PEST MANAGEMENT SCIENCE
·
2019
·Vol. 76
·期 4
·1363-1370
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IF 3.255
Chengfeng Lei,Shili Yang,Wenxiao Lei,Innocent Nyamwasa,Jia Hu,Xiulian Sun
BACKGROUND Baculoviruses provide long-lasting control of crop pests and are harmless to humans and non-target animals, making them attractive bioinsecticides. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has a wide-host range and is one such commercial bioinsecticide, but its low infectivity to older larvae and less-sensitive species precludes its large-scale application. We sought to improve the infectivity of AcMNPV. RESULTS Two enhancing factors, the truncated enhancin from Agrotis segetum granulovirus and GP37 from Cydia pomonella granulovirus, were expressed in fusion with the N-terminal and middle domain of the polyhedrin envelope protein of AcMNPV. Western blotting and immunoelectron microscopy analysis indicated that the enhancing factors were expressed on the occlusion bodies of the resulting AcMNPV variants. Bioassays showed that the median lethal doses of the recombinant viruses were 3.9-fold to 7.4-fold lower than those of the wild-type virus against the second and fourth instar of Spodoptera exigua larvae. The yields of occlusion bodies from the two recombinants in S. exigua larvae were comparable with those of the wild-type virus both in vitro and in vivo . Further bioassays showed that the AcMNPV variants fusing the enhancing factors were incapable of infecting the second instar larvae of S. litura , Helicoverpa armigera , and Pyrausta nubilalis , which were not sensitive to the wild-type AcMNPV. CONCLUSION These genetically modified AcMNPV variants exhibited an enhanced infectivity and may offer better baculovirus control of crop pests. © 2019 Society of Chemical Industry
研究领域:
病毒学
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昆虫学
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生物技术
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DOI
PubMed
高影响因子
Insects
·
2020
·Vol. 11
·期 11
·777
·
IF 2.22
Chengfeng Lei,Jian Yang,Jia Wang,Jia Hu,Xiulian Sun
Simple SummaryThe fall armyworm is a notorious lepidopteran pest that consumes many economically important crops. Its recent invasion into China threatens crops in over 19 provinces. This species is susceptible to its homologous nucleopolyhedrovirus (Spodoptera frugiperdamultiple nucleopolyhedrovirus, or SfMNPV). Selection of indigenous isolates that are adaptable in each geographical region is important for developing a virus-based pesticide. In this study, an SfMNPV field isolate was obtained from a natural population of the fall armyworm in Hubei, China. Two genotypes were cloned from the field isolate, and one genotype, SfHub-A, which had similar activity to the field isolate and produced significantly more progeny viruses, was considered to be a suitable strain for the commercial production of SfMNPV. This information will be valuable for developing a virus-based pesticide against fall armyworm in China.AbstractThe fall armyworm,Spodoptera frugiperda, is a new invading pest in China. The baculovirusSpodoptera frugiperdamultiple nucleopolyhedrovirus (SfMNPV) is a pathogenic agent of the fall armyworm and a potential agent for its control in integrated pest management strategies. In this work, we analyze the molecular and biological characteristics of an SfMNPV isolate collected from maize in China (SfMNPV-Hub). Two genotypes were further isolated from SfMNPV-Hub by an in vivo cloning method. The PstI profile of one genotype (SfHub-A) was similar to genotype A of the SfMNPV Colombian isolate, and the other (SfHub-E) was similar to genotype E of the Colombian isolate. The bioactivity of SfHub-A against second-instarS. frugiperdalarvae was not significantly different from that of SfMNPV-Hub, whereas SfHub-E was 2.7–5.5 fold less potent than SfMNPV-Hub. The speed of kill of SfHub-E was quicker than SfMNPV-Hub, while SfHub-A acted slower than SfMNPV-Hub. Occlusion body (OB) production of SfHub-A in anS. frugiperdacadaver was significantly higher than that of SfMNPV-Hub, while SfHub-E yielded far fewer occlusion bodies (OBs) in the host larvae. These results provide basic information for developing a virus-based pesticide against the invading pestS. frugiperda.Keywords:SfMNPV;field isolate;genotype;molecular characterization;biological activity;OB yield
研究领域:
昆虫学
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病毒学
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农学
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DOI
PubMed
高影响因子
ACS Nano
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2022
·
IF 18.027
Haoguan Gui,Tiantian Yang,Li-Li Li,Fuxin Liang,Zhenzhong Yang
A fabrication strategy for multifunctional organohydrogels is proposed, which combines phase-change microinclusions within a double-network (DN) hydrogel under the stabilization of Janus particles. Janus particles possess reactivity and colloidal stability for more robust organohydrogels, while the interstice among Janus particles enhances the mass transfer between the phase interfaces. Moreover, DN hydrogels are achieved through dynamic cross-linking networks, endowing organohydrogels with injectability and self-healing performance. Phase-change microinclusions are beneficial to the organohydrogels with temperature-responsive mechanical property and temperature-programed shape-memory performance. Organohydrogels can be employed for temperature therapy through the melting-crystallization process of phase-change microinclusions. Simultaneously, the payloads within microinclusions can be released for antibacteria upon melting the encapsulated wax. The organohydrogels can be served as an ideal dressing with temperature-responsive mechanical property, temperature therapy effectiveness, and temperature-triggered antibacterial ability.
研究领域:
材料科学
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生物医学工程
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DOI
PubMed
高影响因子
CRYSTAL GROWTH & DESIGN
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2020
·
IF 4.089
Peng Zhang,Qihao Sun,Yadong Xu,Xiang Li,Lin Liu,Guodong Zhang,Xutang Tao
All-inorganic perovskite CsPbBr3 has attracted intense attentions due to its inspiring optoelectronic properties and excellent stability. Growing large-size single crystals with high quality is vital both for the intrinsic property investigation and the high-performance device fabrication. Here, large-size CsPbBr3 single crystals (ϕ 30 mm × 100 mm) were grown by the modified Bridgman method. The surface morphologies of the as-grown CsPbBr3 single-crystal wafers were characterized by SEM, and inclusions with size of 1–2 μm were observed in the first-time grown crystal (labeled as CPB-1). By adopting a slower growth rate (0.2 mm/h) and cooling rate (5 °C/h) than that of CPB-1, the inclusions were eliminated in subsequent growth (labeled as CPB-2). The hole mobility-lifetime products were measured to be 3.92 × 10–3 and 1.46 × 10–2 cm2·V–1 for CPB-1 and CPB-2, respectively. The carrier mobility of CPB-2 was enhanced 1 order of magnitude from 10.1 ± 0.3 cm2·V–1·s–1 (CPB-1) to 101.3 ± 4.2 cm2·V–1·s–1 due to the elimination of inclusions. In addition, CPB-2 exhibited excellent α particles detection ability with the optimal energy resolution of 15.1% at −60 V bias. We provide an effective way to enhance the optoelectronic properties and device performance of melt-grown CsPbBr3 single crystal by preventing the formation of the inclusions.
研究领域:
材料科学
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光电材料
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晶体生长技术
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DOI
高影响因子
Molecular Catalysis
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2021
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IF 5.062
Yu Han,Xinying Zhang,Liangyu Zheng
In our previous study, the soluble expression of recombinant Candida antarctica lipase B ( r CalB) in E. coli is achieved through fusion with polyamino acid tails. The present study showed that even in the form of inclusion bodies ( r CalB-IBs), r CalB-IBs were still active towards enzyme-catalyzed reactions. r CalB-IBs could be directly crosslinked by the oxidized dextran in the presence of Fe 3 O 4 nanoparticles to obtain magnetic aggregates ( r CalB-mclIBs), and an activity recovery of 113.9 % ± 2.2 % could be achieved under optimized conditions. The obtained r CalB-mclIBs exhibited excellent thermal and storage stabilities, and a good tolerance in changing pH environmental. r CalB-mclIBs could be effectively recycled and could retain 85.4 % ± 2.5 % of its initial activity, and an e.e. p of 84.4 % ± 2.2 % in ( R , S )-NEMPA-ME hydrolysis was observed after the 10th cycle. After the application of r CalB-mclIBs into a promiscuous carbon–carbon bond-forming reaction, the MBH and the Aldol products could be obtained simultaneously. This study provides a simple and inexpensive strategy to highlight the application potential of r CalB-IBs as a biocatalyst.
研究领域:
生物化学
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酶工程
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合成生物学
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DOI
高影响因子
Viruses-Basel
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2019
·Vol. 11
·期 4
·314
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IF 3.811
Gaobo Zhang,Jian Yang,Fujun Qin,Congrui Xu,Jia Wang,Chengfeng Lei,Jia Hu,Xiulian Sun
Dendrolimus punctatuscypovirus (DpCPV), belonging to the genusCypoviruswithin the familyReoviridae, is considered the most destructive pest of pine forests worldwide. DpCPV has a genome consisting of 10 linear double-stranded RNA segments. To establish a reverse genetics system, we cloned cDNAs encoding the 10 genomic segments of DpCPV into three reverse genetics vectors in which each segment was transcribed under the control of a T7 RNA polymerase promoter and terminator tagged with a hepatitis delta virus ribozyme sequence. We also constructed avp80-knockoutAutographa californicamultiple nucleopolyhedrovirus bacmid to express a T7 RNA polymerase codon-optimized for Sf9 cells. Following transfection of Sf9 cells with the three vectors and the bacmid, occlusion bodies (OBs) with the typical morphology of cypovirus polyhedra were observed by optical microscopy. The rescue system was verified by incorporation of aHindIII restriction enzyme site null mutant of the 9th genomic segment. Furthermore, when we co-transfected Sf9 cells with the reverse genetics vectors, the bacmid, and an additional vector bearing anegfpgene flanked with the 5′ and 3′ untranslated regions of the 10th genomic segment, aggregated green fluorescence co-localizing with the OBs was observed. The rescued OBs were able to infectSpodopetra exigualarvae, although their infectivity was significantly lower than that of wild-type DpCPV. This reverse genetics system for DpCPV could be used to explore viral replication and pathogenesis and to facilitate the development of novel bio-insecticides and expression systems for exogenous proteins.Keywords:DpCPV;reverse genetics system;vp80-knockout;foreign protein
研究领域:
病毒学
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分子生物学
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昆虫学
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生物技术
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DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2014
·Vol. 172
·期 5
·2540-2551
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IF 1.687
Jia Dongxu,Yang Yu,Peng Zhengcong,Zhang Dongxu,Li Jianghua,Liu Long,Du Guocheng,Chen Jian
Poly(vinyl alcohol) dehydrogenase (PVADH, EC 1.1.99.23) is an enzyme which has potential application in textile industry to degrade the poly(vinyl alcohol) (PVA) in waste water. Previously, a 1,965-bp fragment encoding a PVADH from Sphingopyxis sp. 113P3 was synthesized based on the replacement of the rare codons in Escherichia coli ( E. coli ). In this work, the deduced mature PVADH ( mPVADH ) gene of 1,887 bp was amplified by polymerase chain reaction (PCR) and inserted into the site between Nco I and Hin dIII in pET-32a(+). The constructed recombinant plasmid was transformed into E. coli Rosetta (DE3). In shake flask, the fusion protein of thioredoxin (Trx)-mPVADH was expressed precisely; however, Trx-mPVADH was found to accumulate mainly as inclusion bodies. After isolating, dissolving in buffer containing urea, purification, dialysis renaturation, and digesting with recombinant enterokinase/His (rEK/His), the bioactive mPVADH fragments were obtained with protein concentration of 0.56 g/L and enzymatic activity of 194 U/mL. The K
m and V
max values for PVA 1799 were 2.33 mg/mL and 15.7 nmol/(min·mg protein), respectively. 1 H-NMR and infrared (IR) spectrum demonstrated that its biological function was oxidizing hydroxyl groups of PVA 1799 to form diketone, and PVA 1799 could be degraded completely by successive treatment with mPVADH and oxidized PVA hydrolase (OPH).
研究领域:
生物技术
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酶学
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环境生物技术
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蛋白质工程
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产品关联
DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2022
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IF 2.025
Zhiting Lao,Shuanqi Li,Jinhui Liang,Jingyi Su,Xin Gong,Xiping Cui,Suqing Zhao
Glypican-3 (GPC3) has a promise to be the diagnostic biomarker as well as therapeutic target for hepatocellular carcinoma (HCC). Nanobody have the great potential in clinical diagnosis and treatment for their characteristics of small size, high solubility, stability, manipulability, binding advantages, and ease of production. In this study, the recombinant glypican-3-N terminal (GPC3-N) protein was expressed as inclusion body in E. coli BL21(DE3)pLysS cells and then purified, which is then used as the immunogen to construct nanobody phage library. The positive clone (named MF15) was obtained by four rounds of bio-panning, and then transformed into the E. coil TOP10F’ cells to express nanobody protein, with the molecular weight of 19 kDa. Both Western blot and immunofluorescence analysis revealed that bacterially expressed GPC3-N protein is biologically active, and MF15 could specifically recognized native GPC3 expressed in HepG2 cells. The results in this study would provide the technical support for the development of diagnostic kits and antibody drugs targeting GPC3.
研究领域:
分子生物学
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癌症研究
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生物技术
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DOI
PubMed
高影响因子
PROTEIN EXPRESSION AND PURIFICATION
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2024
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IF 1.4
Dezheng Peng,Yang Li,Linlin Si,Bo Zhu,Peng Wu,Yibang Li,Dongfang Tang,Yu Liu,Yunxiao Zhang
Semaglutide is currently the most promising antidiabetic drug, especially for the treatment of type 2 diabetes mellitus, due to its excellent efficacy in glycemic control and weight loss. However, the production of semaglutide remains high cost, and high yield, low cost, and high purity still remains a challenge. Herein, we reported a convenient and high-yield strategy for the preparation of semaglutide through fragmented condensation coupling, involving solid-phase peptide synthesis of tetrapeptide and on-column refolding and on-column enzyme cleavage based inclusion body expression of Lys 26 Arg 34 GLP-1 (11–37) with fused protein tags in an X-Y-D4K-G pattern. The optimized N-terminal protein tag significantly boosts inclusion body expression level, while on-column refolding and on-column enzyme cleavage avoid precipitation, enhancing efficiency and yield together with one-step purification. The successful preparation of semaglutide is expected to achieve large-scale industrial production with low cost, high yield and high purity.
研究领域:
药学
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生物技术
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化学工程
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DOI
PubMed
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2021
·
IF 3.493
Xiaolin Pei,Jiapao Wang,Haoteng Zheng,Qinjie Xiao,Anming Wang,Weike Su
The catalytically-active inclusion bodies (CatIBs) represent a promising strategy for immobilizing enzyme without additional carriers and chemicals, which has aroused great attention in academic and industrial communities. In this work, we discovered two natural parallel right-handed coiled-coil tetramer peptides from PDB database by a structural mining strategy. The two self-assembling peptides, NSPdoT from rotavirus and HVdoT from human Vasodilator-stimulated phosphoprotein, efficiently induced the CatIBs formation of a ( R )-Hydroxynitrile lyase from Arabidopsis thaliana ( At HNL) in Escherichia coli cells. This is convenient to simultaneously purify and immobilize the target proteins as biocatalysts. As expected, HVdoT- At HNL and NSPdoT- At HNL possessed drastically increased tolerance toward lower pH values, which will be very critical to synthesize cyanohydrins under acidic condition for suppressing the non-enzymatic side reaction. In addition. At HNL-CatIBs are produced at high yield in host cells as bioactive microparticles, which exhibited high thermal and pH stabilities. Therefore, the CatIBs method represent a promising application for the immobilization of enzymes in the biocatalysis field.
研究领域:
生物催化
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酶工程
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蛋白质科学
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生物技术
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2025
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IF 8.5
Bo Zhang,Tong Wang,Siyu Ni,Yaping Zong,Nan Wang,Yunlong Si,Yong Li
Recovery of bioactive proteins from insoluble inclusion bodies (IB) is a critical challenge when purifying recombinant proteins expressed in E. coli . One practical solution is to enhance the renaturability or refolding capability of the target protein after the insoluble proteins are solubilized with denaturants such as urea. While some methods, such as using chemical additives and manipulating the physical environment, have been reported to assist in protein refolding, their effectiveness remains limited. This work evaluated the potential of a highly conserved domain found in five biotin-dependent carboxylases as a fusion tag for purifying IB proteins. We found that this tag, comprised of 67 amino acids (named P67) as eight anti-parallel β-sheets that collectively form a flattened barrel structure, significantly improved the refolding process of its IB protein partner. The yield of bioactive proteins fused with this tag recovered from the dialysis stage was markedly higher than untagged ones. We further proved that this effect relies on the structural integrity of the tag's spatial conformations. Under natural conditions, soluble proteins can be purified using the P67 tag via the biotin/streptavidin affinity system. In conclusion, this tag offers a promising methodology for purifying both insoluble and soluble recombinant proteins.
研究领域:
分子生物学
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蛋白质科学
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生物技术
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生物化学
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
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2024
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IF 2.8
Yiwen Zhang,Yang Guo,Liang Song,Wenshuai Liu,Rui Nian,Xiying Fan
This study introduces an efficient on-column refolding and purification method for preparing nanobodies (Nbs) expressed as inclusion bodies and fusion proteins. The HisTrap TM FF system was successfully employed for the purification of the fusion protein FN1-ΔI-CM-2D5. The intein ΔI-CM cleavage activity was activated at 42 °C, followed by incubation for 4 h. Leveraging the remarkable thermal stability of Nbs, 2D5 was further purified through heat treatment at 80 °C for 1h. This method yielded up to 107.2 mg of pure 2D5 with a purity of 99.2 % from just 1L of bacterial culture grown in a shaker flask. Furthermore, this approach successfully restored native secondary structure and affinity of 2D5. Additionally, the platform was effectively applied to the refolding and purification of a polystyrene-binding nanobody (B2), which exhibited limited expression in the periplasmic and cytoplasmic spaces of E. coli . This endeavor resulted in the isolation of 53.2 mg of pure B2 Nb with a purity exceeding 99.5 % from the same volume of bacterial culture. Significantly, this approach restored the native secondary structure of the Nbs, highlighting its potential for addressing challenges associated with expressing complex Nbs in E. coli . Overall, this innovative platform provides a scientifically rigorous and reproducible method for the efficient preparation of Nbs, offering a valuable tool for antibody research and development.
研究领域:
生物技术
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蛋白质工程
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分子生物学
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抗体研究
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）