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Construction of a universal recombinant expression vector that regulates the expression of human lysozymein milk

Shen LIU, Shengzhe SHANG, Xuezhen YANG, Huihua ZHANG, Dan LU, Ning LI

《农业科学与工程前沿（英文）》 2018年 第5卷 第3期   页码 382-389 doi: 10.15302/J-FASE-2018211

摘要：

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (mWAP) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (hLZ) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb mWAP 5′ promoter, 4.8 kb hLZ genomic DNA, and 8.0 kb mWAP 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L−1, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.

关键词： BAC recombinant methods     gene expression     human lysozyme     transgenic mice     milk expression vector    

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Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Dan LU,Shengzhe SHANG,Shen LIU,Ying WU,Fangfang WU,Tan TAN,Qiuyan LI,Yunping DAI,Xiaoxiang HU,Yaofeng ZHAO,Ning LI

《农业科学与工程前沿（英文）》 2014年 第1卷 第3期   页码 179-184 doi: 10.15302/J-FASE-2014020

摘要： Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organophosphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the gene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF-hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76±12 to 159±28 mg·L . Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE. Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.

关键词： recombinant human butyrylcholinesterase (rhBCHE)     human lactoferrin bacterial artificial chromosome (hLF BAC)     transgenic mice     milk    

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Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Youguo HAO, Min ZHANG, Jinzhi XU, Bitao BU, Jiajun WEI

《医学前沿（英文）》 2009年 第3卷 第2期   页码 141-147 doi: 10.1007/s11684-009-0041-6

摘要： Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome-to- -Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed Lentivirus encoding Rab9. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The Lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1- , pPACKH1- and pVSV- into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 hours, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with Lentivirus encoding Rab9, which evidenced a satisfactory increasing effect of this virus. Administration of Lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulating the death rate of Purkinje neurons. It is concluded that the packaging of Lentivirus encoding Rab9 was successful. Lentivirus encoding Rab9 can increase the expression of Rab9 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

关键词： Rab9     lentivirus     gene therapy     gene transfer    

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Construction of eukaryotic expression vector of human arresten gene and its secreted expression in HEK

Wei LI PhD , Siming GUAN MM , Zifang SONG PhD , Qichang ZHENG PhD , Jun XIONG PhD , Dan SHANG PhD , Xiaogang SHU PhD ,

《医学前沿（英文）》 2009年 第3卷 第3期   页码 297-302 doi: 10.1007/s11684-009-0058-x

摘要： The eukaryotic expression vector of human arresten gene was constructed and its secretive expression human embryonic kidney (HEK 293) cells was detected. Human arresten gene was amplified from recombinant plasmid pGEM-Arr by polymerase chain reaction (PCR), and then digested with restriction endonucleases I and I. The target fragment was inserted into the I and I restriction sites of eukaryotic expression vector pSecTag2 to construct pST-AT. Restriction analysis and DNA sequencing indicated that the arresten gene was successfully inserted into pSecTag2. The recombinant plasmid was subsequently transfected into HEK 293 cells with LipofectAMINETM2000 Reagent, and the expression of the target gene was detected. RT-PCR revealed that the mRNA of the target gene was transcribed in the transfected HEK 293 cells. Western Blot analysis verified that the recombinant protein in supernatants was correct. The supernatants of transfected cells were prepared, and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was carried out to assess their effect on the proliferation of human umbilical vein endothelial cells, which showed that the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells . These results provided a solid foundation to explore the usage of arresten in tumor anti-angiogenic gene therapy.

关键词： angiogenesis inhibitor     arresten     eukaryotic expression     HEK 293 cells     endothelial cells    

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Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

Yuanyuan LIN MD, Xiaoming CHENG MS, Yuhu SONG MD, Peiyuan LI MD, Ying CHANG MM, Jinjian YAO MD, Jusheng LIN MD, PhD, Li ZHOU PhD, Leiming XU PhD,

《医学前沿（英文）》 2009年 第3卷 第4期   页码 396-402 doi: 10.1007/s11684-009-0056-z

摘要： Virus tagged with green fluorescent protein (GFP) contributes to the visualization and study of the virus in living cells. However, the hepatitis B virus (HBV) particle, which is a compact virion with limited internal space, cannot be incorporated with GFP tag as a large fragment. It was recently reported that protein genetically inserted with a smaller size tetracysteine (TC) tag could be specially labeled by a biarsenical fluorescent dye in living cells. In this study, we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion. TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis. Western blot and enzyme-linked immunosorbent assay (ELISA) analysis showed that the TC-tagged core protein, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) could be expressed in cells transfected with the recombinant HBV vector, which is similar to the cells transfected with wild-type HBV vector. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree, but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins. Taken together, the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to be fluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.

关键词： hepatitis B virus     vector     tetracysteine tag     core protein    

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Perfluoroalkane acids in human milk under the global monitoring plan of the Stockholm Convention on Persistent

《环境科学与工程前沿（英文）》 2022年 第16卷 第10期 doi: 10.1007/s11783-022-1541-8

摘要：

● Perfluorooctanesulfonic acid and perfluorooctanoic acid highest in human milk.

关键词： Human biomonitoring     Human breast milk     LC-MS/MS analysis     Lifestyle parameters    

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Global warming potential associated with Irish milk powder production

William Finnegan, Jamie Goggins, Aksana Chyzheuskaya, Xinmin Zhan

《环境科学与工程前沿（英文）》 2017年 第11卷 第3期 doi: 10.1007/s11783-017-0949-z

摘要： Climate change is an ever growing issue and a major concern worldwide. Both producers and processors need to address the issue now by reducing their carbon footprint. Additionally, if Ireland is to meet their climate and energy targets, as outlined in Food Harvest 2020, which outlines a range of objectives for the Irish agricultural sector, the efficient use of resources and fuels within the industry will need to be increased. In Ireland, agriculture accounts for 29.2% of the total greenhouse gas emissions (58.5 million tonnes CO eq). Therefore, in this paper, a single agri-food product, milk powder, is examined in order to estimate the global warming potential (GWP) associated with its manufacture using life cycle assessment. A cradle-to-processing factory gate analysis, which includes raw milk production, raw milk transportation to the processing factory, its processing into each product and product packaging, is assessed in this study using data collected circa 2013. The factories surveyed processed approximately 24% of the total raw milk processed in the Republic of Ireland in 2013, which was 5.83 billion liters. The average total GWP associated with the manufacture of milk powder is 9.731 kg CO eq·kg milk powder, which has a standard deviation of 2.26 kg CO eq·kg milk powder, for the life cycle stages analyzed in this study. The most significant contributor to GWP is raw milk production (84%), followed by dairy processing (14%), with the remainder of the life cycle stages contributing approximately 2%.

关键词： Dairy     Global warming potential     Ireland     Life cycle assessment     Milk powder     Milk production    

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Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Feng JIANG PhD , Xuezhen WANG PhD , Zheng XUE MD , Suming ZHANG PhD , Siyu FANG BM , Min ZHANG MD, PhD ,

《医学前沿（英文）》 2009年 第3卷 第3期   页码 287-291 doi: 10.1007/s11684-009-0050-5

摘要： In order to explore the role of cyclin-dependent kinase 4 (cdk4) in neurodegenerative diseases, lentiviral-delivered RNA interference (RNAi) was used to silence the expression of the murine cdk4 gene . Three cdk4-shRNAs of mouse and a negative sequence were designed. After synthesis and annealing, double strand oligonucleotides were cloned into a linearized pSIH1-H1-copGFP shRNA vector. It was confirmed by polymerase chain reaction (PCR) and sequencing that three pairs of cdk4-shRNAs and a negative shRNA were correctly inserted into the pSIH1-H1-copGFP vector. The above recombinants were transfected by lipofectamine into BV-2 cells. The gene silencing efficacy rates of the 3 targets were compared by Western blotting. The cdk4-siRNA2 was the most effective in silencing cdk4. The optimized pSIH1-cdk4-siRNA2 and pSIH-negative-siRNA were co-transfected into 293T cells with the lentiviral packaging plasmids respectively. The culture supernatant was harvested and condensed at the 24th and 48thh after transfection. Interference efficiency of the lentivirus expressing cdk4-siRNA was determined by reverse transcriptase-PCR (RT-PCR) and Western blotting in BV-2 cells. Lentivector-mediated RNAi could efficiently down-regulate the expression of the murine cdk4 gene , which provides a potential tool for studying and treating cdk4-related diseases.

关键词： cyclin-dependent kinase 4     RNA interference     plasmid     lentiviral vector    

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UsingKinect for real-time emotion recognition via facial expressions

Qi-rong MAO,Xin-yu PAN,Yong-zhao ZHAN,Xiang-jun SHEN

《信息与电子工程前沿（英文）》 2015年 第16卷 第4期   页码 272-282 doi: 10.1631/FITEE.1400209

摘要： Emotion recognition via facial expressions (ERFE) has attracted a great deal of interest with recent advances in artificial intelligence and pattern recognition. Most studies are based on 2D images, and their performance is usually computationally expensive. In this paper, we propose a real-time emotion recognition approach based on both 2D and 3D facial expression features captured by Kinect sensors. To capture the deformation of the 3D mesh during facial expression, we combine the features of animation units (AUs) and feature point positions (FPPs) tracked by Kinect. A fusion algorithm based on improved emotional profiles (IEPs) and maximum confidence is proposed to recognize emotions with these real-time facial expression features. Experiments on both an emotion dataset and a real-time video show the superior performance of our method.

关键词： Kinect     Emotion recognition     Facial expression     Real-time classification     Fusion algorithm     Support vector machine (SVM)    

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CH4 and N2O emissions from double-rice cropping system as affected by Chinese milk

Heshui XU,Dengyun LI,Bo ZHU,Kai ZHANG,Yadong YANG,Chen WANG,Ying JIANG,Zhaohai ZENG

《农业科学与工程前沿（英文）》 2017年 第4卷 第1期   页码 59-68 doi: 10.15302/J-FASE-2017137

摘要： Chinese milk vetch (CMV) and rice straw (RS) were incorporated into soil to substitute for synthetic N fertilizers and to maintain soil fertility. However, little is known about the integrated impacts of CMV and RS incorporation on CH and N O emissions in double-rice cropping systems in southern China. A field experiment was conducted to estimate the integrated impacts of CMV and RS incorporation in the early- and late-rice seasons on CH and N O emissions. All treatments received uniform N inputs, 6%–37% of which was replaced by CMV and RS crop residue. CMV and/or RS incorporation produced equivalent or slightly more grain yield, while reducing N O emissions by 3%–43%. However, both CMV and RS incorporation increased CH emissions. Annual CH emissions ranged from 321 to 614 kg·hm from CMV and RS amendment treatments, which were 1.5–2.9 times higher than that from synthetic N. Compared with single synthetic N fertilizer, incorporation of CMV and/or RS increased GWP and yield-scaled GWP by 45%–164% and 45%–153%, respectively. Our results demonstrate CMV and RS amendments replacing N fertilizer, maintained stable yield, mitigated N O emission, but enhanced CH emission. Further study is needed on crop residue management in double-cropping rice systems.

关键词： Chinese milk vetch     CH4     double-rice cropping system     grain yield     N2O     rice straw    

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Liquefaction prediction using support vector machine model based on cone penetration data

Pijush SAMUI

《结构与土木工程前沿（英文）》 2013年 第7卷 第1期   页码 72-82 doi: 10.1007/s11709-013-0185-y

摘要： A support vector machine (SVM) model has been developed for the prediction of liquefaction susceptibility as a classification problem, which is an imperative task in earthquake engineering. This paper examines the potential of SVM model in prediction of liquefaction using actual field cone penetration test (CPT) data from the 1999 Chi-Chi, Taiwan earthquake. The SVM, a novel learning machine based on statistical theory, uses structural risk minimization (SRM) induction principle to minimize the error. Using cone resistance ( ) and cyclic stress ratio ( ), model has been developed for prediction of liquefaction using SVM. Further an attempt has been made to simplify the model, requiring only two parameters ( and maximum horizontal acceleration ), for prediction of liquefaction. Further, developed SVM model has been applied to different case histories available globally and the results obtained confirm the capability of SVM model. For Chi-Chi earthquake, the model predicts with accuracy of 100%, and in the case of global data, SVM model predicts with accuracy of 89%. The effect of capacity factor ( ) on number of support vector and model accuracy has also been investigated. The study shows that SVM can be used as a practical tool for prediction of liquefaction potential, based on field CPT data.

关键词： earthquake     cone penetration test     liquefaction     support vector machine (SVM)     prediction    

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A review of traditional and novel detection techniques for melamine and its analogues in foods and animal feed

Mengshi LIN,

《化学科学与工程前沿（英文）》 2009年 第3卷 第4期   页码 427-435 doi: 10.1007/s11705-009-0244-z

摘要： Melamine, a nitrogen-rich chemical, has received much attention in recent years due to a series of highly publicized food safety incidents, including 2007 pet food recalls in North America and 2008 melamine contamination in milk, infant formula, and other milk-derived products in China. Current analytical methods for testing melamine are time-consuming, expensive, and labor-intensive. Therefore, there is an increasing interest in the food science and analytical chemistry field to develop simple, quick, sensitive, and cost-effective methods for detection of melamine and its analogues (e.g., cyanuric acid, melamine cyanurate, ammelide, and ammeline) in food ingredients, processed food, and animal feed. This review aims to summarize traditional and novel analytical techniques that have been used or show great potential to detect, characterize, and quantify melamine and its analogues in foods and animal feed.

关键词： milk-derived     Melamine     labor-intensive     cyanurate     time-consuming    

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A modified neural learning algorithm for online rotor resistance estimation in vector controlled induction

A. CHITRA,S. HIMAVATHI

《能源前沿（英文）》 2015年 第9卷 第1期   页码 22-30 doi: 10.1007/s11708-014-0339-1

摘要： Online estimation of rotor resistance is essential for high performance vector controlled drives. In this paper, a novel modified neural algorithm has been identified for the online estimation of rotor resistance. Neural based estimators are now receiving active consideration as they have a number of advantages over conventional techniques. The training algorithm of the neural network determines its learning speed, stability, weight convergence, accuracy of estimation, speed of tracking and ease of implementation. In this paper, the neural estimator has been studied with conventional and proposed learning algorithms. The sensitivity of the rotor resistance change has been tested for a wide range of variation from -50% to+50% on the stability of the drive system with and without estimator. It is quiet appealing to settle with optimal estimation time and error for the viable realization. The study is conducted extensively for estimation and tracking. The proposed learning algorithm is found to exhibit good estimation and tracking capabilities. Besides, it reduces computational complexity and, hence, more feasible for practical digital implementation.

关键词： neural networks     back propagation (BP)     rotor resistance estimators     vector control     induction motor    

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Unconfined compressive strength prediction of soils stabilized using artificial neural networks and support vector

Alireza TABARSA, Nima LATIFI, Abdolreza OSOULI, Younes BAGHERI

《结构与土木工程前沿（英文）》 2021年 第15卷 第2期   页码 520-536 doi: 10.1007/s11709-021-0689-9

摘要： This study aims to improve the unconfined compressive strength of soils using additives as well as by predicting the strength behavior of stabilized soils using two artificial-intelligence-based models. The soils used in this study are stabilized using various combinations of cement, lime, and rice husk ash. To predict the results of unconfined compressive strength tests conducted on soils, a comprehensive laboratory dataset comprising 137 soil specimens treated with different combinations of cement, lime, and rice husk ash is used. Two artificial-intelligence-based models including artificial neural networks and support vector machines are used comparatively to predict the strength characteristics of soils treated with cement, lime, and rice husk ash under different conditions. The suggested models predicted the unconfined compressive strength of soils accurately and can be introduced as reliable predictive models in geotechnical engineering. This study demonstrates the better performance of support vector machines in predicting the strength of the investigated soils compared with artificial neural networks. The type of kernel function used in support vector machine models contributed positively to the performance of the proposed models. Moreover, based on sensitivity analysis results, it is discovered that cement and lime contents impose more prominent effects on the unconfined compressive strength values of the investigated soils compared with the other parameters.

关键词： unconfined compressive strength     artificial neural network     support vector machine     predictive models     regression    

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Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

LIU Hongchun, CAO Zhongwei, JIN Jianjun, WANG Jiyao

《医学前沿（英文）》 2008年 第2卷 第2期   页码 166-170 doi: 10.1007/s11684-008-0030-1

摘要： This study aims to demonstrate that blocking the receptor-interacting protein2 (Rip2) expression can decrease inflammatory cytokine production by macrophage and protect mice from endotoxin lethality. Murine Rip2 small interfering RNA (siRNA) plasmids were constructed and transfected into macrophage and Rip2 expression was detected with reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cell proliferation was assayed with MTT. TNF-? concentration was assayed with ELISA and high-mobility group box 1 protein (HMGB1) level with semi-quantitative western blot after lipopolysaccharide (LPS) stimulation. LPS challenge was given after the plasmids were injected into mice and the survival rate was calculated. Rip2 siRNA plasmid could block the mRNA and protein expression of Rip2 and promote cell proliferation. Blocking Rip2 could attenuate LPS-induced TNF-? and HMGB1 production. The HMGB1 expression in the liver decreased to (40.21 ± 11.03) pg/g, and serum TNF-? level decreased to (300.43 ± 59.26) ng/L ( < 0.05). The survival rate of mice from endotoxemia was also improved ( < 0.05). The results demonstrate that Rip2 siRNA plasmid can block the expression of Rip2, decrease the production of TNF-? and HMGB1 and protect mice from fatal endotoxemia.
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标题 作者 时间 类型 操作

Construction of a universal recombinant expression vector that regulates the expression of human lysozymein milk

Shen LIU, Shengzhe SHANG, Xuezhen YANG, Huihua ZHANG, Dan LU, Ning LI

期刊论文

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Dan LU,Shengzhe SHANG,Shen LIU,Ying WU,Fangfang WU,Tan TAN,Qiuyan LI,Yunping DAI,Xiaoxiang HU,Yaofeng ZHAO,Ning LI

期刊论文

Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Youguo HAO, Min ZHANG, Jinzhi XU, Bitao BU, Jiajun WEI

期刊论文

Construction of eukaryotic expression vector of human arresten gene and its secreted expression in HEK

Wei LI PhD , Siming GUAN MM , Zifang SONG PhD , Qichang ZHENG PhD , Jun XIONG PhD , Dan SHANG PhD , Xiaogang SHU PhD ,

期刊论文

Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

Yuanyuan LIN MD, Xiaoming CHENG MS, Yuhu SONG MD, Peiyuan LI MD, Ying CHANG MM, Jinjian YAO MD, Jusheng LIN MD, PhD, Li ZHOU PhD, Leiming XU PhD,

期刊论文

Perfluoroalkane acids in human milk under the global monitoring plan of the Stockholm Convention on Persistent

期刊论文

Global warming potential associated with Irish milk powder production

William Finnegan, Jamie Goggins, Aksana Chyzheuskaya, Xinmin Zhan

期刊论文

Lentivector-mediated RNAi efficiently downregulates expression of murine cdk4 gene

Feng JIANG PhD , Xuezhen WANG PhD , Zheng XUE MD , Suming ZHANG PhD , Siyu FANG BM , Min ZHANG MD, PhD ,

期刊论文

UsingKinect for real-time emotion recognition via facial expressions

Qi-rong MAO,Xin-yu PAN,Yong-zhao ZHAN,Xiang-jun SHEN

期刊论文

CH4 and N2O emissions from double-rice cropping system as affected by Chinese milk

Heshui XU,Dengyun LI,Bo ZHU,Kai ZHANG,Yadong YANG,Chen WANG,Ying JIANG,Zhaohai ZENG

期刊论文

Liquefaction prediction using support vector machine model based on cone penetration data

Pijush SAMUI

期刊论文

A review of traditional and novel detection techniques for melamine and its analogues in foods and animal feed

Mengshi LIN,

期刊论文

A modified neural learning algorithm for online rotor resistance estimation in vector controlled induction

A. CHITRA,S. HIMAVATHI

期刊论文

Unconfined compressive strength prediction of soils stabilized using artificial neural networks and support vector

Alireza TABARSA, Nima LATIFI, Abdolreza OSOULI, Younes BAGHERI

期刊论文

Blockage of receptor-interacting protein 2 expression by small interfering RNA in murine macrophages

LIU Hongchun, CAO Zhongwei, JIN Jianjun, WANG Jiyao

期刊论文