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Gene cloning,protein expression,and enzymatic characterization of a double-stranded RNA degrading enzyme in Apolygus lucorum

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Insect Science 2024年第1期31卷 119-133页

作者： Jie-Yu Zhang Jing Zhao Keyan Zhu-Salzman Qin-Qin Ji Yi-Ping Jiang Liu-Bin Xiao De-Jin Xu Guang-Chun Xu Lin-Quan Ge Yong-An Tan College of Plant Protection Yangzhou UniversityYangzhouJiangsu ProvinceChina Institute of Plant Protection Jiangsu Academy of Agricultural SciencesNanjingJiangsu ProvinceChina Department of Entomology Texas A&M UniversityCollege StationTXUSA Taizhou Customs of the People's Republic of China TaizhouJiangsu ProvinceChina

RNA interference(RNAi)is a powerful tool that post-transcriptionally silences target genes in eukaryotic ***,silencing efficacy varies greatly among different insect ***,we met with little success when attempting to knock down genes in the mirid bug Apolygus lucorum via dsRNA *** disappearance of double-stranded RNA(dsRNA)could be a potential factor that restricts RNAi ***,we found that dsRNA can be degraded in midgut fluids,and a dsRNase of ***(AldsRNase)was identified and *** alignment indicated that its 6 key amino acid residues and the Mg2+-binding site were similar to those of other insects’*** signal peptide and endonuclease non-specific domain shared high sequence identity with the brown-winged green stinkbug Plautia stali *** showed high salivary gland and midgut expression and was continuously expressed through the whole life cycle,with peaks at the 4th instar ecdysis in the whole *** purified AldsRNase protein obtained by heterologously expressed can rapidly degrade *** comparing the substrate specificity of AldsRNase,3 specific substrates(dsRNA,small interfering RNA,and dsDNA)were all degraded,and the most efficient degradation is ***,immunofluorescence revealed that AldsRNase was expressed in the cytoplasm of midgut *** cloning and functional study of AldsRNase,the enzyme activity and substrate specificity of the recombinant protein,as well as the subcellular localization of nuclease,the reason for the disappearance of dsRNA was explained,which was useful in improving RNAi efficiency in *** and related species.

关键词： Apolygus lucorum cloning dsRNase enzymatic activity protein expression

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Proteomic identification of ruminal epithelial protein expression profiles in response to starter feed supplementation in pre-weaned lambs

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Animal Nutrition 2021年第4期7卷 1271-1282页

作者： Daming Sun Shengyong Mao Weiyun Zhu Junhua Liu Jiangsu Key Laboratory of Gastrointestinal Nutrition and Animal Health Laboratory of Gastrointestinal MicrobiologyCollege of Animal Science and TechnologyNanjing Agricultural UniversityNanjing 210095Jiangsu ProvinceChina National Center for International Research on Animal Gut Nutrition Nanjing Agricultural UniversityNanjing 210095China National Experimental Teaching Demonstration Center of Animal Science Nanjing Agricultural UniversityNanjing 210095China

The pre sent study aimed to comparatively characterize the ruminal epithelial protein expression profiles in lambs fed ewe milk or milk plus starter diet using proteome *** new-born lambs were randomly divided into a group receiving ewe milk(M,n=10)and a group receiving milk plus starter diet(M+S,n=10).From 10 d old,M group lambs remained with the ewe and suckled ewe milk without receiving the starter *** lambs in the M+S group were separated from the ewe and received starter *** lambs were slaughtered at 56 d *** rumen epithelia samples(4 per group)were collected to characterize their protein expression profiles using proteomic *** analysis showed that 31 upregulated proteins and 40 downregulated proteins were identified in the rumen epithelium of lambs in response to starter diet *** results showed that starter feeding regulates a variety of biological processes in the epithelium,especially blood vessel development and extracellular matrix protein ***,the expression of proteins associated with synthesis and degradation of ketone bodies,butanoate metabolism,and citrate cycle signaling transduction pathway were upregulated in the group with starter diet supplementation,including 3-hydroxy-3-methylglutaryl coenzyme A synthase(HMGCS2,fold change[FC]=1.93),3-hydroxybutyrate dehydrogenase 1(BDH1,FC=1.91),and isocitrate dehydrogenase 1(IDH1,FC=8.12).The metabolic processes associated with ammonia detoxification and antioxidant stress were also affected by starter diet supplementation,with proteins,microsomal glutathione S-transferase 3(MGST3,FC=2.37)and IDH1,linked to the biosynthesis of glutamate and glutathione metabolism pathway being upregulated in the group with starter diet *** addition,starter feeding decreased the expression of Rasrelated protein rap-1 A(RAP1 A,FC=0.48)enriched in Rap1 signaling pathway,Ras signaling pathway,cyclic adenosine monophosphate(cAMP)signaling pa

关键词： Starter Rumen epithelium protein expression Proteomics Lamb

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Proteomics Profiling of Host Cell Response via protein expression and Phosphorylation upon Dengue Virus Infection

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Virologica Sinica 2019年第5期34卷 549-562页

作者： Meng Miao Fei Yu Danya Wang Yongjia Tong Liuting Yang Jiuyue Xu Yang Qiu Xi Zhou Xiaolu Zhao State Key Laboratory of Virology College of Life SciencesWuhan UniversityWuhan 430072China Hubei Key Laboratory of Cell Homeostasis College of Life SciencesWuhan UniversityWuhan 430072China Laboratory of RNA Virology Wuhan Institute of VirologyChinese Academy of ScienceWuhan 430071China

Dengue virus(DENV)infection is a worldwide public health *** date,the knowledge about the pathogenesis and progression of DENV infection is still *** global profiling based on proteomic analysis together with functional verification analysis is a powerful strategy to investigate the interplay between the virus and host *** the present study,quantitative proteomics has been applied to evaluate host responses(as indicated by altered proteins and modifications)in human cells(using K562 cell line)upon DENV-2 infection,as DENV-2 spreads most widely among all DENV *** analysis was performed to define differentially expressed proteins in the infected cells compared to the mock-control,and it revealed critical pathogen-induced changes covering a broad spectrum of host cellular compartments and *** also discovered more dramatic changes(>20%,160 regulated phosphoproteins)in protein phosphorylation compared to protein expression(14%,321 regulated proteins).Most of these proteins/phosphoproteins were involved in transcription regulation,RNA splicing and processing,immune system,cellular response to stimulus,and macromolecule *** blot analysis was also performed to confirm the proteomic *** roles of these altered proteins were *** present study provides valuable large-scale protein-related information for elucidating the functional emphasis of host cell proteins and their post-translational modifications in virus infection,and also provides insight and protein evidence for understanding the general pathogenesis and pathology of DENV.

关键词： Dengue virus(DENV) Quantitative proteomics Virus-host interaction protein expression protein phosphorylation

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Using the SELDI proteinChip System to Detect Changes in protein expression in Vero Cells after Infection

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中国病毒学 2007年第1期22卷 68-73页

作者： Zhi-jun LIU Bin WANG Zhi-yong YAN Xu-xia SONG Dong-meng QIAN Zhi-qiang BAI Department of Microbiology Qingdao Medical College and Key Laboratory of Medicine and Biotechnology of Qingdao Qingdao University Medical College Qingdao 266021China

人的疱疹单一的病毒(HSV-1 ) 1 引起美容，眼睛，并且 encephalitic 疾病并且与潜伏的感染和癌症被联系。这里，我们开发了由使用 SELDI 蛋白质薄片在在 vitro 有教养的 Vero 房间检测蛋白质表示的变化在蛋白质水平学习 HSV-1 感染的致... 详细信息

人的疱疹单一的病毒(HSV-1 ) 1 引起美容，眼睛，并且 encephalitic 疾病并且与潜伏的感染和癌症被联系。这里，我们开发了由使用 SELDI 蛋白质薄片在在 vitro 有教养的 Vero 房间检测蛋白质表示的变化在蛋白质水平学习 HSV-1 感染的致病的一个工具。在有为 12， 24 或 48 h 的 HSV-1 和文化的感染以后，房间被收获并且 lysed。IMAC3 数组被用于 SELDI-TOF-MS 在感染前后检测 proteomic 差别。薄片检测了一系列差别表示蛋白质山峰。有趣地，在 16 912 Da 的山峰和 17 581 Da 与 ISG15 的分子的团精确相应，它可以在感染的过程期间参予抗病毒的活动。因此，我们获得了的结果能用作一个基础学习在病毒和它的主人之间的 HSV-1 和相互作用的致病。另外，他们能为 HSV-1 感染的治疗在新治疗学的目标的发现帮助。关键词 SELDI 蛋白质薄片 - Vero 房间 - HSV-1 - 蛋白质表达式 CLC 数字 R373 基础项目：中国(30540075 ) 的国家自然科学基础；

关键词： SELDI protein Chip Vero Cells HSV-1 protein expression

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E protein Prokaryotic expression of Avian Infectious Bronchitis Virus

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Journal of Northeast Agricultural University(English Edition) 2008年第3期15卷 31-35页

作者： WEI Ping ZHANG Fang MING Xiaobo ZENG Xiangwei ZHU Yuqing WANG Lin College of Veterinary Medicine Northeast Agricultural University Harbin 150030 China College of Wild Life Resource Northeast Forestry University Harbin 150040 China

The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed into ***. BL21 and induced by IPTG. SDS-PAGE result showed that when objective protein fused with GST （about 20 ku）, the relative molecular mass of fusion protein was 38 ku. It indicated that objective protein was about 12.4 ku. The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.

关键词： avian infectious bronchitis virus (IBV) coronavirus small envelope protein (E) protein expression

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Encouraging Solution to the Problem of Synthesizing protein α-Thioester

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Chinese Journal of Chemistry 2024年第10期42卷 1114-1120页

作者： Xinliang Liu Zijun Gao Jie Zhao Farong Ye Ping Huang Ping Wang Center for Chemical Glycobiology Shanghai Key Laboratory for Molecular Engineering of Chiral DrugsFrontiers Science Center for Transformative MoleculesSchool of Chemistry and Chemical EngineeringZhangjiang Institute for Advanced StudyShanghai JiaoTongUniversityShanghai 200240China Shenzhen Research Institute of Shanghai Jiao Tong University ShenzhenGuangdong 518057China

Expressed protein ligation(EPL)provides a powerful tool to access large-size proteins with precise *** methods for constructing the critical protein thioester for EPL have predominantly relied on the recombinant intein fusion expressed in Escherichia coli(***).Despite its powerful applications,the expression of thioester derived from eukaryotic protein in *** inherently suffers from its limited solubility,the inactivity of intein,premature hydrolysis and low *** overcome these obstacles,we present herein the facile one-flask synthesis of inaccessible proteinα-thioester via a SUMO-protein-intein(SPI)sandwich *** utility of SUMO enhances the protein fusion yield and solubility,prevents premature hydrolysis and simplifies the purification *** inaccessible protein thioester with internal Cys residues can be readily produced and is compatible with the EPL-desulfurization protocol used to prepare complex proteins,which is otherwise difficult to obtain using traditional *** utility has been highlighted through the synthesis of human granulocyte colony-stimulating factor(G-CSF).

关键词： Desulfurization Glycoproteins Glycopeptides protein expression Peptide thioester

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Oligomerization of drug transporters:Forms,functions,and mechanisms

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Acta Pharmaceutica Sinica B 2024年第5期14卷 1924-1938页

作者： Chunxu Ni Mei Hong College of Life Sciences South China Agricultural UniversityGuangzhou 510642China Guangdong Provincial Key Laboratory of protein Function and Regulation in Agricultural Organisms South China Agricultural UniversityGuangzhou 510642China

Drug transporters are essential players in the transmembrane transport of a wide variety of clinical *** broad substrate spectra and versatile distribution pattern of these membrane proteins infer their pharmacological and clinical *** our accumulating knowledge on the three-dimensional structure of drug transporters,their oligomerization status has become a topic of intense study due to the possible functional roles carried out by such kind of post-translational modification(PTM).In-depth studies of oligomeric complexes formed among drug transporters as well as their interactions with other regulatory proteins can help us better understand the regulatory mechanisms of these membrane proteins,provide clues for the development of novel drugs,and improve the therapeutic *** this review,we describe different oligomerization forms as well as their structural basis of major drug transporters in the ATP-binding cassette and solute carrier superfamilies,summarize our current knowledge on the influence of oligomerization for protein expression level and transport function of these membrane proteins,and discuss the regulatory mechanisms of ***,we highlight the challenges associated with the current oligomerization studies and propose some thoughts on the pharmaceutical application of this important drug transporter PTM.

关键词： Drug development Drug transporters Oligomerization protein expression protein–protein interaction Regulatory mechanism Structural basis Transport function

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expression of non-structural protein NS3 gene of Bombyx mori densovirus (China isolate)

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Journal of Genetics and Genomics 2008年第4期35卷 239-244页

作者： Huijuan Yin Qin Yao Zhongjian Guo Fang Bao Wei Yu Jun Li Keping Chen Institute of Life Sciences Jiangsu University 301# Xuefu Road Zhenjiang 212013 China

The invertebrate parvovirus Bombyx mori Densonucleosis Virus type 3 （China isolate）, named BmDNV-3, is a kind of bidensovirus. It is a new type of virus with unique replication mechanisms. To investigate the effects of the NS3 gene during viral DNA replication, a pair of primers was designed for amplifying NS3 gene of Bombyx mori densovirus （China isolate）. Gene NS3 amplified was cloned into a prokaryotic expression vector pET-30a and the donor plasmid pFastBacHTe, respectively. The NS3 protein was expressed in Escherichia coli BL21. The pFastBacHTe-NS3 was transformed to E. coli DHIOBac. The recombinant bacmid baculoviruses （rBacmid-EGFP-NS3） isolated from the white colonies were transfected into BmN-4 cells using a transfection reagent. BraN-4 cells were infected with recombinant virus to express fusion proteins. The expression of fusion protein around 30 kDa in E. coli BL21 was identified by SDS-PAGE, Western blotting, and mass spectrometry. The expressed NS3 protein by B. mori nucleopolyhedrovirus bacmid system was confirmed by Western blotting using an anti-NS3 polyclonal antibody. And about 45 kDa protein was found. The expressed fusion protein was smaller than the expected size of EGFP-NS3, 55 kDa. Western blotting analysis indicated that EGFP-NS3 protein was expressed in infected larvae with smaller molecular size.

关键词： BmDNV NS3 protein expression

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expression and characterization of a new valosin-containing protein from silkworm

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Insect Science 2012年第5期19卷 549-558页

作者： Ping Chen Ruo-Ran Wang Zhen Peng Qiong Liu Jia-Zuan Ni Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun College of Life Sciences Shenzhen University Shenzhen Guangdong Province Graduate University of the Chinese Academy of Sciences Chinese Academy of Sciences Changchun China

Valosin-containing protein （VCP） is a type-II adenosine triphosphatase （ATPase） wih extensive biological function in organisms. Silkworm is the second in- sect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP-like gene was amplified from the fat body of silkworm follow- ing genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction （RT-PCR） and 3＇-RACE （rapid amplification of complementary DNA ends） methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP-like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP-like protein with ATPase activity. The expression of silkworm VCP-like protein was also confirmed by Western blotting and mass spectromet- ric analyses. Distribution of the VCP-like gene in various tissues of the silkworm was also studied by real-time PCR. Results showed that the messenger RNA （mRNA） of VCP-like protein is widely expressed in fat body, reproductive organs （testis or ovary）, silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP-like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP-like protein.

关键词： ATPase activity Bombyx mori gene cloning protein expression real-timPCR valosin-containing protein

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Cell apoptosis in perihematomal brain regions and expression of Caspase-3 protein in patients with hypertensive intracerebral hemorrhage

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Neural Regeneration Research 2006年第5期1卷 420-423页

作者： Xinqing Zhang Xiaoliang Yin Kun Zhang Zhimin Zhang Hui Cai Honglan Xu Department of Neurosurgery Beijing Chuiyangliu Hospital Beijing 100022 China Department of Pathology Beijing Chuiyangliu Hospital Beijing 100022 China

BACKGROUND： In patients with intracerebral hemorrhage （ICH）, besides the space-occupying effect of hematoma, hematomal component also causes the pathological changes of perihematomal region, including the death of neurons and glial cells, vasogenic brain edema, the destruction of blood brain barrier and so on, which are the important factors to influence the prognosis of patients. Therefore, it is necessary to perform further investigation and study on the pathological characteristics of injury and death of brain nerve cells. OBJECTIVE： To observe the pathological changes of apoptosis and Caspase-3 expression in perihematomal brain regions in patients with hypertensive ICH （HICH） in different stages of onset, and analyze their relationship. DESIGN ： Case-control observation. SETTING： Departments of Neurosurgery and Pathology of Beijing Chuiyangliu Hospital.PARTICIPANTS： Totally 19 patients with HICH, including 12 male, 7 female, aged （58.3±12.8） ranging from 49 to 78 years, whose mean volume of hemorrhage was （48.6±16.4） mL, were involved . All the cases conformed to the diagnostic criteria of intracerebral hemorrhage formulated in the 4^th National Cerebrovascular Diseases Conference and were confirmed by skull CT scanning. Informed consents of operation and specimens were obtained from the patients and relatives. METHODS： ①Patients with HICH who had undergone surgical evacuation of an intracerebral hematoma by traverse temporal lobe approach in the Department of Neurosurgery, Beijing Chuiyangliu Hospital from January 2004 to July 2005 were involved. Nineteen specimens of brain tissue from perihematomal region of HICH patients in different phases served as patient group. Five specimens were obtained from distant regions of patients in the super-early phase as the control group. According to the time from onset to operation, the 19 cases were divided into 3 groups： 6 cases in super-early phase（onset 〈 8 hours）, 8 cases in early phase （onset about 8 to 24 h

关键词： intracerebral hemorrhage cell apoptosis correlation analysis pathological characteristics immunohistochemical staining visual field signal transduction diagnostic criteria blood brain barrier protein expression measurement data temporal lobe nerve cells CT scanning brain edema

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