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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

The codon-optimized capsid gene of duck circovirus can be highly expressed in yeast and self-assemble into virus-like particles

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Journal of Integrative Agriculture 2017年第7期16卷 1601-1608页

作者： YANG Cui XU Yu JIA Ren-yong liu Si-yang WANG Ming-shu ZHU De-kang CHEN Shun liu ma-feng ZHAO Xin-xin SUN Kun-feng JING Bo YIN Zhong-qiong CHENG An-chun Avian Disease Research Centre Sichuan Agricultural UniversityChengdu 611130P.R.China Institute of Preventive Veterinary Medicine Sichuan Agricultural UniversityChengdu 611130P.R.China Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu 611130P.R.China

The capsid （Cap） protein, which is the only structural protein of duck circovirus （DuCV）, is the most important antigen for the development of vaccines against DuCV and the virus＇s serological diagnostic methods. In order to use yeast expression system to produce a large quantities of DuCVCap protein which is close to its natural form to display the antigen peptides perfectly, the Cap gene was optimized into the codon-optimized capsid （Opt-Cap） gene towards the preference of yeast firstly. Then, the genes of Cap and Opt-Cap were separately cloned into pPIC9K plasmid and transformed into Picha pas- toris GSl15. The strains that displayed the phenotype of Mut~ and contained multiple inserts of expression cassette were selected from those colonies. After the induction expression, the secretory type of Cap protein, which was about 43 kDa, was best expressed under 0.5% （v/v） methanol and sorbitol induction. Compared with the Cap gene, the expression level of Opt-Cap gene was much higher. What＇s more, the purified Cap protein had a good reactivity to its specific polyclone antibody and DuCV-positive serum, and it was able to self-assemble into virus-like particles （VLPs）. These VLPs, with a diameter of 15-20 nm and without a nucleic acid structure, showed a high level of similarity to DuCV particles in size and shape. All of the resultsdemonstrated that, based on the codon-optimization, it is suitable to use the P. pastoris expression system to produce DuCV VLPs on a large scale. It is the first time that a large amounts of DuCV VLPs were produced successfully in P. pastoris, which might be particularly useful for the further studies of serological diagnosis and vaccines of DuCV.

关键词： capsid gene codon'optimization duck circovirus virus-like particles

Role of duck plague virus glycoprotein C in viral adsorption:Absence of specific interactions with cell surface heparan sulfate

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Journal of Integrative Agriculture 2017年第5期16卷 1145-1152页

作者： JING Yan-chun WU Ying SUN Kun-feng WANG Ming-shu CHENG An-chun CHEN Shun JIA Ren-yong ZHU De-kang liu ma-feng YANG Qiao JING Bo CHEN Xiao-yue Institute of Preventive Veterinary Medicine Sichuan Agricultural UniversityChengdu 611130P.R.China Key Laboratory of Animal Diseases and Human Health of Sichuan Province Chengdu 611130P.R.China Avian Diseases Research Center College of Veterinary MedicineSichuan Agricultural UniversityChengdu 611130P.R.China

many mammalian herpes viruses utilize heparan sulfate （HS） moieties present on cell surface proteoglycans as receptors for cell entry, and this process also requires viral glycoprotein C （gC） homologues. However, our understanding of the role of gC in facilitating attachment of other alpha-herpes viruses such as the duck plague virus （DPV） remains preliminary. To study the role of gC during DPV infection, we used a gC-deleted mutant virus （DPV-AgC-EGFP）. Examination of the viral copy number by real-time PCR, as well as time course studies of viral adsorption and proliferation revealed that gC was involved in the viral binding to the cell surface. The affinity of viral glycoproteins （gB-DPV, gC-DPV, and gE-DPV） to HS was assessed using a prokaryotic expression system and HJTrapTM HeparJn HP column chromatography. In addition, to confirm that gC played a role in the interaction between DPV and HS, viruses were treated with the HS analogue heparin and host cells were treated with its inhibitors heparinase prior to exposure to DPV-△gC-EGFP or wild-type strain Chinese virulent duck plague virus （DPV-CHv）. The effects of heparin and heparinase on virus infectivity demonstrated that function of gC on Viral adsorption is independent of interactions between gC and heparin sulfate on cell surface. All in all, this study demonstrated that the gC of DPV can mediate viral adsorption in an HS-independent manner, which distinguish it from the gC of some other alpha-herpes viruses. Future studies will be required to identify the receptors involved in gC protein binding to cells. This work provides us a foundation for further studies of examining the roles of gC in the adsorption during duck plague virus infection.

关键词： duck plague virus (DPV) glycoprotein C (gC) heparan sulfate (HS) viral adsorption

Immunogenicity and protective efficacy of DHBV DNA vaccines expressing envelope and capsid fusion proteins in ducks delivered by attenuated Salmonella typhimurium

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Journal of Integrative Agriculture 2018年第4期17卷 928-939页

作者： liu Si-yang JIA Ren-yong LI Qing-qing feng Dai-shen SHEN Hao-yue YANG Cui WANG Ming-shu ZHU De-kang CHEN Shun liu ma-feng ZHAO Xin-xin YIN Zhong-qiong JING Bo CHENG An-chun Avian Disease Research Center Sichuan Agricultural University Chengdu 611130 P.R. China Institute of Preventive Veterinary Medicine Sichuan Agricultural University Chengdu 611130 P.R. China Key Laboratory of Animal Disease and Human Health of Sichuan Province Chengdu 611130 P.R.China

Duck hepatitis B virus(DHBV) shares many basic characteristics with hepatitis B virus(HBV) and is an attractive model for vaccine development. In this study, DHBV DNA vaccines were designed to express envelope and capsid fusion proteins to enhance the breadth of immune response in ducks. Attenuated Salmonella typhimurium(SL7207) was used as a carrier and adjuvant to boost the magnitude of immune response. Based on this strategy, novel DNA vaccines(SL7207-p VAX1-LC and SL7207-p VAX1-SC) were generated. Growth kinetics, genetic stabilities and relative transcription levels of the L, S and C genes introduced by these vaccine strains were measured before inoculation to guarantee safety and efficacy. The relative transcript levels of the CD4 and CD8 T genes and the antibody levels(Ig Y) in ducks receiving the vaccines were higher than those in single gene delivered groups. Additionally, the copy number of covalently closed circular DNA in hepatocytes after DHBV challenge also provided evidence that our fusion vaccines could enhance the protective efficiency against DHBV infection in ducks.

关键词： fusion DNA genes attenuated Salmonella typhimurium(SL7207) hepatitis B virus(HBV) duck hepatitis B virus(DHBV) oral immunization

The mechanisms of pH regulation on promoting volatile fatty acids production from kitchen waste

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Journal of Environmental Sciences 2025年第1期147卷 414-423页

作者： feng liu Tingting Wang Leiyu feng Yinguang Chen College of Environmental Science and Engineering Tongji UniversityShanghai 200092China TUS-Environmental Science and Technology Development Co. Ltd.Beijing 100084China

The anaerobic acid production experiments were conducted with the pretreated kitchen waste under pH *** results showed that pH 8 was considered to be the most suitable condition for acid production,especially for the formation of acetic acid and propionic *** average value of total volatile fatty acid at pH 8 was 8814 mg COD/L,1.5 times of that under blank *** average yield of acetic acid and propionic acid was 3302 mg COD/L and 2891 mg COD/L,*** activities of key functional enzymes such as phosphotransacetylase,acetokinase,oxaloacetate transcarboxylase and succinylcoA transferase were all *** further explore the regulatory mechanisms within the system,the distribution of microorganisms at different levels in the fermentation system was obtained by microbial sequencing,results indicating that the relative abundances of Clostridiales,Bacteroidales,Chloroflexi,Clostridium,Bacteroidetes and Propionibacteriales,which were great contributors for the hydrolysis and acidification,increased rapidly at pH 8 compared with the blank ***,the proportion of genes encoding key enzymes was generally increased,which further verified the mechanism of hydrolytic acidification and acetic acid production of organic matter under pH regulation.

关键词： Kitchen waste Acid fermentation Volatile fatty acids pH regulation Functional genes

The future of zebrafish research:highlights from the 18th International Zebrafish Conference

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Journal of Genetics and Genomics 2025年第1期52卷 1-3页

作者： Chenyang Bian Dongyuan ma feng liu State Key Laboratory of Membrane Biology Institute for Stem Cell and RegenerationInstitute of ZoologyUniversity of Chinese Academy of SciencesChinese Academy of SciencesBeijing 100101China School of Life Sciences Shandong UniversityQingdaoShandong 266000China

The International Zebrafish Conference(IZFC)has served as the premier gathering for the zebrafish research community for over 20 *** numerous plenaries and concurrent sessions,the conference showed the latest advancements in zebrafish research and provided attendees with insights into the most innovative research tools and technologies currently available.

关键词： fish gathering insight

Influence of insulin resistance on long-term outcomes in patients with coronary artery disease after sirolimus-eluting stent implantation

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Chinese Medical Journal 2010年第6期123卷 651-657页

作者： ZHAO Liang-ping LU An-kang SHEN Wei-feng liu Hai-feng ma Xiao-ye FAN Xiao-ming ZHANG Qi Department of Cardiology Ruijin HospitalShanghai Jiao Tong University School of MedicineShanghai 200025China

Background Insulin resistance（IR）is significantly associated with coronary artery disease and cardiovascular events in patients with or without type 2 diabetes *** study aimed to evaluate the influence of IR on long-term outcomes of patients undergoing percutaneous coronary intervention（PCI）with sirolimus-eluting stent（SES）*** A total of 467 consecutive patients undergoing SES-based PCI were divided into lR group（n=104）and non-IR group（n=363）.The patients were followed up for one *** rate of major adverse cardiac events（maCEs） including death, non-fatal myocardial infarction and recurrent angina pectoris was compared by the log-rank test,and the independent risk factors were identified by the Cox regression *** maCEs occurred more frequently,and cumulative survival rate was lower in the IR group than in the non-IR group during the follow-up （all P〈0.05）.IR was an independent risk factor for the occurrence of cardiac death and non-fatal myocardial infarction（OR=2176,95% CI=1.35-5.47,P=0.034）.Old age,diabetes,and multi-vessel disease were determinants for recurrent angina pectoris after PCI（P〈0.05）.Subgroup analysis revealed that IR（OR=3.35,95% CI=1.07-13.59,P=0.013）and multi-vessel disease（OR=2.19,95%CI=1.01-5.14,P=0.044）were independent risk predictors for recurrent angina pectoris in patients with diabetes after *** IR is associated with reduced maCE-free survival and remains an independent predictor for recurrent angina pectoris after PCI with SES implantation.

关键词： insulin resistance coronary artery disease sirolimus-eluting stent long-term prognosis

Differential-geometry-based multi-dimensional joint position-velocity estimation using Crab pulsar profile distortion

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Chinese Journal of Aeronautics 2025年第1期38卷 551-567页

作者： Jin liu Huanzi ZHANG Xiaolin NING Xin ma School of Electronic Information Wuhan University of Science and TechnologyWuhan 430081China School of Instrumentation and Optoelectronic Engineering Beihang UniversityBeijing 100191China Hefei National Laboratory Hefei 230088China

The traditional orbit determination method based on pulsar profile distortion can determine the six elements of the ***,the estimation accuracies of these methods are limited and the computational load of a six-dimensional search is *** solve this problem,the differential-geometry-based Multi-dimensional Joint Position-Velocity Estimation(MJPVE)using Crab pulsar profile distortion is proposed in this ***,through theoretical analysis,it is found that the pulsar profile distortion caused by the initial state error in some joint positionvelocity directions is very *** other words,the accuracies of estimation in these directions are very ***,the search dimension can be reduced,which in turn greatly reduces the computational ***,we construct the chi-squared function of the pulsar profile with respect to the estimation error in joint position-velocity direction and use differential geometry to find the joint position-velocity directions corresponding to different degrees of ***,we utilize the grid search based on directory folding in these joint position-velocity directions corresponding to large degrees of distortion to obtain the joint position-velocity *** experimental results show that compared with the grouping bi-chi-squared inversion method,MJPVE has high precision and extensive navigation information.

关键词： Joint Position-Velocity Estimation Pulsars Profile Distortion Orbit Determination Differential Geometry

Conversion-type cathode materials for high energy density solid-state lithium batteries

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Journal of Energy Chemistry 2025年第1期100卷 409-425页

作者： Yuhao ma Shihong Qing Hongyu liu Chuntao ma Yuan Yu Chuang Yu Liping Wang School of materials and Energy University of Electronic Science and Technology of ChinaChengdu 611731SichuanChina Zhejiang Provincial Key Laboratory of Fiber materials and manufacturing Technology Zhejiang Sci-Tech UniversityHangzhou 310018ZhejiangChina State Key Laboratory of Advanced Electromagnetic Engineering and Technology School of Electrical and Electronic EngineeringHuazhong University of Science and TechnologyWuhan 430074HubeiChina

Solid-state lithium batteries(SSLBs)are regarded as an essential growth path in energy storage systems due to their excellent safety and high energy *** particular,SSLBs using conversion-type cathode materials have received widespread attention because of their high theoretical energy densities,low cost,and *** the great progress in research and development of SSLBs based on conversiontype cathodes,their practical applications still face challenges such as blocked ionic-electronic migration pathways,huge volume change,interfacial incompatibility,and expensive processing *** review focuses on the advantages and critical issues of coupling conversion-type cathodes with solid-state electrolytes(SSEs),as well as state-of-the-art progress in various promising cathodes(e.g.,FeS_(2),CuS,FeF_(3),FeF_(2),and S)in ***,representative research on conversion-type solid-state full cells is discussed to offer enlightenment for their practical ***,the energy density exhibited by the S cathode stands out impressively,while sulfide SSEs and halide SSEs have demonstrated immense potential for coupling with conversion-type ***,perspectives on conversion-type cathodes are provided at the material,interface,composite electrode,and battery levels,with a view to accelerating the development of conversion-type cathodes for high-energy–density SSLBs.

关键词： Conversion-type cathode Lithium-free cathode Solid-state electrolyte Solid-state lithium battery High energy density