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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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Morphological and pathologic changes of experimental chronic atrophic gastritis (CAG)and the regulating mechanism of protein expression in rats

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2006年第8期7卷 634-640页

作者： WANG Liang-jing CHEN Shu-jie CHEN Zhe CAI Jian-ting SI Jian-min Department of Gastroenterology the Second Affiliated Hospital School of Medicine Zhejiang University Hangzhou 310009 China Lab of Digestive Disease Clinical Institute Sir Run Run Shaw Hospital School of Medicine Zhejiang University Hangzhou 310016 China

Objective： To study the pathologic change and molecular regulation in cell proliferation and apoptosis of gastric mucosa in rats with chronic atrophic gastritis （CAG）, and evaluate the possible mechanisms. Methods： Rats were administered with 60% alcohol or 2% salicylate sodium, 20 mmol/L deoxycholate sodium and 0.1% ammonia water to establish chronic atrophic gastritis （CAG） models. The gastric specimens were prepared for microscopic view with hematoxylin and eosin （H-E） and alcian blue （A-B） stain. The number of infiltrated inflammatory cells, the thickness of the mucosa gland layer （μm） and the number of gastric glands were calculated. The damage of barrier in mucosa with erosion or ulceration, and the thickness of mucin were examined by scanned electron microscope （SEM）. The levels of PGE2, EGF （epiderminal growth factor） and gastrin in the serum were measured with radioimmunoassay or ELISA method. The immunohistochemistry method was used to observe the number of G cells, the expression of protein of EGFR （EGF receptor）, C-erbB-2, p53, p6 and bcl-2 in gastric tissue. Results： Under SEM observation, the gastric mucosa was diffused erosion or ulceration and the thickness of mucin was decreased. Compared with normal rats, the grade of inflammatory cell infltration in CAG rats was elevated, whereas the thickness and number of gastric gland were significantly lower （P〈0.05）. Compared with normal level of （0.61±0.28） μg/L, EGF in CAG （2.24±0.83） μg/L was significantly higher （P〈0.05）. The levels of PGEz and gastrin in serum were significantly lower in CAG rats than that in normal rats （P〈0.05）. Immunohistochemistry detection showed that the number of G cell in antrum was lower in CAG group （P〈0.05）. Imrauno-stain showed EGFR protein expression in the basal and bilateral membrane, and the cytoplasma in atrophic gastric gland, while negative expression was observed in normal gastric epithelial cells. Positive staining of p53 and p 16 protein was localized in the nucleus

关键词： Chronic atrophic gastritis (CAG) Rat protein expression

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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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protein expression of Goat Follicular Fluid （GFF）

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Journal of Agricultural Science and Technology(A) 2012年第7期2卷 917-920页

作者： Sri Wahjuningsih Mochammad Sasmito Djati Faculty of Animal Husbandry University of Brawoaya Malang 65142 Indonesia Faculty of Sciences University of Brawo'aya Malang 65142 Indonesia

Oocyte maturation process is very complex. Until now synthesis and protein function as well as pathway mechanism in oocyte maturation process are not yet well understood. The mechanism of goat follicular fluid （GFF） in maturation in vitro medium needs to be deeply studied before it is implemented widely. The aim of the research was analyzing the GFF to know protein expression of ERK1, ERK 2, p90rsk, Cyclin-Bl and cdc25A by Western Blotting method. GFF collection by aspiration from small follicle （〈 4 mm） and big follicle （4-8 mm） by using needle with spuit size of 18 G. It was concluded that there was protein expression of ERK2 and p90rsk, at 42 kDa and 22 kDa respectively.

关键词： protein expression follicular fluid goat ERK2 p90rsk.

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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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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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An Improved Strategy for Efficient expression and Purification of Soluble HIV-1 Tat protein in ***

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Virologica Sinica 2009年第6期24卷 518-528页

作者： Shi-meng ZHANG Rong FAN Tian-yi YANG Yi SUN Jing-yun LI Qin-zhi XU Ping-kun ZHOU The Department of Radiation Toxicology and Oncology Beijing Institute of Radiation Medicine Beijing100850 China Institute of Microbiology and Epidemiology Beijing 100071 China

Although the endogenous function of Tat has been elucidated in the past twenty years, the study of its exogenous activity has been hampered due to the difficulty of large scale preparation of the active Tat protein. To express the full-length Tat protein in ***, the tat gene was cloned from an HIV infected patient by overlapping PCR. Rare codon usage analysis showed that rare *** codons, especially consecutive rare codons for Arg, account for 14% (14 of 101) rare *** codons in the tat gene. The expression of the HIV-1 tat gene was verified to be very poor in strain BL21 (DE3) due to the abundance of rare codons; however, tat gene expression was found to be very efficient in the host strain of Rosetta-gami B (DE3), which was supplemented with six rare tRNAs for Arg, Leu, Ile and Pro. Subsequent purification revealed that the proteins are soluble and unusually, the tagged Tat can form dimers independent of cystine disulfide bonds. The purity, integrity and molecular weight of the Tat protein were demonstrated by MALDI-TOF mass spectrometry. Reporter gene activating assay was further confirmed by investigating the transactivation activity of the recombinant Tat protein. Our improved strategy for efficient high level expression and purification of soluble Tat protein has paved the way to fully investigate its exogenous function.

关键词： HIV tat gene E.coli protein expression Codon usage

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