Name: a method for production of recombinant mcd1d protein in insect cells.
Uploaded: 2007-12-10T22:00:00
Description: Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

一，解冻的细胞 TN5细胞的冷冻瓶，并在37℃水浴解冻。 25厘米2训练班烧瓶中加入5毫升昆虫快递培养基（Invitrogen公司的产品目录号10486）。注意表达五年没有谷氨酰胺;需要添加100X谷氨酰胺笔链球菌单独或谷氨酰胺1 LT媒体10毫升。添加到TN5细胞，并在29 ° C培养箱中孵育30分钟。然后，吸用DMSO中轻轻互不干扰烧瓶底部的细胞。添加新鲜培养基5毫升。 二?...

<ol>
	<li>Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+glycolipipd+antigen-specific+T+cells+using+fluorecent+CD1d+tetramers.">In vivo identification of glycolipipd antigen-specific T cells using fluorecent CD1d tetramers.</a> J. Exp. Med. 191, 1895-1903 (2000).</li><li>Kinjo, Y., Wu, D., Kim, G., Xing, G. W., Poles, M. A., Ho, D. D., Tsuji, M., Kawahara, K., Wong, C. H., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+bacterial+glycosphingolipids+by+natural+killer+T+cells.">Recognition of bacterial glycosphingolipids by natural killer T cells.</a> Nature. 434, 520-525 (2005).</li><li>Naidenko, O. V., Maher, J. K., Ernst, W. A., Sakai, T., Modlin, R. L., Kronenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+and+antigen+presentation+of+ceramide-containing+glycolipids+by+soluble+mouse+and+human+CD1d+molecules.">Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.</a> J. Exp. Med. 190, 1069-1080 (1999).</li><li>Porcelli, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD1+family:+A+third+lineage+of+antigen-+presenting+molecules.">The CD1 family: A third lineage of antigen- presenting molecules.</a> Adv. Immunol. 59, 1-98 (1995).</li><li>Sidobre, S., Kronemberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD1+tetramers:+A+powerful+tool+for+the+analysis+of+glycolipid-reactive+T+cells.">CD1 tetramers: A powerful tool for the analysis of glycolipid-reactive T cells.</a> J. Immunol. 169, 1340-1348 (2002).</li></ol>

在此过程中，它是如何启动，成长，感染昆虫细胞，以及如何滴度使用这些细胞的杆状病毒。在此过程中最重要的方面是细胞的维护和知道确切的杆状病毒滴度。一旦你已经产生了CD1的四聚体，它们可以被用来作为糖脂反应性T细胞的功能强大的分析工具。杆状病毒系统也广泛用于生产重组蛋白，包括MHC II类四聚体，所以此过程有一个更广泛的适用性。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Express Five SFM</td>
<td> Serum Free Medium</td>
<td>Invitrogen</td>
<td>10486025</td>
<td>&nbsp;</td>
<tr>
<td>High Five TM</td>
<td> Insect Cells</td>
<td>Invitrogen</td>
<td>B855-02</td>
<td>&nbsp;</td>
<tr>
<td>Glutamine Pen strep</td>
<td>Antibiotics</td>
<td>Invitrogen</td>
<td>10378-016</td>
<td>&nbsp;</td>
<tr>
<td>25cm2 TC Flask</td>
<td> Plastic</td>
<td>Fisher</td>
<td>10-126-28</td>
<td>&nbsp;</td>
<tr>
<td>175 cm2 TC Flask</td>
<td> Plug seal flask</td>
<td>Fisher</td>
<td>10-126-8</td>
<td>&nbsp;</td>
<tr>
<td>Erlenmeyer Flask</td>
<td> Cell Culture Flask</td>
<td>Fisher</td>
<td>07 200 672</td>
<td>&nbsp;</td>
<tr>
<td>YM 30 Concenterator</td>
<td>30,000 MWCO</td>
<td>Millipore</td>
<td>UFC 903096</td>
<td>&nbsp;</td>
<tr>
<td>FPLC equipment</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>BrA Enzyme and Buffer</td>
<td> Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Ni-NTA Agarose Column</td>
<td>His Tag Protein Purification Column</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Desalting column</td>
<td> To get Rod of Salt from Protein</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MonoQ Column</td>
<td>Anion Exchange Chromatgraphy</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>S200 Column</td>
<td> Size Exclusion Chromatography</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>alpha-Galcer</td>
<td> Glycolipid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PE- Streptavidin</td>
<td> For conjugating Protein</td>
<td>Molecular Probe</td>
<td> S-866</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a method for production of recombinant mcd1d protein in insect cells.

CD1的蛋白质构成的抗原提呈分子的第三类。他们是细胞表面的糖蛋白，约50 kDa的糖基化，非共价与β2-微球蛋白重链表达。他们结合血脂，而不是肽。虽然它们的结构确认的CD1蛋白MHC I类和II类抗原呈现分子的相似性，mCD1d槽比较窄，深，高疏水性和它有两项具有约束力的口袋，而不是为经典所描述的几个浅口袋MHC编码的抗原提呈分子。根据其氨基酸序列，这样一个hydrobphobic槽提供了一个理想的环境脂质抗原结合。 自然杀伤T（NKT）细胞使用他们的TCR识别势必会或CD1d糖脂。已参与对保护自身免疫性疾病和传染病和肿瘤排斥反应的T细胞CD1所提出的血脂。因此，辨别能力，净化，并跟踪CD1反应NKT细胞的反应是非常重要的。 α-半乳糖神经酰胺（A - Galcer）与CD1d四聚体的生成NKT细胞生物学的重要见解。从其他CD1的分子构造的四聚体也已产生，这些新的试剂，极大地扩大了监测脂质为基础的疫苗的反应的潜在使用，并在自身免疫性疾病和其他的诊断的脂质反应性T细胞的功能的知识，治疗。

Watch this Scientific Journal Video about A Method For Production of Recombinant mCD1d Protein in Insect Cells. at JoVE.com

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

一个方法，准备昆虫细胞和感染杆状病毒重组mCD1d mCD1d四聚体的proteinand生产的目的。

重组昆虫细胞中的mCD1d的蛋白质的生产方法。

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

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