<meta charset="utf8"></head><body>使用 Fibrinogen-PAGE 揭示花生蠕虫中的纤维蛋白原溶解剂

<ol>
	<li>Bikdeli, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COVID-19+and+thrombotic+or+thromboembolic+disease:+Implications+for+prevention,+antithrombotic+therapy,+and+follow-up.">COVID-19 and thrombotic or thromboembolic disease: Implications for prevention, antithrombotic therapy, and follow-up.</a> J Am Coll Cardiol. 75 (23), 2950-2973 (2020).</li><li>Tsivgoulis, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolysis+for+acute+ischaemic+stroke:+current+status+and+future+perspectives.">Thrombolysis for acute ischaemic stroke: current status and future perspectives.</a> Lancet Neurol. 22 (5), 418-429 (2023).</li><li>Tang, M., Hu, C., Lin, H., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrinolytic+drugs+induced+hemorrhage:+mechanisms+and+solutions.">Fibrinolytic drugs induced hemorrhage: mechanisms and solutions.</a> Blood Coagul Fibrinolysis. 34 (5), 263-271 (2023).</li><li>Lu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification,+characterization,+and+chemical+modification+of+Bacillus+velezensis+SN-14+fibrinolytic+enzyme.">Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme.</a> Int J Biol Macromol. 177, 601-609 (2021).</li><li>Abu-Tahon, M. A., Abdel-Majeed, A. M., Ghareib, M., Housseiny, M. M., Abdallah, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombolytic+and+anticoagulant+efficiencies+of+purified+fibrinolytic+enzyme+produced+from+Cochliobolus+hawaiiensis+under+solid-state+fermentation.">Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.</a> Biotechnol Appl Biochem. 70 (6), 1954-1971 (2023).</li><li>Lin, W., Lin, H., Xin, P., Yan, H., Kang, B., Tang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+approach+to+analyze+the+cell+components+of+cerebral+blood+clots.">A Comprehensive approach to analyze the cell components of cerebral blood clots.</a> J Vis Exp. (197), e65791 (2023).</li><li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus.">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> J Vis Exp. (196), e65631 (2023).</li><li>. Plasmin affinity purification method based on agarose gel and plasmin and application thereof Available from: <a target="_blank" href="https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470">https://patents.google.com/patent/CN116103270A/en?oq=CN202310108470</a> (2023)</li><li>Walton, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fibrin+plate+method+for+the+assay+of+plasminogen+activators.">An improved fibrin plate method for the assay of plasminogen activators.</a> Clinica Chimica Acta. 13 (5), 680-684 (1966).</li><li>Ngere, J. B., Ebrahimi, K. H., Williams, R., Pires, E., Walsby-Tickle, J., McCullagh, J. S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-exchange+chromatography+coupled+to+mass+spectrometry+in+life+science,+environmental,+and+medical+research.">Ion-exchange chromatography coupled to mass spectrometry in life science, environmental, and medical research.</a> Anal Chem. 95 (1), 152-166 (2023).</li><li>Kato, S., Takeuchi, K., Iwaki, M., Miyazaki, K., Honda, K., Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitin-+and+streptavidin-mediated+affinity+purification+systems:+A+screening+platform+for+enzyme+discovery.">Chitin- and streptavidin-mediated affinity purification systems: A screening platform for enzyme discovery.</a> Angew Chem Int Ed Engl. 62 (31), e202303764 (2023).</li><li>Sharma, N., Sharma, R., Rajput, Y. S., Mann, B., Singh, R., Gandhi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+methods+for+milk+proteins+on+polyacrylamide+gel+electrophoresis:+Critical+analysis+and+options+for+better+resolution.">Separation methods for milk proteins on polyacrylamide gel electrophoresis: Critical analysis and options for better resolution.</a> International Dairy Journal. 114, 104920 (2021).</li><li>. Preparation and application of natural fibrinolytic enzyme from peanut worm Available from: <a target="_blank" href="https://patents.google.com/patent/CN109295042A/en">https://patents.google.com/patent/CN109295042A/en</a> (2019)</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#160; M Tris-HCl (pH 6.8)</td>
			<td>Solarbio</td>
			<td>T1020</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 M Tris-HCl (pH 8.8)</td>
			<td>Solarbio</td>
			<td>T1010</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/Bis-acrylamide</td>
			<td>Biosharp</td>
			<td>BL513B</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>XiLONG SCIENTIFIC</td>
			<td>7727-54-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>PYREX</td>
			<td>2-9425-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tube (1.5 mL)</td>
			<td>Biosharp</td>
			<td>BS-15-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Constant Temperature Incubator</td>
			<td>JINGHONG</td>
			<td>JHS-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomas Brillant Blue Stainning solution</td>
			<td>Beyotime</td>
			<td>P0017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Analytical Balance</td>
			<td>DENVER</td>
			<td>TP-213</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Solarbio</td>
			<td>F8051</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading pipette tips, Bulk</td>
			<td>Biosharp</td>
			<td>BS-200-GTB</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>AHS</td>
			<td>ATS-1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal rotation oscillator</td>
			<td>NuoMi</td>
			<td>NMSP-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Milli-Q Reference</td>
			<td>Millipore</td>
			<td>Z00QSV0CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Cell</td>
			<td>BIO-RAD</td>
			<td>165-8000~165-8007</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;,N&#39;-Tetramethylethylenediamine</td>
			<td>Sigma</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (1 mL)</td>
			<td>Axygene</td>
			<td>T-1000XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (10 &#181;L)</td>
			<td>Axygene</td>
			<td>T-10XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tip (200 &#181;L)</td>
			<td>Axygene</td>
			<td>T-200XT-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (1 mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY18723</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (10&#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZX98775</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (200 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY20280</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettor (50 &#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>ZY15331</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>cence</td>
			<td>H1650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>V900859</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Solarbio</td>
			<td>T8060</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Solarbio</td>
			<td>T8230</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8200</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid separation and display of active fibrinogenolytic agents in sipunculus nudus through fibrinogen-polyacrylamide gel electrophoresis

In recent years, due to the continued rise of thrombotic diseases, thrombotic diseases have become a new major global health problem1. At present, antithrombotic drugs are classified into three groups: anti-platelet aggregation drugs, anticoagulants, and thrombolytic drugs. Among them, thrombolytic drugs, such as urokinase (UK), tissue plasminogen activator (tPA), etc., are the only clinically used drugs that can hydrolyze thrombus2. Meanwhile, more safe and effective thrombolytic drugs are being developed by the identification of novel thrombolytic agents3.
However, the identification of novel thrombolytic agents is time-consuming and labor-intensive, which mainly involves the separation/purification and characterization/checking stages. The former is to separate each component, and the latter is to display their fibrino(geno)lytic activities4,5. In previous studies, despite the fact we have successfully isolated a novel fibrino(geno)lytic enzyme (sFE) from the peanut worm (Sipunculus nudus) by affinity chromatography and fibrin(ogen) plate assay6,7,8, these processes are such time and labor-consuming. First, it is necessary to determine whether the peanut worm homogenates have fibrino(geno)lytic activity by the fibrin(ogen) plate method and reaction products based on PAGE9. Then, a series of chromatography, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, and other methods, have to be carried out for separation and purification10,11. Subsequently, the fibrin plate assay is performed again to check the fibrinogenolytic activity of each separated component. Finally, native-PAGE and sodium dodecyl sulfate (SDS)-PAGE are performed to determine the molecular weight of the active fibrinogenolytic agents12. Therefore, it is critical to rapidly separate and display the active fibrinogenolytic agents.
To rapidly separate and display the active fibrinogenolytic agents in the peanut worm homogenates, a new fibrinogen-PAGE method was developed by combining the PAGE and fibrinogen plate methods, i.e., the substrates of fibrinogenolytic agents fibrinogen were added to the native-PAGE gel. After native-PAGE, the components were separated by their molecular weight. Meanwhile, each active fibrinogenolytic component can be displayed by staining. By this method, we successfully detected more than one active fibrinogenolytic agent of peanut worm&#160;homogenate within 6 h. Moreover, this fibrinogen-PAGE method is time and cost-friendly. Furthermore, this method could be used to study the fibrinogenolytic agents of the other organisms.

<meta charset="utf8"></head><body>作者聚焦：用于纤维蛋白原溶解研究的时间和成本效益的纤维蛋白原-PAGE

通过纤维蛋白原-聚丙烯酰胺凝胶电泳快速分离和展示 Sipunculus nudus 中的活性纤维蛋白原溶解剂

Our research is focused on the fibrinolytic system. In this study, we are trying to simplify the mass for screening of fibrinogenolytical agents in peanut worms using Fibrinogen-PAGE technique. We have developed a new Fibrinogen-PAGE method by combining the PAGE and Fibrinogen plate methods.Compared to the other technics currently used, our protocol consists of a 10 year study separate and display the active fibrinogenolytic genes within the same pathogen. Overall, this mass is time and cost effective. With our findings, the discovery of neuro-fibrinogenolytic agents will become simple and can speed up the development of novel drugs that can be used to treat a variety of cardiovascular and other diseases.Our laboratory will focus on studying the coagulation and the fibrinogenolytic systems in different organisms to isolate and screen for novel fibrinogenolytic agents that can be useful for therapeutics.

Read this Scientific Article Protocol about Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis at JoVE.com

Fibrinogenolytic agents that can dissolve fibrinogen directly have been widely used in anti-coagulation treatment. Generally, identifying new ...

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Research

JoVE Journal

Biochemistry

Rapid Separation and Display of Active Fibrinogenolytic Agents in Sipunculus nudus through Fibrinogen-Polyacrylamide Gel Electrophoresis

226 次观看.  Quanzhou First Hospital Affiliated to Fujian Medical University. 我们的研究重点是纤溶系统。在这项研究中，我们试图简化使用纤维蛋白原-PAGE 技术筛选花生蠕虫中纤维蛋白原溶解剂的质量。我们通过结合 PAGE 和纤维蛋白原板方法开发了一种新的纤维蛋白原-PAGE 方法。与目前使用的其他技术相比，我们的方案包括一项为期 10 年的研究，分离并显示同一病原体中的活性纤维蛋白原溶解基因。总体而言，这种?...