Yazarlar RWSN PB2-Strep virüs için Nada Naffakh ve Marie-Anne Rameix-Welti (Institut Pasteur) teşekkür etmek istiyorum.

<html> Protokolünün şematik akış şeması Şek. 1 ve reaktifleri bir tablo aşağıda sunulmuştur. 1. Enfeksiyon (16 - 24 saat) <ol><li> Dulbecco&#39;nun değiştirilmiş Eagle ortamı (DMEM)-yüksek bunlar ertesi gün (yaklaşık 5 x 10 hücre 8) yaklaşık% 80 konfluent ulaşacak örneğin glikoz, orta, 5 mm 150 plastik hücre kültür tabaklarında HeLa hücreleri tohumu. Bu hücreler% 100 birbirine karışmış ulaşamamaktadır olması önemlidir. </li><li> Ertesi gün, içine Strep-etiketli grip virüsü stoku seyreltik fosfat-tamponlu salin (PBS) içeren% 0.3 bovin (BSA) 3 arasında bir enfeksiyon çokluğu için serum albümini. </li><li> Hücrelerinden büyüme medyumu kaldırmak, bir kez PBS ile yıkanır ve 10 ml PBS hücrelere virüs içeren ekleyin. Oda sıcaklığında 1 saat süre ile inkübe edilir. </li><li> DMEM-yüksek glukoz ile enfeksiyon orta değiştirin ve 37 inkübasyon devam ° C </li></ol>&quot;&gt; Inkübasyon süresi (Tartışma bakınız) üzerinde çalışma yapılacak olan enfeksiyon faz bağlıdır. 2. Subselüler Fraksiyonu (3 saat) <ol><li> Soğuk PBS ile bir defa yıkanır ve ardından hücreler bir lastik sıyırıcı ve 50 ml konik tüpe transferi ile soğuk PBS içerisinde askıya hücreleri. </li><li> 1000 rpm&#39;de 5 dakika boyunca bir masa üstü santrifüj ile ve daha sonra pelet hücreler PBS aspire. </li><li> 10 ml soğuk sükroz tampon içinde Pastör pipetiyle hücre peleti (10 mM Hepes pH 7.9, 10 mM KCI, 2 mM Mg asetat (MgOAc), 3 mM CaCl2, 340 mM sukroz, 1 mM ditiyotreitol (DTT),% 1 proteaz inhibitörü karışımı) . Süspansiyon sonra, hücre kümeleri bozmak için bir 10 ml plastik pipet serum takılı bir 200 ul pipet aracılığıyla hücrelere geçerler. </li><li> Buz üzerinde 10 dk inkübe edin. Yavaşça hücreleri tekrar süspansiyon periyodik ters. </li><li> 15 sn için yüksek hızda% 0.5 ve girdaplı bir son konsantrasyon elde etmek Nonidet P-40 (NP-40) eklenir. Derhal bir masa üstü centrifu içinde 4 ° C&#39;de 3.500 x g&#39;de 10 dakika boyunca santrifüjge. </li><li> 4 at süpernatan ve mağaza çıkarın ° C Bu sitoplazmik bölümüdür. Pelet orijinal hücre peleti daha küçük ve daha beyaz olmalıdır. Bütün fraksiyonlar 4 ° C&#39;de en az 4 saat boyunca saklanabilir </li><li> Adım 2,5 gibi 5 ml soğuk sükroz tampon, tekrar santrifüj ile pelet yıkanır ve süpernatant atılır. </li><li> 1.5 ml soğuk nucleoplasmic ekstraksiyon tamponu (50 mM Hepes pH 7.9, 150 mM KOAc, 1,5 mM MgCl2,% 0.1 NP-40, 1 mM DTT,% 1 proteaz inhibitörü karışımı) içinde pelet (çekirdekler içeren) tekrar süspansiyon. </li><li> Bir dar havaneli ile soğutulmuş, tamamen cam 4 ml Dounce Homojenizatör transfer ve 20 vuruş ile çekirdekleri homojenize. Homojenizasyon sırasında köpük kaçının. Direnci yaklaşık 10 vuruş sonrası artırmalıdır. Verimli homojenleştirme bir faz kontrast mikroskop altında teyit edilebilir. </li><li> 1.5 ml mikrosantrifüj tüpü homojenize çekirdekleri aktarın ve 4 dönen bir tekerlek üzerinde 20 dk inkübe °C. </li><li> 10 dakika süre ile 4 ° C&#39;de bir mikrosantrifüj içinde 16,000 x g&#39;de santrifüje. </li><li> 4 at süpernatan ve mağaza çıkarın ° C Bu nucleoplasmic bölümüdür. </li><li> 1.5 ml sindirim tamponu (50 mM Hepes pH 7.9, 10 mM NaCl, 1.5 mM MgCl2, 1 mM DTT,% 1 proteaz inhibitörü karışımı, 100 U / ml Benzonase (RNA analizi için, 20 U / ml RNase ile ikame in Pastör pipetiyle pelet içermeyen Dnaz i)) 37 ön ° C&#39;a ısıtılmış ve 37 &#39;de 10 dakika inkübe ° C. </li><li> 5 M NaCl 42 ul (150 mM NaCl nihai konsantrasyon) ekleyin ve 4 de 20 dakika daha inkübe ° C. </li><li> 10 dakika süre ile 4 ° C&#39;de bir mikrosantrifüj içinde 16,000 x g&#39;de santrifüje. </li><li> 4 at süpernatan ve mağaza çıkarın ° C Bu ch150 bölümüdür. </li><li> Pastör pipetiyle pelet 1.5 ml soğuk yüksek tuz tamponu (50 mM Hepes pH 7.9, 500 mM NaCI, 1.5 mM MgCl2,% 0.1 NP-40, 1 mM DTT,% 1 proteaz inhibitörü karışımı) ve buz üstünde 20 dakika inkübe edilir. </li><li> 16.000 az x g &lt;santrifüjleyin/ Em&gt; 10 dakika süre ile 4 ° C&#39;de bir mikrosantrifüj içerisinde. </li><li> 4 at süpernatan ve mağaza çıkarın ° C Bu ch500 bölümüdür. </li></ol> 3. Strep-etiket Arıtma (2 saat) <ol><li> Sitoplazmik fraksiyon (150 mM NaCl nihai konsantrasyon) ile 5 M NaCl 300 ul ekleyin. </li><li> 15 ml konik tüp fraksiyon başına paketlenmiş Strep-Tactin Sepharose boncuk kısım 100 ul. </li><li> Karıştırmak ve bir masa üstü, 1.000 x g hızında 5 dakika santrifüj tüpü, invert boncuklara Strep yıkama tamponu ile 10 hacim (20 mM Hepes pH 7.9, 150 mM NaCl (ch500 numuneler için 500 mM NaCl), 1 mM EDTA) ekleyin santrifüj. </li><li> , Süpernatant atın iki kez adımı 3.3 tekrarlayın ve sonra Strep yıkama tamponu eşit hacmi boncuk pelletini. </li><li> Her bir fraksiyon (nuceloplasmic için 1.5 ml&#39;lik tüplere mikrosantrifüj, ch150 ve ch500 fraksiyonlar ve sitoplazmik fraksiyon için bir 15 ml konik tüpe) ile yıkanmış, Strep-Tactin boncuk bulamacın 200 ul ekleyin. </li><li> Döndürmek4 at tüpleri 1s ° dönen bir tekerlek üzerinde C. </li><li> Tüpler 4 az 1000 x g&#39;de 1 dk ° C&#39;de santrifüje Süpernatant atın. </li><li> Her tüpe 1 ml soğuk Strep yıkama tamponu ekleyin. 15 ml tüp (sitoplazmik fraksiyonu) 1.5 ml tüp boncuk aktarın. 4 de, tüm tüpler 1 dk ° C, adım 3,7 gibi sonra santrifüj yıkanır. Iki kez bu yıkama işlemi tekrarlayın. </li><li> Son yıkama adımdan sonra, düz bir pipet kullanarak yıkama tamponu tüm izlerini silmek. 100 ul yıkama tamponu (Strep yıkama tamponu 2.5 mM desthiobiotin içeren) ekleyin ve hafifçe karıştırın. </li><li> Buz üzerinde 15 dk inkübe edin. Yavaşça tüplerin alt dokunarak bazen boncuk karıştırın. </li><li> Santrifüj tüpünde 4 az 1000 x g&#39;de 1 dk ° C de mikrosantrifüj. Süpernatant içeren Yıkanan Strep-etiketli vRNPs toplayın. </li></ol> 4. Temsilcisi Sonuçlar Fraksiyonasyon etkinliğinin en antib kullanılarak parçalandı lizatlarının Western blot analizi ile karar verilirSubselüler belirteçler (Şekil 2) için belirli bir odies. Spesifik olarak, başarılı bir çekirdek içi fraksiyonasyon nucleoplasmic içinde çok az veya hiç hücresel RNA polimeraz II (Pol II), ya da çözünebilen Nükleer fraksiyonu 4, ve en çok 150 mM NaCl 5 ile ekstre edilmelidir göstermelidir. Pol II&#39;nin bir dejenerasyonu da tekrarlanabilir literatürde 6 ile tutarlıdır enfekte olmamış hücrelere, kıyasla influenza virüs ile enfekte olmuş hücrelerin içinde gözlenmiştir. Şekil bir sitoplazmik eluat için gösterildiği gibi vRNPs saflaştırılması iyi, gümüş veya Coomassie boyama tarafından değerlendirilecektir. 3. Bizim deneyimlerimize göre, en Strep-PB2 tam şekillendirilmiş vRNP bir parçası olarak, yani biraz çözünür polimeraz yakalanır arınmışsa. Gümüş veya Coomassie boyama tarafından gözlemlenen PA/PB1/PB2 oranı: Bu yüksek NP yansır. Birkaç yerde RNases (örneğin, RNaz A gibi) dissosiyatif olarak böyle bir şekilde çatlatma viral RNA&#39;nın bilinmektedir olarak daha düşük bir oran, RNases ile tampon kirlenme anlaşılacağıNP multimerleri 7 polimeraz yedik. İlginçtir, sadece Benzonase tedavisi, viral RNA sindirmek için yeterli iken, vRNP proteinlerin stokiyometrik oranları üzerinde hiçbir etkiye sahip olduğu görülmektedir. Bu fenomeni açıklamak değil de, biz bu Benzonase düşündüren diğer RNases (veriler gösterilmemiştir) ile sindirim sonrası vRNPs bozulması bazı önemli yapısal RNA bölgeleri bozulmadan bırakabilir gözlenmiştir. Sitoplazma ve nükleoplazmanın (nükleaz sindirim yapılmadan) gelen vRNPs saflaştırılması sonra, yüksek molekül ağırlıkları az 8 sönük ama farklı bantlar 8 grip genom segment tahmin molekül ağırlıkları karşılık gümüş boyama, (ref bakın. 3 ile takip edilebilir ). 9 saat, enfeksiyondan sonraki her fraksiyonda arındırılan vRNPs nispi miktarları Şekil &#39;de gösterilmiştir. 4. VRNPs dağılımı erken zaman noktalarında ch150 fraksiyonunda güçlü birikimi ile, enfeksiyon boyunca değişir veGeç zaman noktalarında nükleoplazmanın ve sitoplazma artmış birikimi. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="Şekil 1"/> Subselüler fraksiyonlama Şekil 1. Akış çizelgesi. Ref uyarlanmıştır. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="Şekil 2"/> Şekil 2. Influenza virüsü ile enfekte hücrelerden Subselüler fraksiyonları Western blot analizi. 10 9 HeLa hücrelerinde hücre içi fraksiyon önce 9 saat influenza virüs suşu WSN ile enfekte edildi. Toplam protein Eşit miktarlarda her kulvarın yüklenen ve gösterilen antikorlar kullanılarak analiz edildi. RNA polimeraz II (Pol II) C-terminal alanı (hypophosphorylated bandı en önemli olduğu), her türlü tanır klonu 8WG16 kullanılarak tespit edildi. Ref uyarlanmıştır. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="Şekil 3"/> Şekil 3.VRNPs saf gümüş leke analizi. HeLa hücreleri RWSN (negatif kontrol olarak) veya sitoplazmik fraksiyondan Strep saflaştırma takiben 9 saat boyunca RWSN-PB2-Strep ile enfekte edilmiştir. Asterisk RNaz sindirim sonra görünmez bantları ifade eder. Ref uyarlanmıştır. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="Şekil 4"/> Şekil 4. Farklı hücresel fraksiyonları arınmış vRNPs Gümüş leke analizi. 10 9 HeLa hücrelerinde 9 RWSN PB2-Strep veya RWSN ile enfekte olan h Subselüler fraksiyon ve Strep arıtma izledi. Her fraksiyonu eluat Eşit miktarlarda yüklendi. Ref uyarlanmıştır. 3.

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Çıkar çatışması ilan etti.

Birçok çalışmada son zamanlarda bireysel protein ya da grip virüsü enfeksiyonu 8 katılan hücresel ağları tespit ederken, bu etkileşimlerin çoğunluğunun fonksiyonel önemi belirsizdir. Grip virüsü RNA sentezi ve çekirdeğinde 9 karmaşık biofiziksel ve biyokimyasal doğaya kromatin bazlı fonksiyonların mutlak bağımlılığı göz önüne alındığında, yeni teknikler bu fonksiyonları aydınlatmak için gerekli olacaktır. VRNPs bir afinite saflaştırma ile birleştiğinde b...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> DMEM-yüksek glukoz </td><td> Gibco </td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td> Sigma </td><td> A9418 </td><td></td></tr><tr><td> Proteaz inhibitörü Mix G </td><td> Serva </td><td> 39101 </td><td></td></tr><tr><td> Benzonase Nükleaz </td><td> Novagen </td><td> 71206 </td><td> 25 U / ml </td></tr><tr><td> DNaz I, RNaz-free </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / ml </td></tr><tr><td> Homojenizatör Dounce </td><td> Wheaton </td><td> 432-1271 </td><td> &quot;B&quot; havaneli yazın kullanın </td></tr><tr><td> Strep-Tactin Sepharose </td><td> IBA GmbH </td><td> 2-1201-025 </td><td> % 50 süspansiyon sütun biçiminde de kullanılabilir </td></tr><tr><td> Desthiobiotin </td><td> IBAGmbH </td><td> 2-1000-002 </td><td></td></tr></tbody></table>

affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells

Tüm negatif dallı RNA virüsleri gibi, influenza virüs genomu tek zincirli genom nükleoprotein (NP) tarafından kapsül içine, ve oluşan trimeric polimeraz kompleksi ile ilişkili olduğu viral ribonükleoprotein kompleksleri (vRNP), formunda paketlenmiş PA, PB1 ve PB2 alt birimlerinin. Bununla birlikte, çoğu RNA virüslerinin aksine, influenza virüs enfekte olmuş hücreler, çekirdeklerinde viral RNA&#39;nın sentezi gerçekleştirmek. İlginçtir, viral mRNA sentezi primer olarak hücresel öncesi mRNA&#39;ların kullanır ve bu süreç kromatin 1 gerçekleşir ileri sürülmektedir. Viral polimeraz ve konak RNA polimeraz II yanı sıra, NP ve ev sahibi arasındaki nükleozom arasındaki etkileşimlerin da 1,2 karakterize edilmiştir. Yakın zamanda, bir tek Strep-Tag kodlayan rekombinant influenza virüsünün üretimi genetik olarak viral polimeraz PB2 alt biriminin C-terminali (RWSN-PB2-Strep 3) kadar olan kaynaşmıştr nitelendirdi. Bu rekombinant virüsler enfekte olmuş hücreler arası vRNPs dahil olmak üzere PB2-içeren kompleksleri, bir saflaştırma izin verir. VRNPs saflaştırılmış elde etmek için, enfekte hücre kültürleri ve vRNPs bu hücreler elde lizatları arındırılan afinitesi vardır edilir. Ancak, bugüne kadar kullanılan parçalama işlemleri genel bir nükleaz varlığına rağmen, çoğu zaman sadece verimsiz kromatin bağlı materyali çıkarmak, bir adım deterjan lizis dayalı olarak yapılmıştır. Bizim ön çalışma nükleer vRNPs büyük bir kısmı geleneksel bir hücre parçalanması sırasında elde olmadığını düşündürmüştür ve bu nedenle afinite saflaştırılmış olamazdı. Bu ekstraksiyon verimini artırmak için, ve non-kromatin bağlı nükleer vRNPs gelen kromatin-bağlı ayırmak için, biz grip virüsü ile enfekte olmuş hücrelerde bir adım bilge Subselüler çıkarma protokolü uyarlanmıştır. Kısaca, bu prosedür ilk hücreden çekirdek ayırır ve daha sonra eriyen nükleer proteinler (burada &quot;nucleoplasmic&quot; fraksiyon olarak adlandırılır) ayıklar.Kalan çözünmeyen nükleer malzeme ardından Benzonase, iki tuz çıkarımı adımları takip belirsiz bir DNA / RNA nükleaz ile sindirilir: İlk 150 mM NaCl (&quot;ch150&quot; olarak adlandırılır) kullanılarak, daha sonra 500 mM NaCl (&quot;ch500&quot;) (Şekil 1 ). Bu tuz ekstraksiyon adımları 500 mM NaCl henüz hala yakınlık matrisi tagged vRNPs bağlayıcı izin nükleer vRNPs% 85&#39;inden çözünürleştirmek için yeterli olduğu konusunda bizim gözlemlerimize göre seçilmiştir. Enfekte olmuş hücrelerin hücre içi fraksiyonasyon sonra, her bir fraksiyondan afinite purify PB2-etiketli vRNPs mümkündür ve protein ve sırasıyla, Western blot ve astar uzantısı, RNA kullanılarak analiz bileşenler. Son zamanlarda, 500 mM NaCl (ch500) 3 ile ekstrakte kromatin fraksiyonu üzerine enfeksiyon sonrası geç noktaları sırasında bu vRNP ihracat kompleksleri şeklinde keşfetmek için bu yöntem kullanılmıştır.

Watch this Scientific Journal Video about Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells at JoVE.com

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Grip virüsleri konak hücre kromatin ile birlikte kendi RNA genomu çoğaltmak. Burada, enfekte olmuş hücrelerin kromatin gelen sağlam virüs ribonükleoprotein kompleksleri arındırmak için bir yöntem sunulmaktadır. Viral kompleksleri Arıtılmış sırasıyla, Western blot ve protein ve RNA içerikleri astar uzantısı hem de analiz edilebilir.

Enfekte Hücre Kromatin gelen Gribi ribonükleoprotein Komplekslerinin Affinity Arıtma

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

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JoVE Journal

Immunology and Infection

13.0K Görüntüleme.  Universitätsklinikum Freiburg. Grip virüsleri konak hücre kromatin ile birlikte kendi RNA genomu çoğaltmak. Burada, enfekte olmuş hücrelerin kromatin gelen sağlam virüs ribonükleoprotein kompleksleri arındırmak için bir yöntem sunulmaktadır. Viral kompleksleri Arıtılmış sırasıyla, Western blot ve protein ve RNA içerikleri astar uzantısı hem de analiz edilebilir.

Video: Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Plazmid DNA Rekombinant Grip Virüsü Üretimi

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

İmmünoloji ve Enfeksiyon

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Gribi için yüksek verimlilik Tespit Yöntemi

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

Patojen Vakuoller saflaştırılması gelen<em&gt; Legionella</em&gt;-Enfekte Fagositler

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

Bakülovirüs İfade Sistemi ile Novel H7N9 influenza virüs hemaglutinin Fonksiyonel rekombinant ve Neuraminidase Proteinlerin İfadesi

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Embriyone Tavuk Yumurta Grip Virüsü Yayılım

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

İnfluenza A virüsü nükleoproteini Fare MX1 Protein Co-immunopresipitasyonu

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Domuz influenza A virüs Algılama ve İzolasyonu için Burun Mendil

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Fenotipik ve Kronik Lenfositik koryomenenjit Virüs ile enfekte Fare İzole Aktif Düzenleyici T hücrelerinin Fonksiyonel Analiz

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

Bakteri ve mantar virüsle enfekte olmuş hücreler üzerinde Biyofilm Başlatma Belirlenmesi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...