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Development and optimization of a double antibody sandwich ELISA for the detection of goose T cell surface CD8α molecule

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Journal of Integrative Agriculture 2016年第10期15卷 2363-2368页

作者： ZHANG Wei CHENG Bei-bei CHEN Shun WANG Ming-shu JIA Ren-yong ZHU De-kang LIU Ma-feng LIU Fei SUN Kun-feng YANG Qiao WU Ying CHEN Xiao-yue CHENG An-chun Institute of Preventive Veterinary medicine Sichuan Agricultural University Avian disease Research Center College of Veterinary Medicine Sichuan Agricultural University key laboratory of Animal disease and human Health of Sichuan Province Sichuan Agricultural University

CD8, a glycoprotein on the surface of T cells, is involved in the defense against viral infection and plays significant roles in antigen presentation and in the antiviral immune response. CD8 is composed of two chains. Of these, the CD8α chain was chosen for the detection because it involved in both the CD8αα homodimer and the CD8αβ heterodimer. Here, we established a double antibody sandwich enzyme-linked immunosorbent assay（DAS-ELISA） for specific detection of goose CD8α（go CD8α）. The results showed that the optimal coated antibody and antigen dilutions were 1：50（the antibody titer was 1：12 800） and 1：32（0.3 ng m L^–1）, respectively, while the optimal capture antibody and horseradish peroxidase（HRP）-labelled goat anti-rabbit Ig G dilutions were 1：50（the antibody titer was 1：51 200） and 1：4 000（the antibody titer was 1：5 000）, respectively. The optimal blocking buffer was 5% bovine serum albumin（BSA）. The best incubating condition was overnight at 4℃, the best blocking time was 120 min and the best anti-capture antibody working time was 150 min. In addition, the minimum dose detectable by DAS-ELISA was 5×10^–3 ng m L^–1. Most importantly, go CD8α expression levels in goose spleen mononuclear cells（MNCs） post-Goose parvoviruse（GPV） infection were found to be significantly up-regulated using the DAS-ELISA method, which was consistent with previous results obtained using real-time quantitative PCR. In conclusion, the DAS-ELISA method reported here is a novel, specific technique for the clinical detection of go CD8α.

关键词： T cells goose CD8α polyclonal antibody double antibody sandwich ELISA

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Anti-apoptotic protein BCL-XL as a therapeutic vulnerability in gastric cancer

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Animal Models and Experimental medicine 2023年第3期6卷 245-254页

作者： Yumin Wei Liping Zhang Chao Wang Zefeng Li Mingjie Luo Guomin Xie Xingjiu Yang Mengyuan Li Shuyue Ren Dongbing Zhao Ran Gao Jia-Nan Gong National human diseases Animal Model Resource Center The Institute of Laboratory Animal ScienceChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijingChina NHC Key Laboratory of Human Disease Comparative Medicine Beijing Engineering Research Center for Experimental Animal Models of Human Critical DiseasesBeijingChina Beijing Engineering Research Center for Experimental Animal Models of human Critical diseases BeijingChina Department of Pancreatic and Gastric Surgical Oncology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina State key laboratory of Ophthalmology Zhongshan Ophthalmic CenterSun Yat-Sen UniversityGuangzhouChina

Background: New therapeutic targets are needed to improve the outcomes for gastric cancer(GC) patients with advanced disease. Evasion of programmed cell death(apoptosis) is a hallmark of cancer cells and direct induction of apoptosis by targeting the pro-survival BCL2 family proteins represents a promising therapeutic strategy for cancer treatment. Therefore, understanding the molecular mechanisms underpinning cancer cell survival could provide a molecular basis for potential therapeutic interventions. Method: Here we explored the role of BCL2L1 and the encoded anti-apoptotic BCL-XL in GC. Using Droplet Digital PCR(ddPCR) technology to investigate the DNA amplification of BCL2L1 in GC samples and GC cell lines, the sensitivity of GC cell lines to selective BCL-XL inhibitors A1155463 and A1331852, pan-inhibitor ABT-263, and VHL-based PROTAC-BCL-XL was analyzed using(CellTiter-Glo) CTG assay in vitro. Western Blot(WB) was used to detect the protein expression of BCL2 family members in GC cell lines and the manner in which PROTAC-BCL-XL kills GC cells. Coimmunoprecipitation(Co-IP) was used to investigate the mechanism of A1331852 and ABT-263 kills GC cell lines. DDPCR, WB, and real-time PCR(RTPCR) were used to investigate the correlation between DNA, RNA, protein levels, and drug activity. Results: The functional assay showed that a subset of GC cell lines relies on BCL-XL for survival. In gastric cancer cell lines, BCL-XL inhibitors A1155463 and A1331852 are more sensitive than the pan BCL2 family inhibitor ABT-263, indicating that ABT-263 is not an optimal inhibitor of BCL-XL. VHL-based PROTAC-BCL-XL DT2216 appears to be active in GC cells. DT2216 induces apoptosis of gastric cancer cells in a time-and dose-dependent manner through the proteasome pathway. Statistical analysis showed that the BCL-XL protein level predicts the response of GC cells to BCL-XL targeting therapy and BCL2L1 gene CNVs do not reliably predict BCL-XL expression.Conclusion: We identified BCL-XL as a promising therapeutic target in a subset o

关键词： apoptosis BCL2L1(BCL-XL) gastric cancer(GC) PROTAC-BCL-XL selective BCL-XL inhibitors

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MicroRNA-122-functionalized DNA tetrahedron stimulate hepatic differentiation of human mesenchymal stem cells for acute liver failure therapy

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Bioactive Materials 2023年第10期28卷 50-60页

作者： Hongyan Wei Fenfang Li Tiantian Xue Haixia Wang Enguo Ju Mingqiang Li Yu Tao laboratory of Biomaterials and Translational medicine Center for NanomedicineThe Third Affiliated HospitalSun Yat-sen UniversityGuangzhou510630China Guangdong Provincial key laboratory of Liver disease Research Guangzhou510630China

As the most abundant liver-specific microRNA, microRNA-122 (miR122) played a crucial role in the differentiation of stem cells into hepatocytes. However, highly efficient miR122 delivery still confronts challenges including poor cellular uptake and easy biodegradation. Herein, we for the first time demonstrated that the tetrahedral DNA (TDN) nanoplatform had great potential in inducing the differentiation of human mesenchymal stem cells (hMSCs) into functional hepatocyte-like cells (HLCs) by transferring the liver-specific miR122 to hMSCs efficiently without any extrinsic factors. As compared with miR122, miR122-functionalized TDN (TDN-miR122) could significantly up-regulate the protein expression levels of mature hepatocyte markers and hepatocyte-specific marker genes in hMSCs, indicating that TDN-miR122 could particularly activate the hepatocyte-specific properties of hMSCs for developing cell-based therapies in vitro. The transcriptomic analysis further indicated the potential mechanism that TDN-miR122 assisted hMSCs differentiated into functional HLCs. The TDN-miR122-hMSCs exhibited hepatic cell morphology phenotype, significantly up-regulated specific hepatocyte genes and hepatic biofunctions in comparison with the undifferentiated MSCs. Preclinical in vivo transplantation appeared that TDN-miR122-hMSCs in combination with or without TDN could efficiently rescue acute liver failure injury through hepatocyte function supplement, anti-apoptosis, cellular proliferation promotion, and anti-inflammatory. Collectively, our findings may provide a new and facile approach for hepatic differentiation of hMSCs for acute liver failure therapy. Further large animal model explorations are needed to study their potential in clinical translation in the future.

关键词： Hepatocyte differentiation Tetrahedral framework nucleic acids Cell therapy Transcriptomics Acute liver failure treatment

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Predictive value of Th17 and Treg cells at baseline for HBsAg loss in chronic hepatitis B patients with low HBsAg quantification treated with pegylated interferon and nucleos(t)ide analogue

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Liver Research 2023年第2期7卷 136-144页

作者： Li-Li Wu Xiao-Yan Li Kai Deng Bing-Liang Lin Hong Deng Dong-Ying Xie Geng-Lin Zhang Qi-Yi Zhao Zhi-Shuo Mo Yue-Hua Huang Zhi-Liang Gao Department of Infectious diseases The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouGuangdongChina Guangdong Provincial key laboratory of Liver disease Research The Third Affiliated Hospital of Sun Yat-sen UniversityGuangzhouGuangdongChina Institute of human Virology Key Laboratory of Tropical Disease Control of Ministry of EducationZhongshan School of MedicineSun Yat-sen UniversityGuangzhouGuangdongChina key laboratory of Tropical disease Control(Sun Yat-sen University) Ministry of EducationGuangzhouGuangdongChina

Background and aims:The primary goal of chronic hepatitis B(CHB)treatment is to reduce hepatitis B surface antigen(HBsAg).T helper 17(Th17)and regulatory T(Treg)cells are essential for the development of CHB.However,how Th17 and Treg cells contribute to HBsAg loss is still unknown.Therefore,this study aimed to search for the predictive value of Th17 and Treg cells for HBsAg loss in CHB patients with low HBsAg quantification.Methods:The study included 99 hepatitis B e antigen(HBeAg)-negative CHB patients who had completed a year of nucleos(t)ide analogue(NA)monotherapy and had received both NA and pegylated interferon(PEG-IFN)treatment for less than 96 weeks(96 wk).In the cured group,48 patients lost HBsAg within 48 wk,while 51 patients did not(uncured group).Blood samples and clinical data were collected for research.Results:During PEG-IFN and NA combination therapy,the proportion of Th17 cells in the cured group increased significantly,while the proportion of Treg cells in the uncured group increased.From 0 to 24 wk,the proportion of Th17 cells in the cured group was significantly higher than in the uncured group,while the opposite was true for Treg cells.Patients with alanine aminotransferase(ALT)2.5 upper limit of normal(ULN)at 12 wk had a higher proportion of Th17 cells and a lower proportion of Treg cells than those with ALT<2.5 ULN at 12 wk.Additionally,the proportion of Th17 cells is inversely associated with the level of HBsAg,whereas the level of Treg cells is positively related to HBsAg quantification.The clinical cure index,including age,HBsAg quantification,and the proportions of Th17 and Treg cells,had a higher area under the curve(0.957)for predicting HBsAg loss when compared to the proportions of Th17 and Treg cells and HBsAg quantification alone.Conclusions:Combined with quantification of HBsAg,the proportions of Th17 cells and Treg cells at baseline can be used as good predictors of HBsAg loss in patients with low HBsAg quantification treated with NA and PEG-IFN.

关键词： Chronic hepatitis B(CHB) T helper 17(Th17)cell Regulatory T(Treg)cell Hepatitis B surface antigen(HBsAg)loss Clinical cure Pegylated interferon(PEG-IFN) Nucleos(t)ide analogue(NA)

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Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

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Nano Research 2023年第4期16卷 5383-5390页

作者： Guobin Mao Yang Yang Shijie Cao Silu Ye Yifang Li Wei Zhao Hongwei An Yingxia Liu Junbiao Dai Yingxin Ma CAS key laboratory of Quantitative Engineering Biology Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic GenomicsShenzhen Institute of Synthetic BiologyShenzhen Institutes of Advanced TechnologyChinese Academy of SciencesShenzhen 518055China Guangxi University of Chinese medicine Nanning 530001China Shenzhen key laboratory of Pathogen and Immunity National Clinical Research Center for infectious diseaseState Key Discipline of Infectious DiseaseShenzhen Third People’s HospitalSecond Hospital Affiliated to Southern University of Science and TechnologyShenzhen 518112China

The global pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus has necessitated rapid,easy-to-use,and accurate diagnostic methods to monitor the virus infection.Herein,a ratiometric fluorescence enzyme-linked immunosorbent assay(ELISA)was developed using Si-fluorescein isothiocyanate nanoparticles(FITC NPs)for detecting SARSCoV-2 nucleocapsid(N)protein.Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane(APTES)-FITC as the Si source.This method did not need post-modification and avoided the reduction in quantum yield and stability.The p-nitrophenyl(pNP)produced by the alkaline phosphatase(ALP)-mediated hydrolysis of pnitrophenyl phosphate(pNPP)could quench Si fluorescence in Si-FITC NPs via the inner filter effect.In ELISA,an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody.ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs.The change in fluorescence intensity ratio could be used for detecting N protein,with a wide linearity range(0.01-10.0 and 50-300 ng/mL)and low detection limit(0.002 ng/mL).The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum.Moreover,this proposed method can accurately distinguish coronavirus disease 2019(COVID-19)and non-COVID-19 patient samples.Therefore,this simple,sensitive,and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection.

关键词： Si-fluorescein isothiocyanate(FITC)nanoparticles ratiometric fluorescent probe severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) inner filter effect enzyme-linked immunosorbent assay(ELISA)

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Peripheral ApoE4 Leads to Cerebrovascular Dysfunction and Aβ Deposition in Alzheimer's disease

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神经科学通报:英文版 2023年第8期39卷 1330-1332页

作者： Rui Sun Chunming Xie Department of Neurology School of MedicineAffiliated Zhongda HospitalSoutheast UniversityNanjing 210009China Institute of Neuropsychiatry Affiliated Zhongda HospitalSoutheast UniversityNanjing 210009China The key laboratory of Developmental Genes and human disease Southeast UniversityNanjing 210009China

Alzheimer's disease(AD)is a neurodegenerative disease characterized by progressive cognitive dysfunction and behavioral impairment that occurs in old age and pre-old age[1].Apolipoprotein4(ApoE4)is the most important genetic risk factor for late-onset AD[2].Peripheral apoE4 is separated from that in the CNS by the blood-brain barrier(BBB)and is abundantly produced by the liver and macrophages and released into the blood to modulate lipid-related events.

关键词： ApoE4 Alzheimer impairment

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导致狐狸重症腹泻疫情的新型犬冠状病毒的发现与鉴定

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Science Bulletin 2023年第21期68卷 2598-2606,M0006页

作者：刘雨婷邓瑶牛胜朱娜宋敬东张晓爽苏伟婷佴文慧陆柔剑 David MIrwin 高福王文玲王奇慧谭文杰张树义 College of Animal Science and Veterinary medicine Shenyang Agricultural UniversityShenyang 110866China NHC key laboratory of Biosafety National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China College of Veterinary medicine Shanxi Agricultural UniversityJinzhong 030801China National key laboratory of Intelligent Tracking and Forecasting for Infectious diseases National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Tianjin Zoo Tianjin 300381China Kunming Cell Bank State Key Laboratory of Genetic Resources and EvolutionKunming Institute of ZoologyChinese Academy of SciencesKunming 650223China Department of laboratory medicine and Pathobiology University of TorontoToronto ON M5S 1A8Canada CAS key laboratory of Pathogen Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China School of laboratory medicine and Life Sciences Wenzhou Medical UniversityWenzhou 325035China

2019年末,中国东北地区狐狸群中暴发大规模疫情,发病狐狸表现出急性腹泻症状,并出现大量狐狸死亡.通过对该疫源地发病狐狸肛拭子、血液与发病组织的检测分析,获得了多条全长新型犬冠状病毒(CCoV)基因组序列,研究团队将该病毒命名为VuCC... 详细信息

2019年末,中国东北地区狐狸群中暴发大规模疫情,发病狐狸表现出急性腹泻症状,并出现大量狐狸死亡.通过对该疫源地发病狐狸肛拭子、血液与发病组织的检测分析,获得了多条全长新型犬冠状病毒(CCoV)基因组序列,研究团队将该病毒命名为VuCCoV,并确认VuCCoV是引起该疫情的病原.进一步分析表明,VuCCoV基因组的4个结构基因(S,E,M,N)中的3个(E,M,N)与犬冠状病毒(CCoV)对应基因具有超过90%的核苷酸同一性,但S基因核苷酸序列表现出较大的差异.VuCCoV的RNA依赖的RNA聚合酶与蝙蝠冠状病毒PB025的对应蛋白仅存在2~3个氨基酸差异,系统进化分析提示VuCCoV很可能来源于蝙蝠.表面等离子体共振分析实验表明,VuCCoV刺突蛋白受体结合区(RBD)可与犬和狐狸的氨肽酶N(APN)结合,但不与人APN结合,假病毒感染及病毒分离培养实验进一步证实该VuCCoV不易感染人源细胞.蝙蝠等野生动物宿主的病毒跨物种传播对人类和家畜健康构成了显著威胁,VuCCoV的发现和溯源证实蝙蝠作为疫情出现的潜在驱动因素的重要性.积极监控蝙蝠及其他犬科动物中的病毒感染,发现、鉴定对人兽健康构成潜在威胁的新型冠状病毒并研究其进化变异特征与感染特性,对于防控新发人兽共患传染病、建设国家生物安q全体系具有重要意义.

关键词：犬冠状病毒人兽共患传染病病毒感染表面等离子体共振犬科动物动物宿主氨肽酶N 狐狸

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Dynamic Observation of Cartilage Damage and Serum IL-1β, TNF-α, IL-6 and IL-17 in Post-traumatic Rat Osteoarthritis Model

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Journal of Northeast Agricultural University(English Edition) 2022年第3期29卷 59-65页

作者： Lv Liang-yu Ma Tian-wen Yu Yue Jia Li-na Xu Xin-yu Zhao Ming-chao Chen Hong Sheng Xuan-bo Li Ting Xiao Jian-hua Gao Li College of Veterinary medicine Northeast Agricultural UniversityHeilongjiang Key Laboratory Animals and Comparative MedicineHarbin 150030China

The pathogenesis of animal osteoarthritis(OA)is not clear.The aim of this study was to investigate the dynamic changes of cartilage lesions and serum IL-1β,TNF-α,IL-6 and IL-17 inflammatory factors in a post-traumatic osteoarthritis model in rats,and to explore their values in monitoring the progression of osteoarthritis.Forty male SD rats were randomly divided into the control group(n=20)and the model group(n=20).The OA model was established by anterior cruciate ligament transection combined with partial medial meniscus resection(ACLT+PMMx)in the model group,and sham operation was performed in the control group.On the 0th,10th,20th and 30th days after modeling,the degree of joint swelling and the number of white blood cells in the peripheral blood were evaluated,and tibia samples were taken for macroscopic observation score and pathological observation.The serum levels ofIL-1β,TNF-α,IL-6 and IL-17 concentration were detected by enzyme-linked immunosorbent assay(ELISA).The results showed that the cartilage damage in the tibial surface and pathological observations of rats in the model group gradually increased with time,the eye scores increased and increased significantly on the 20th and 30th days(P<0.01),and the joints of the rats were swollen.And the swelling was extremely significant on the 30th day(P<0.01).Compared with the control group,the number of white blood cells in the peripheral blood of the model group gradually increased,and the increase was extremely significant on the 20th and 30th days(P<0.01).The serum IL-17 concentration in the model group tended to increase,and the increase was significant on the 20th day(P<0.05)and extremely significant on the 30th day(P<0.01);the serum TNF-αand IL-1βconcentrations in the model group showed an increasing trend,and the increase was extremely significant on the 10th,20th and 30th days(P<0.01);IL-6 concentration in the serum of the model group showed an increasing trend,and it increased significantly on the 30th day(P<0.05).The results showed that in the rat mod

关键词： osteoarthritis inflammatory factor cartilage injury rat

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The challenge of emerging SARS-CoV-2 mutants to vaccine development

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Journal of Genetics and Genomics 2021年第2期48卷 102-106页

作者： Rong Li Jun Liu Hui Zhang Institute of human Virology Key Laboratory of Tropical Disease Control of Ministry of EducationGuangdong Engineering Research Center for Antimicrobial Agent and ImmunotechnologyZhongshan School of MedicineSun Yat-sen UniversityGuangzhouGuangdong 570080China

Since the outbreak of the pandemic,waves of epidemics caused by severe acute respiratory syndrome coronavirus 2(SARS-Co V-2)variants that harbor novel mutations have never paused.Globally,it undergoes rapid mutations that involve single-nucleotide polymorphism(SNP)dominantly,whereas ORF1ab and spike genes contain the most of more than 20,000 mutation sites reported within a year(Fang et al.,2021).Mutations inside spike protein are highly concerned for their potential impact on viral transmissibility and immune evasion,as spike protein is responsible for the interaction with the viral receptor angiotensin-converting enzyme 2(ACE2)to mediate viral entry to the target cells.D614G identified in early 2020 is a globally dominant mutation(Korber et al.,2020).In late 2020,several variants were reported,which had caused continental and eventually worldwide epidemics.These notable variants include B.1.1.7 lineage(501Y.V1,Variant of Concern[VOC]202012/01),501Y.V2 variant(known as B.1.351 lineage),and P.1 lineage(also named 501Y.V3).In comparison with the D614G and D614 lineages identified in early 2020,they contain a large number of mutations within spike protein(Fig.1).

关键词： respiratory acute eventually

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Transformation of sulfamethazine during the chlorination disinfection process: Transformation,kinetics, and toxicology assessment

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Journal of Environmental Sciences 2019年第2期31卷 48-56页

作者： Yunjia Yang Jiachen Shi Yi Yang Jie Yin Jing Zhang Bing Shao Beijing key laboratory of Diagnostic and Traceability Technologies for Food Poisoning Beijing Center for Disease Prevention and Control Beijing Research Center for Preventive medicine

Various disinfection byproducts(DBPs) form during the process of chlorination disinfection,posing potential threats to drinking water safety and human health. Sulfamethazine(SMT),the most commonly used and frequently detected veterinary antibiotic, was investigated in detail with regard to its transformation and kinetics in reactions with free available chlorine(FAC). Using liquid chromatography coupled to quadrupole time-of-flight tandem mass spectrometry, several DBPs were identified based on different confidence levels, and a variety of reaction types, including desulfonation, S–N cleavage, hydroxylation, and chlorine substitution, were proposed. The kinetic experiments indicated that the reaction rate was FAC-and pH-dependent, and SMT exhibits low reactivity toward FAC in alkaline conditions. The DBPs exhibited a much higher acute toxicity than SMT, as estimated by quantitative structure activity relationship models. More importantly, we observed that the FAC-treated SMT reaction solution might increase the genotoxic potential due to the generation of DBPs. This investigation provides substantial new details related to the transformation of SMT in the chlorination disinfection process.

关键词： LC-QTOF Sulfamethazine Transformation products Chlorination Toxicity

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