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.

Esophageal cancer is a common malignant tumor of digestive tract.Remarkable regional difference is a prominent feature of the clinical epidemiology of esophageal cancer.They are mainly manifested in incidence rate,incidence type,onset age,and gene mutation.These differences may be related to dietary habits,lifestyle,and environmental factors.In recent years,research on the regional differences in esophageal cancer has gradually deepened.This article summarizes the differences in incidence rate,incidence type,gene mutations,epigenetics,risk factors,and prognosis of esophageal cancer in different regions,including Asia(China,India,Japan,and other countries),Europe,America(the United States),Africa,and other regions.Understanding these differences can help doctors and public health experts understand the risk factors and causes of esophageal cancer and further develop highly effective prevention and treatment strategies to reduce the occurrence and mortality rate of this malignancy.

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 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 To investigate the effect of Andrographis paniculata Nees(APN)extract on Coxsackievirus A16(CVA16)in vitro.Methods African green monkey kidney-derived Vero cells(Vero cells)were treated with APN extract at the concentration of 500.0,250.0,125.0,60.0,30.0,15.0,7.5 and 3.8 μg/mL,the cytotoxicity was determined with cell counting Kit-8 and the IC50was calculated by Probit unit regression method.Direct inactivating activity on CVA16,blocking of CVA16 adsorbing Vero cells and inhibition of CVA16 replication in Vero cells were determined and compared between Ribavirin(RBV) and APN extract with CVA16-infected Vero cells.SPSS 24.0 software was used to analyze the data.Results The selected concentrations of APN extract and RBV for experiment were 15.0, 7.5 and 3.8 μg/mL according to cytotoxicity test.Both of APN extract and RBV had neither direct inactivation on CVA 16 nor blocking of CVA16 adsorbing at the concentration of 15.0,7.5 and 3.8 μg/mL(F=1.54,1.52 and 0.67, 1.68,all P>0.05).However,both drugs had the capability of inhibiting CVA 16 replication in Vero cells at the concentration of 15.0 and 7.5 μg/mL(t=6.87,11.76 and 7.71,12.84,all P<0.05).Conclusion Experimental result shows that APN extract can effectively inhibit CVA 16 replication in Vero cells in vitro.

Objective To develop a novel anti-Enterovirus 71 (EV71) vaccine as recombinant virus-like particles.Methods By utilizing the foreign antigen presentation and virus-like particles forming features of Norovirus casipid VP1 P domain (NoVP), two pET-28a (+)-based recombinant expression plasmids containing either NoVP alone or NoVP with three specific epitopes SP55, SP70 and VP2-28 of EV71 capsid proteins tandemly inserted at the surface loop site were constructed and transferred to Escherichia coli.The recombinant fusion proteins of NoVP + EV71-SP55-SP70-VP2-28 and NoVP were induced expression and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot and transmission electron microscopy (TEM) observation.BALB/C mice were randomly divided into three groups:group A immunized with the recombinant fusion protein, group B immunized with NoVP and group C injected with 10 mmol/L Tris plus 20 mmol/L NaCl (pH 9.0).Enzymelinked immunosorbent assay (ELISA) was used to test specific antibodies in the serum of the mice, besides, the serums were mixed with the EV71 H3-TY strain and Vero cells, then specific antibody titer was examined by microneutralization test.One way ANOVA and Bonferroni test were used to analyze data.Results Both recombinant fusion protein and NoVP were expressed in Escherichia coli in inclusion bodies form.SDS-PAGE demonstrated that the relative molecular weights of recombinant fusion protein and NoVP protein were approximately 43 × 103 and 36 × 103, respectively;positive protein band of about 43 × 103 (relative molecular mass) was detected in recombinant fusion protein by Western Blot.Virus-like particles derived from the recombinant fusion proteins were observed under TEM.ELISA showed that absorbance 490 (A490) of mice serum added in SP70 peptide was significantly higher than those of group B and C (F =13.860,P ＜0.05).And microneutralization test demonstrated that the serum from group A was able to neutralize EV71 at a geometric mean titer above 1:38.Conclusion A novel virus-like particles vaccine against EV71 with good antigenicity and specificity has been prepared, which is able to induce high titer of neutralizing antibody against EV71 in mice.

Objective To establish a simple and reliable experimental rodent model sensitive to coxsackievirus A16 ( CVA16) .Methods Mongolian gerbils with different age were selected and inoculated intraperitoneally with live CVA16, and the gerbils were observed daily until 14 days postinoculation to screen for the most optimal ages sensitive to the virus.The dose-dependent symptoms were evaluated and the 50% lethal dose (LD50) was determined.The virus titers were measured in blood and various tissues of CVA 16-infected Mongolian gerbils 3 days post-infecton.Finally, the gerbils were immunized twice with inactivated CVA 16 vaccine at day 1 and day 11, respectively, followed by challenge with the virus with a dose of LD50 at day 14.The gerbils were then observed for another 2 weeks to record their body weight , symptom and mortality rate .Their blood samples were collected from the eyes , and CVA16-specific neutralizing antibodytiters and total antibody titers was checked by microneutralization test and ELISA , respectively .Results Various clinical symptoms, such as inactivity, hind limb weakness, paralysis and even death occurred in gerbils following CAV 16 infection. 7-day-old and 14-day-old gerbils are susceptible to CVA 16 infection whereas 28-day-old gerbils are resistant .The most sensitive and appropriate age is 14-day-old.The 50%lethal dose was determined to be 1×104.5 CCID50.High titers of the virus were confirmed in blood and various tissues of Mongolian gerbils contracted CAV 163 days post-infecton.The survival rate is 87.5%for 14-day-old gerbils preimmunized with two doses of inactivated CVA 16 vaccine and challenged with the virus.The geometric mean titers ( GMTs) of neutralizing antibody was 28.14, and the seroprevalence was 87.5%.Conclusions Mongolian gerbils is sensitive to CVA16 and the virus reproduces actively in Vivo.Thus, it can be used as a reliable small animal model for studies of CVA 16 pathogenesis , vaccine development and drug evaluation .