted to the Department of Burn and Plastic Surgery, Karamay Central Hospital of Xinjiang. The patients include 12 males and 3 females, aged 12 to 48 years, with the mean age of 31 years. The scars were at 9 cm×6 cm-14 cm×11 cm in size. The tissue expander of 100-300 ml was placed subcutaneously, in the normal skin area on neck, on one or each side at the first stage operation. The first expander infusion was performed 10-14 days after surgery. The tissue expansion remains for 3-9 months, with an interval of 10 days of each infusion. After the tissue fully expanded, the expander was maintained for 1 month. At the second stage, the expander was removed, and the expanded flap was transferred to repair the wound. Results: The expander exposure due to friction occurred in 2 patients. The final therapeutic effect was not affected, because of iodine gauze bandage. Blood supply of expanded flaps was good in other patients. The size of the expanded flaps was 12 cm×8 cm-16 cm×15 cm. All flaps survived after the second stage surgery. Patients were followed up for 0.5-5 years after surgery. The color and texture of flaps was similar to adjacent normal skin. Conclusions Skin and soft tissue expansion is a safe and effective method in repairing neck scar.

Objective: To study infection of coxsackievirus A6 (CV-A6) in Mongolian gerbils. Methods: To screen the optimal ages of Mongolian gerbils, five groups with different ages were infected with 1×10⁵ TCID₅₀ dose of CV-A6 XS45 strain by intraperitoneal, and symptom scores of Mongolian gerbils were collected. Then to estimate the dose-effect, three doses of virus were injected to the Mongolian gerbils. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry(IHC) were used to determine virus load and tissues infection in muscle, brain and intestinal tract. Results: Mongolian gerbils infected with 1×10⁵ TCID₅₀ dose CV-A6 consistently exhibited clinical signs, and the morbidity (death) rates of five age groups were up to 100%. There was a positive correlation between the trend of symptom scores changes and ages. The morbidity (death) rates of three doses (1×10³ TCID₅₀, 1×10⁴ TCID₅₀, 1×10⁵ TCID₅₀) also were up to 100% in 28 days Mongolian gerbils. The correlation between the trend of symptom scores changes and doses were negative. Virus loads were detected in muscle, brain and intestinal tract of pathogenesis animal. The virus loads of muscle were higher than others. IHC results showed virus infection and cytopathic effects in three tissues. Conclusions Mongolian gerbils had high susceptibility to CV-A6, and were best for animal model of CV-A6 infection.

Objective Discuss medical health integration continuance management mode in the prevention of elderly patients after discharge in bed household application effect of pressure ulcers. Methods To 120 cases of elderly patients in bed in hospital time order is divided into control group and experimental group,by the medical health outreach group respectively in the hospital two days before the assessment of patients and family rehabilitation plan,the control group given conventional discharge and telephone follow-up after discharge,the experimental group according to the medical health integration management mode,made up of medical health outreach team to stay in bed for elderly patients after discharge pressure ulcer risk factors assessment,targeted prevention of pressure sores rehabilitation plan,group management,remote care joint family supervision,timely follow up the capa and the exami-nation of the effect,the pressure ulcer management and quality of life scale to compare two groups of patients at discharge,6 months after hospital discharge,the quality of life of 12 months after discharge and the incidence of pressure ulcers in a year. Results The experimental group was lower than those of control group,the incidence of pressure ulcers was statistically significant difference(P<0.05); 6 months and 12 months after discharge physiological field,psychological field in the quality of life score were higher than control group,the difference was statistically significant(P<0.05); Score compared two groups of environmental and social sciences has no statistical significance (P>0.05). Conclusion Medical health integration continuance management can effectively reduce the incidence of pressure ulcers that occupy the home stay in bed for elderly patients,improve their quality of life.

Objective To identify immunodominant B linear cell epitopes in capsid proteins VP1 to VP3 of Coxsackievirus A16 (CVA16) strain YY157.Methods The protean algorithms of bioinformatic software Lasergene were used to analyze antigenicity, hydrophilicity and surface probability of amino acid sequence of CVA16 capsid proteins VP1 to VP3.Multiple regions containing potential lineal B cell epitopes were predicted and their corresponding average indexes were calculated by BepiPred 1.0 Server.Corresponding peptides were synthesized and examined in peptide-enzyme-linked immunosorbent assay (ELISA) individually to check whether it reacted positively or negatively toward sera from children with confirmed CVA16 infection.Results Totally 21 possible B cell linear epitopes were predicted according to their relatively strong antigenicity, hydrophilicity and surface probability.The corresponding synthetic peptides reacted positively with sera of CVA16-infected children in a varying extent.Conclusion Immunodominant B cell linear epitopes of capsid proteins VP1 to VP3 of CVA16 strain YY157 are successfully predicted and confirmed.