Name: purification of a high molecular mass protein in streptococcus mutans
Uploaded: 2019-09-14T14:00:00
Description: Watch this Scientific Journal Video about Purification of a High Molecular Mass Protein in Streptococcus mutans at JoVE.com

这项工作得到了日本科学促进会（JSPS）（赠款号16K15860和19K10471至T.M.、17K12032至M.I.和18K09926到N.H.）和SECOM科学技术基金会（赠款号2018.09.10号）的支持。

注：在生成S. mutans _gtfC（其中 gtfC基因的整个编码区域被替换为spcr）之前，必须在执行这些协议之前完成。有关第5代 的详细信息，请参阅已发表的文章。
1. 底漆设计
<ol><li>
准备引力为S.mutans他-gtfC的建设。 注：本协议中使用的引性序列如表1所示。<strong ...

<ol>
	<li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual 3rd Edition. , (2001).</li><li>Murata, T., Ishikawa, M., Shibuya, K., Hanada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+functional+analysis+of+a+gene+of+interest+in+Streptococcus+mutans:+gene+disruption+followed+by+purification+of+a+polyhistidine-tagged+gene+product.">Method for functional analysis of a gene of interest in Streptococcus mutans: gene disruption followed by purification of a polyhistidine-tagged gene product.</a> Journal of Microbiological Methods. 155, 49-54 (2018).</li><li>Horton, R. M., Cai, Z., Ho, S. M., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+splicing+by+overlap+extension:+tailor-made+genes+using+the+polymerase+chain+reaction.">Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.</a> Biotechniques. 54 (3), 129-133 (2013).</li><li>Murata, T., Hanada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+chloride+channel+permease+to+fluoride+resistance+in+Streptococcus+mutans.">Contribution of chloride channel permease to fluoride resistance in Streptococcus mutans.</a> FEMS Microbiology Letters. 363 (11), 101 (2016).</li><li>Murata, T., Okada, A., Matin, K., Hanada, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+gene-disrupted+Streptococcus+mutans+strain+without+gene+cloning.">Generation of a gene-disrupted Streptococcus mutans strain without gene cloning.</a> Journal of Visualized Experiments. (128), (2017).</li><li>Hanada, N., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+the+Streptococcus+mutansgtfC+gene,+coding+for+synthesis+of+both+soluble+and+insoluble+glucans.">Isolation and characterization of the Streptococcus mutansgtfC gene, coding for synthesis of both soluble and insoluble glucans.</a> Infection and Immunity. 56 (8), 1999-2005 (1988).</li><li>LeBlanc, D. J., Lee, L. N., Inamine, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+nucleotide+base+sequence+analysis+of+a+spectinomycin+adenyltransferase+AAD(9)+determinant+from+Enterococcus+faecalis.">Cloning and nucleotide base sequence analysis of a spectinomycin adenyltransferase AAD(9) determinant from Enterococcus faecalis.</a> Antimicrobial Agents and Chemotherapy. 35 (9), 1804-1810 (1991).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> Journal of Visualized Experiments. (63), e3998 (2012).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR:+The+Polymerase+Chain+Reaction.">PCR: The Polymerase Chain Reaction.</a> Journal of Visualized Experiments. , (2019).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Gel+Electrophoresis.">DNA Gel Electrophoresis.</a> Journal of Visualized Experiments. , (2019).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel+Purification.">Gel Purification.</a> Journal of Visualized Experiments. , (2019).</li><li>Wingfield, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+precipitation+using+ammonium+sulfate.">Protein precipitation using ammonium sulfate.</a> Current Protocols in Protein Science. 84 (1), (2016).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separating+Protein+with+SDS-PAGE.">Separating Protein with SDS-PAGE.</a> Journal of Visualized Experiments. , (2019).</li><li>Database, J. S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Western+Blot.">The Western Blot.</a> Journal of Visualized Experiments. , (2019).</li><li>Smith, P. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Perry, D., Wondrack, L. M., Kuramitsu, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+putative+cariogenic+properties+in+Streptococcus+mutans.">Genetic transformation of putative cariogenic properties in Streptococcus mutans.</a> Infection and Immunology. 41 (2), 722-727 (1983).</li><li>Lau, P. C., Sung, C. K., Lee, J. H., Morrison, D. A., Cvitkovitch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+ligation+mutagenesis+in+transformable+streptococci:+application+and+efficiency.">PCR ligation mutagenesis in transformable streptococci: application and efficiency.</a> Journal of Microbiological Methods. 49 (2), 193-205 (2002).</li><li>Ge, X., Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+gene+deletions+in+Streptococcus+sanguinis+by+high+throughput+PCR.">Genome-wide gene deletions in Streptococcus sanguinis by high throughput PCR.</a> Journal of Visualized Experiments. (69), (2012).</li></ol>

引体的设计是协议中最关键的一步。gtfC-反向和spcr-前引基的序列根据gtfC的3°端区域和spcr的5°端区域的顺序自动确定。每个引基包括 24 个互补基库，这些基础对 GS 链接器进行编码，并在其 5' 区域对 His-tag 编码序列进行编码。通过在 3° 端添加 His 标记编码序列，可以避免位于上游侧翼区域的本机调控序列中断。DNA停止科顿必须从gtfC-反向引基器中...

对基因功能的分析通常涉及将野生型菌株的类特征与被破坏的菌株进行比较。一旦产生基因中断的菌株，基因产物的外源性添加允许功能恢复。
获得后续恢复测定所需的纯化基因产物的最常见方法是在大肠杆菌1中执行异体表达。然而，膜蛋白或高分子质量蛋白的表达往往很难使用这个系统1。在这些情况下，目标蛋白通常从通过一系列复杂的步骤原生合成蛋白质的细胞中分离出来，这可能导致基因产物的丧失。为了克服这些问题，根据基因破坏方法2、基于PCR的DNA拼接方法3（指定的两步融合PCR）和基因电穿孔，为基因产物纯化开发了一个简单的程序。在链球菌的转化。在基因产品的C-终点区添加多肽标记（His-tag）有助于通过固定金属亲和色谱（IMAC）进行纯化。
为了分离His-tag表达菌株，感兴趣的基因的整个基因组DNA（在这个His-tag表达基因中断的菌株中）被一个抗生素抗药性标记基因所取代。产生His-tag表达应变的过程与产生基因中断的应变的过程几乎相同，如前面描述的4，5。因此，基因破坏和基因产物分离的方法应作为功能分析的序列实验进行。
在本工作中，多聚苯二烯编码序列被附加到gtfC（GenBank locus标记SMU_1005）基因的3°端，在S.mutans6中编码葡聚酰亚胺转移酶-SI（GTF-SI）。然后，对链球菌物种进行表达研究。大肠杆菌实现异源性gtfC表达是困难的，很可能是因为GTF-SI的分子质量很高。这种菌株被命名为S.穆坦斯·克特夫克。图解图中描绘了野生S.mutans（S.mutans WT）中gtfC和光谱素抗基因盒（spcr）7位数的组织及其衍生物。图 1.GTF-SI是一种分泌蛋白，有助于培育造血性牙科生物膜6。在蔗糖的存在下，在WT S.mutans菌株的光滑玻璃表面上观察到粘附生物膜，但在S.mutans gtfC-中断菌株（S.mutans +gtfC）2、5.当重组GTF-SI的外源性添加后，生物膜形成在S.mutans _gtfC中恢复。应变，S.mutans His-gtfC，然后用于生产重组GTF-SI。

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Nippon Genetics</td>
			<td>NE-AG02</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Anaeropack</td>
			<td>Mitsubishi Gas Chemical</td>
			<td>A-03</td>
			<td>Anaerobic culture system</td>
		</tr>
		<tr>
			<td>Anti-His-Tag monoclonal antibody</td>
			<td>MBL</td>
			<td>D291-7</td>
			<td>HRP-conjugated</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td>Measurement of protein concentration</td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>Becton, Dickinson</td>
			<td>237500</td>
			<td>Bacterial culture medium</td>
		</tr>
		<tr>
			<td>CBB R-250</td>
			<td>Wako</td>
			<td>031-17922</td>
			<td>For biofilm staining</td>
		</tr>
		<tr>
			<td>Centrifugal ultrafiltration unit</td>
			<td>Sartorius</td>
			<td>VS2032</td>
			<td>Buffer replacement and protein concentration</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Kubota</td>
			<td>7780II</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromatographic column</td>
			<td>Bio-Rad</td>
			<td>7321010</td>
			<td>For IMAC</td>
		</tr>
		<tr>
			<td>Dialysis membrane clamp</td>
			<td>Fisher brand</td>
			<td>21-153-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dialysis tubing</td>
			<td>As One</td>
			<td>2-316-06</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Takara</td>
			<td>R045A</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr>
			<td>DNA sequencing</td>
			<td>Eurofins Genomics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECL substrate</td>
			<td>Bio-Rad</td>
			<td>170-5060</td>
			<td>For western blotting</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M pH 8.0)</td>
			<td>Wako</td>
			<td>311-90075</td>
			<td>Tris-EDTA buffer preparation</td>
		</tr>
		<tr>
			<td>Electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>1652086</td>
			<td>0.2 cm gap</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>EtBr solution</td>
			<td>Nippon Gene</td>
			<td>315-90051</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Gel band cutter</td>
			<td>Nippon Genetics</td>
			<td>FG-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel extraction kit</td>
			<td>Nippon Genetics</td>
			<td>FG-91202</td>
			<td>DNA extraction from agarose gel</td>
		</tr>
		<tr>
			<td>Imager</td>
			<td>GE Healthcare</td>
			<td>29083461</td>
			<td>For SDS-PAGE and western blotting</td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Wako</td>
			<td>095-00015</td>
			<td>Binding buffer and elution buffer preparation</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>EZ-022</td>
			<td>Temperature setting: 4 &#176;C</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Nippon Medical &#38; Chemical Instruments</td>
			<td>LH-100-RDS</td>
			<td>Temperature setting: 37 &#176;C</td>
		</tr>
		<tr>
			<td>Membrane filter</td>
			<td>Merck Millipore</td>
			<td>JGWP04700</td>
			<td>0.2 &#181;m diameter</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Kubota</td>
			<td>3740</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>NaH2PO4&#183;2H2O</td>
			<td>Wako</td>
			<td>192-02815</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Wako</td>
			<td>198-13765</td>
			<td>Preparation of binding buffer and elution buffer</td>
		</tr>
		<tr>
			<td>(NH4)2SO4</td>
			<td>Wako</td>
			<td>015-06737</td>
			<td>Ammonium sulfate precipitation</td>
		</tr>
		<tr>
			<td>Ni-charged resin</td>
			<td>Bio-Rad</td>
			<td>1560133</td>
			<td>For IMAC</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Eurofins Genomics</td>
			<td>Custom-ordered</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein standard</td>
			<td>Bio-Rad</td>
			<td>161-0381</td>
			<td>For SDS-PAGE and western blotting</td>
		</tr>
		<tr>
			<td>Solvent filtration apparatus</td>
			<td>As One</td>
			<td>FH-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin</td>
			<td>Wako</td>
			<td>195-11531</td>
			<td>Antibiotics; use at 100 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Sterile syringe filter</td>
			<td>Merckmillipore</td>
			<td>SLGV004SL</td>
			<td>0.22 &#181;m diameter</td>
		</tr>
		<tr>
			<td>Streptococus mutans &#916;gtfC</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>gtfC replaced with spcr</td>
		</tr>
		<tr>
			<td>Streptococus mutans UA159</td>
			<td>Stock strain in the lab.</td>
			<td></td>
			<td>S. mutans ATCC 700610, Wild-type strain</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako</td>
			<td>196-00015</td>
			<td>For biofilm development</td>
		</tr>
		<tr>
			<td>TAE (50 &#215; )</td>
			<td>Nippon Gene</td>
			<td>313-90035</td>
			<td>For agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>PTC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (1 M, pH 8.0)</td>
			<td>Wako</td>
			<td>314-90065</td>
			<td>Tris-EDTA buffer preparation</td>
		</tr>
	</tbody>
</table>

purification of a high molecular mass protein in streptococcus mutans

解释基因的功能通常涉及野生型菌株和菌株的类名特征的比较，其中感兴趣的基因已被破坏。基因中断后功能丧失随后通过中断基因产物的外源性添加而恢复。这有助于确定基因的功能。前面描述的方法涉及生成gtfC基因破坏的链球菌菌株。在这里，描述了一种不要求的方法，用于在基因中断后从新生成的S.mutans菌株中纯化gtfC基因产物。它涉及在感兴趣的基因的3°端添加多聚苯乙烯编码序列，允许使用固定金属亲和色谱简单纯化基因产物。在此方法的遗传修饰中，不需要 PCR 以外的酶反应。基因破坏后通过外源性补充恢复基因产物是确定基因功能的有效方法，也可以适应不同的物种。

图 3显示了第一个 PCR（图 3A）和第二 PCR （图 3B） 中每个放大子的大小。如表 1所述，每个放大子的大小与预测大小相对应。图4A显示了用第二种PCR产品转化并镀在含有霉素的BHI琼脂板上的S.mutans菌落。然后，在胶凝剂凝胶上运行殖民地 PCR 产品 （?...

Watch this Scientific Journal Video about Purification of a High Molecular Mass Protein in Streptococcus mutans at JoVE.com

Purification of a High Molecular Mass Protein in Streptococcus mutans

这里描述的是一个简单的方法，用于纯化链球菌中的基因产物。该技术在蛋白质，特别是膜蛋白和高分子质量蛋白的纯化方面可能有利，可用于其他各种细菌物种。

链球菌中高分子质量蛋白的纯化

Elucidation of a gene&#39;s function typically involves comparison of phenotypic traits of wild-type strains and strains in which the gene of interest has ...

这种方法可以帮助回答微生物学领域的关键问题，例如，在E.Coli中难以表达的基因产品如何净化。该技术的主要优点是，除了PCR，没有酶反应是载体，可用于蛋白质纯化的常见应用。虽然这种方法可以提供对链球菌突变体中蛋白质纯化的见解，但它也可以是其他微生物物种的一种信息。演示这个程序的将是山下博士，一个来自我们实验室的研究生。在从链球菌突变体中提取底像设计和基因组DNA提取后，使用野生型链球菌和GFC破坏链球菌突变体基因组作为PCR模板执行第一个PCR。使用准备好的 gtfC 前进和反向底因和 spc r 转发和反向底因放大含有 gtfC 基因下游部分的区域和那些含有物种霉素抗性基因的区域。然后，电泳每个PCR产品在1%的阿加罗斯凝胶。使用凝胶频化器将所需的DNA片段从凝胶中切入约1000个碱基对和2000个碱基对，切入微离心管。在每个管中加入500微升的溶解缓冲液，并在56摄氏度下孵育10分钟，溶解凝胶片。使用硅膜基凝胶提取法净化碎片。使用第一个 PCR 的产品作为 PCR 模板使用嵌套前进和嵌套反向底像执行第二个 PCR。当第二个 PCR 产品无法通过电泳进行确认时，应设计另一个嵌套底像对。电泳中五微升的PCR混合物在阿加罗斯凝胶上。确认通过溴化乙基凝胶染色图像生成约3，000个碱基对的适当安培，然后根据手稿进行。现在，将浓缩第二PCR产品的5微升混合到50微升的冰冷、称职的野生链球菌突变细胞中，以前冷冻过。将混合物加入电穿孔盒中，并将蛋黄酱放入电穿孔装置的保素室。给细胞一个1.8千伏、600欧姆和10微法拉的电脉冲，2.5毫秒。将500微升的脑心输液汤加入到库子中。立即将悬浮液的10至100微升扩散到含有斑点霉素的脑心输液螺旋板上。在37摄氏度下孵育板，直到菌落生长到足以被拾起。根据手稿，在产生链球菌突变多石丁编码序列后，结合菌株和夜间接种无斑点霉素，在4摄氏度的10000倍g下将细菌培养悬浮液分离20分钟。将文化上一层酒转移到三升玻璃烧杯中。要浓缩培养物上清液中的蛋白质，请将两升上清液放在磁搅拌器上，然后开始剧烈搅拌。加入1，122克硫酸铵，让沉淀物在4小时或夜间在4摄氏度下剧烈搅拌。下一个。将硫酸铵沉淀溶液在15，000倍g下离心，在4摄氏度下20分钟。去位。用铲子，收集沉淀物，并转移到200毫升的玻璃烧杯。将颗粒重新在 35 毫升的结合缓冲液中。然后，将悬浮液的每个约25毫升转移到再生的纤维素透析管中。将透析管放入 2.5 升搅拌器上的搅拌器中，在 4 摄氏度下进行回旋，以将悬浮液进行透析。两小时后更换透析溶液，并通宵继续透析。接下来，将管材中的透析悬浮液转移到离心管，并在4摄氏度下以2万次g将管以2万次g放置，10分钟。使用分部过滤设备，将上清液倒入 0.2 微米膜过滤器进行过滤。将滤金物转移到 75 毫升的烧瓶中。要将多石丁标记的 gtfSI 从过滤的悬架中分馏，请先准备固定金属亲和力色谱。根据手稿平衡树脂后，关闭霍夫曼捏公鸡。将过滤的悬浮液的五毫升加入柱中，以制造泥浆。然后，将所有浆料转移到剩余的过滤悬浮液中，并在4摄氏度下轻轻旋转混合物30分钟。将混合物重新加载到列中。打开霍夫曼捏公鸡，以去除重力流的悬架。用20毫升的结合缓冲液清洗IMAC树脂，然后洗洗20毫升的洗脱缓冲液，以获得重组的gtfSI。之后，将埃卢酸盐装入离心超滤管中，将溶液以2000倍g离心，1至5分钟，使溶液浓缩。清空滤液容器，并将 15 毫升的存储缓冲液添加到管中。再次离心样品，再重复两次。重组 gtfSI 溶液最终浓缩到大约 1 毫升。将浓缩物转移到微离心管中，并在摄氏四度时储存。继续执行其余功能恢复协议。在此协议中，agarose凝胶电泳显示，来自第一PCR和第二PCR的每个安培量的大小与预测尺寸相对应。链球菌突变菌落被第二个PCR产品转化，并镀在含有斑点霉素的脑心输液螺旋板上。在阿加罗斯凝胶上运行的殖民地PCR产品表明，每个安培量都具有预测尺寸。SDS-PAGE 将用固定金属亲和力色谱纯化的蛋白质作为单一波段进行观察。使用抗多晶石丁抗体进行的西方印迹证实，观测带是预期的160千吨多石丁标记蛋白。蔗糖衍生的生物膜形成能力只见链球菌突变野生型和链球菌突变体 His-gtfC，在管壁上形成附着生物膜，存在1%蔗糖。这在链球菌三角洲gtfC中未观察到。然而，25微克的重组gtfSI的加入恢复了链球菌三角洲gthFC的粘附生物膜形成能力。由于此方法的成功取决于第二次 PCR 扩增，因此第二个 PCR 产品必须通过电泳进行确认。这种方法获得的组蛋白类型蛋白也与功能性酸（如蛋白质-蛋白质相互作用）有关。该技术在发展之后，结合基因破坏，为微生物学领域的研究人员探索基因功能铺平了道路。

Research

JoVE Journal

Immunology and Infection

Purification of a High Molecular Mass Protein in Streptococcus mutans

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

总蛋白提取和二维凝胶电泳方法<em&gt;鼻疽</em&gt;物种

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

高产净化<em&gt;恶性疟原虫</em&gt;裂殖子适用于Opsonizing抗体检测

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode

一步法负色谱纯化<em&gt;幽门螺杆菌</em&gt;中性粒细胞激活蛋白过度表达<em&gt;大肠杆菌</em&gt;在批处理模式

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

RNA纯化在细胞内生长<em&gt;李斯特菌</em&gt;巨噬细胞

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

A Murine Model of Group B Streptococcus Vaginal Colonization

A Murine Model of Group B Streptococcus Vaginal Colonization

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...