Name: rescue of recombinant newcastle disease virus from cdna
Uploaded: 2013-10-11T23:30:00
Description: Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

लेखक डीआरएस की प्रयोगशालाओं में अतीत और वर्तमान सदस्यों को धन्यवाद देना चाहूंगा. पीटर Palese और NDV के विकास के लिए Adolfo गार्सिया-Sastre आनुवांशिकी तकनीक और तकनीकी सहायता के लिए रिवर्स. NIAD R01AI088770 अनुदान और उत्कृष्टता के होमलैंड सुरक्षा विज्ञान और प्रौद्योगिकी केंद्र के विभाग द्वारा लिए द्वारा एजी-प्रयोगशाला में न्यूकासल रोग वायरस में रिसर्च आंशिक रूप से वित्त पोषित है उभरते और जूनोटिक पशु रोग (CEEZAD, पुरस्कार संख्या 2010-ST-061-AG001). एल एम एस प्रयोगशाला में रिसर्च एनआईएच अनुदान RO1 AI077719, R21NS075611-01, R03AI099681-01A1, इन्फ्लुएंजा अनुसंधान और निगरानी (HHSN266200700008C) के लिए उत्कृष्टता के NIAID केंद्र, और biodefense इम्यून मॉडलिंग के लिए रोचेस्टर सेंटर के विश्वविद्यालय (HHSN272201000055C) द्वारा वित्त पोषित है .

1. स्तनधारी कोशिकाओं की तैयारी (चित्रा -4 ए, दिन 1) स्प्लिट एचईपी-2 या A549 कोशिकाओं 6 अच्छी तरह प्लेटें में अभिकर्मक से पहले दिन. कोशिकाओं के घनत्व अगले दिन 80-90% संगम तक पहुंच जाना चाहिए. आमतौर पर, एक मिल?...

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कई कारकों NDV बचाव करते हुए अच्छे परिणाम प्राप्त करने के लिए विचार किया जाना है. सबसे पहले, प्रयोग की जाने वाली पूर्ण लंबाई सीडीएनए निर्माण NDV जीनोम में नए transgenes / संशोधनों के कार्यात्मक समावेश अनुमति देने क...

न्यूकासल रोग वायरस (NDV), Avulavirus जीनस 1 से संबंधित एक एवियन paramyxovirus, गंभीर रूप से दुनिया भर के सभी पोल्ट्री खेती को प्रभावित कर सकते हैं जो एक आर्थिक रूप से प्रासंगिक और इस तरह व्यापक रूप से शोध और surveilled जूनोटिक एजेंट है. नहीं एक मानव रोगज़नक़ हालांकि, NDV भी अच्छी तरह से एक मॉडल paramyxovirus के रूप में और इसकी वजह से बेहद रोचक, प्राकृतिक oncolytic गुणों से 6 दोनों पशुचिकित्सा क्षेत्र से परे अध्ययन किया गया है. NDV पर अनुसंधान बहुत पहले CONZELMANN और coleagues 2 से रेबीज वायरस के लिए वर्णित एकल असहाय, गैर खंडों नकारात्मक भावना आरएनए वायरस, के लिए रिवर्स आनुवंशिकी तकनीक के विकास से लाभ हुआ. विदेशी जीन या उनके जंगली प्रकार जीनोम के संशोधनों को ले जाने आनुवंशिक रूप से संशोधित NDVs, की एक किस्म व्यापक रूप से कभी के बाद से अध्ययन किया गया है. इन पुनः संयोजक वायरस के साथ काम NDV की बल्कि अन्य प्रासंगिक हुमा की न केवल अलग विषैलापन कारकों को चिह्नित करने के लिए आलोचना की गई हैn इस तरह इन्फ्लूएंजा ए वायरस के रूप में 7 रोगजनकों - या आकस्मिक Nipah वायरस 8. इसके अलावा, विभिन्न अध्ययनों के एक नंबर ज्यादातर वायरस के immunostimulatory गुणों को बढ़ाने के द्वारा, NDV 6,9,10 की जन्मजात antitumoral गतिविधि में सुधार के लिए इन तकनीकों के प्रयोग का पता लगाया है. पुनः संयोजक NDVs पर अनुसंधान के अन्य प्रासंगिक क्षेत्र इन्फ्लूएंजा 5,11,12 के रूप में अन्य वायरल बीमारियों के खिलाफ टीका उम्मीदवारों की पीढ़ी कर दिया गया है, एचआईवी 13, सार्स 15, 14 खसरा, या कि श्वसन sincytial वायरस (आरएसवी) 16 की वजह से. NDV द्वारा प्रदान की विभिन्न उल्लेखनीय फायदे मानव आबादी में प्रतिरक्षा preexisting की कमी कर रहे हैं के बीच, विदेशी आनुवंशिक सम्मिलित करता है, recombinatory गतिविधि की कमी और समग्र aforementioned प्राकृतिक immunostimulatory गुण 17 के साथ संयुक्त एक उच्च सुरक्षा प्रोफाइल की स्थिरता. यह भी पी में पुनः संयोजक बीवालेन्त टीके की क्षमता का उपयोग उल्लेखनीय हैoultry, NDV दोनों के खिलाफ रक्षात्मक और उच्च रोगजनक एवियन इन्फ्लूएंजा 11,12 वायरस. इस प्रकार भी मनुष्य के लिए खतरनाक बर्ड फ्लू के संभावित अंतर - विशिष्ट कूद को रोकने में मदद, पालतू जानवरों के लिए जंगली से फैल बाद के अवसरों को कम करने के लिए एक शानदार तरीका हो सकता है. अंत में, रिपोर्टर व्यक्त NDV सहज प्रतिरक्षा प्रतिक्रिया के मूल्यांकन के साथ ही कई वायरस 18-27 द्वारा इनकोडिंग इंटरफेरॉन प्रतिपक्षी की पहचान के लिए इस्तेमाल किया गया है. एक पुनः संयोजक का बचाव प्रक्रिया, गैर खंडों, नकारात्मक असहाय आरएनए वायरस मूल रूप से कृत्रिम रूप से Ribonucleoprotein या RNP के रूप में जाना न्यूनतम संक्रामक आणविक मशीनरी, चित्रा (2) एन्कोडिंग सीडीएनए transfecting द्वारा एक उत्पादक सेल में एक वायरल प्रतिकृति चक्र मजबूर पर होते हैं. RNPs वायरल पोलीमर्स (पी और एल प्रोटीन) से मिलकर बनता है, nucleoprotein (एनपी) और वायरस के पूर्ण लंबाई antigenomic आरएनए. इस शाही सेना + antigenome वें हैभी वायरल RNP के प्रोटीन के आराम के साथ जुड़ा हुआ है, जो पूरक आरएनए जीनोम की पीढ़ी के लिए आवश्यक ई टेम्पलेट, एक प्राकृतिक वायरस संक्रमण पर सेल के cytoplasm (चित्रा 2A पर रिलीज होगी कि एक ही संक्रामक जटिल स्मरण दिलाता है ). बाद में इस कदम से, वायरल चक्र स्वाभाविक रूप से आगे बढ़ सकते हैं और संशोधित जीनोम encapsidating पुनः संयोजक virions, चित्रा (2 बी) उत्पन्न हो जाएगा. उल्लेखनीय है, बजाय antigenomic सीडीएनए के जीनोमिक सीडीएनए के अभिकर्मक बहुत impairs या पूरी तरह से बचाव दक्षता 2,28-30 समाप्त करता है. Antigenomic सीडीएनए ट्रांसफ़ेक्ट है, तब भी जब कोशिकाओं में RNPs में पुनः संयोजक शाही सेना के encapsidation की दक्षता शायद बहुत कम है. इस वजह से, NDV के लिए बचाव प्रोटोकॉल अक्सर अनुमोदक कोशिकाओं के साथ और / या द्वारा उन्हें coculturing द्वारा मूल रूप से ट्रांसफ़ेक्ट कोशिकाओं से जारी कुछ वायरल कणों के प्रवर्धन के लिए विभिन्न चरणों में शामिलembryonated अंडे का संक्रमण. पिछले बचाव के लिए, सीडीएनए वांछित संशोधनों उत्पन्न करने के क्रम में मानक क्लोनिंग प्रक्रियाओं द्वारा चालाकी से किया जा सकता है. विभिन्न जीन उत्पादों और वायरस के विनियामक दृश्यों के विशिष्ट परिवर्तन straightforwardly इस तरह से प्राप्त किया जा सकता है, पुनः संयोजक NDV शामिल प्रकाशित काम के कई NDV जीनोम में एक नया ट्रांसक्रिप्शनल इकाई के अलावा आवश्यकता है. Paramyxovirus परिवार के अन्य सदस्यों की तरह, NDV जीनोम विभिन्न वायरल जीवन चक्र 1 के लिए महत्वपूर्ण एक कम ढाल में 3 &#39;के अंत तक उनके स्थान सम्मान के आधार पर व्यक्त कर रहे हैं जो छह ट्रांसक्रिप्शनल इकाइयों में आठ विभिन्न प्रोटीन encodes. इस वजह से, जीनोम के भीतर नई ट्रांसक्रिप्शनल इकाई के स्थान को ध्यान से वायरल प्रतिकृति के transgene और हानि की अभिव्यक्ति के बीच एक संतुलन प्राप्त करने के लिए चुना जाना चाहिए. पी और एम जीन के बीच निवेशन सबसे, टी इस्तेमाल किया गया हैHough अन्य साइटों को भी 13,31 परीक्षण किया गया है. (मैं) NDV जीनोम में शामिल होने के लिए किसी भी नए जीन वायरल शाही सेना पर निर्भर शाही सेना के लिए उपयुक्त संकेतों के नियंत्रण के अधीन हो गया है: जो कुछ भी डालने, NDV सीडीएनए में क्लोनिंग एक rescuable निर्माण उत्पन्न करने के लिए कुछ नियमों का पालन करने की जरूरत है पोलीमर्स. इन दृश्यों नया खुला पढ़ने फ्रेम (ओआरएफ) के अपस्ट्रीम में जोड़ा जाना चाहिए ताकि पोलीमर्स एक एकल nucleotide intergenic अनुक्रम (आईजी) से स्थान दिया गया है, जो पिछले जीन (जीई) और नई transgene (जी एस) की शुरुआत के अंत पहचान सकते हैं . Paramyxoviridae परिवार के अधिकांश सदस्यों के लिए के रूप में NDV (ii) कुशल प्रतिकृति, जीनोम लंबाई के कई किया जा रहा है पर निर्भर है, एक वैध कोज़ाक (कश्मीर) यूकेरियोटिक ribosomal अनुवाद में सुधार करने के अनुक्रम के अलावा भी बेहतर विदेशी प्रोटीन अभिव्यक्ति 32 के लिए सिफारिश की है छह 33, इसलिए NDV में किसी भी प्रविष्टि इस &quot;छह के नियम&quot; का पालन किया है. यदि आवश्यक हो, अनुरोधuired अतिरिक्त nucleotides बहाव के नए ओआरएफ जोड़ा जा सकता है, और (iii) transgene की अनुक्रम खोजने के लिए जाँच की जानी चाहिए संभव जीई और बचाव दक्षता, transgene अभिव्यक्ति और / या वायरस व्यवहार्यता प्रभावित हो सकती है, जो दृश्यों की तरह जी एस. अगर मौजूद है, इन दृश्यों चुप mutagenesis के द्वारा हटा दिया जाना चाहिए. aforementioned नियमों का पालन पुनः संयोजक पूरी लंबाई सीडीएनए की पीढ़ी कुशलता से यहाँ विस्तृत रूप में एक आनुवंशिक रूप से संशोधित NDV उत्पादन के क्रम में पहला कदम है. सिस्टम में सभी डीएनए निर्माणों T7 शाही सेना पोलीमरेज़ प्रमोटर (चित्रा 3) के नियंत्रण में हैं. इस cytoplasmic पोलीमर्स एक पुनः संयोजक संशोधित चेचक अंकारा वायरस (एमवीए-T7) 34 के साथ coinfection द्वारा ट्रांस में प्रदान की जाती है. चित्रा 3A पूर्ण लंबाई antigenomic सीडीएनए 5 encodes जो pNDV-B1 प्लाज्मिड, दिखा. चित्रा 3B pTM1 plasmids एनपी एन्कोडिंग से पता चलता है, पी और एल ORFs. , यू encodes जो प्लास्मिड pCITE GFP,nder T7 प्रमोटर, हरी फ्लोरोसेंट प्रोटीन (GFP), और चिकन बीटा actin के प्रमोटर 35 के तहत एक ही ओआरएफ encodes जो pCAGGS GFP 18, नियंत्रण के रूप में इस्तेमाल कर रहे हैं. इस प्रोटोकॉल में हम lentogenic NDV तनाव Hitchner बी 1 से 5 (चित्रा 4) के सीडीएनए से पुनः संयोजक NDV बचाव के लिए प्रक्रिया दिखा.

<table border="1">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>CORNING Cellgro</td>
			<td>10-013-CV</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>OptiMEM</td>
			<td>GIBCO</td>
			<td>31985-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (LPF2000)</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td></td>
		</tr>
		<tr>
			<td>35% Bovine Albumin (BA)</td>
			<td>Sigma</td>
			<td>232-936-2</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>CORNING Cellgro</td>
			<td>25-052-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (PS) 100x</td>
			<td>CORNING Cellgro</td>
			<td>30-002-CI</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td>Any supplier</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>
			Cell lines 
			A549 cells (catalogue number CRL-185), HEp-2 cells (catalogue number CRL-185), chicken embryo fibroblasts (catalogue number CRL-12203) and duck embryo fibroblasts (catalogue number CCL-141) are available from the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA). All cell lines are maintained in a 37 &#176;C incubator with 5% CO2 in DMEM 10% FBS, 1% PS. 
			Embryonated chicken eggs 
			Embryonated chicken eggs can be obtained from Charles River Laboratories, Specific Pathogen Fee Avian Supply (SPAFAS) Avian Products and Services (Franklin Commons, 106 Route 32, North Franklin, CT 06254, USA) and are maintained at 37 &#176;C. Viability of the embryos is assessed with an egg candler. Eggs are infected when they reach 8-10 days old. Both infection and harvest of the allantoic fluid takes place under sterile conditions. All eggs are autoclaved and discarded following standard laboratory biosafety protocols. 
			Turkey red blood cells (RBC) 
			Turkey RBC can be purchased from Truslow Farms (201 Valley Road, Chestertown, Md 21620, USA)and stored at 4 &#176;C. To prepare RBC for HA assay, wash 5 ml of the commercial stock with 45 ml of PBS 1x in a 50 ml conical tube. Centrifuge for 5 min at 1,000 rpm and carefully discard the supernatant. Dilute pelleted RBC 1:1,000 in PBS 1x for a final 0.5-1.0% concentration. Washed RBC can be stored at 4 &#176;C for several days. 
			Plasmids 
			Plasmid preparations are obtained with any commercially available maxiprep kit following manufacturer&#39;s instructions, diluted in ddH20 to a final concentration of 1 &#956;g/&#956;l and stored at -20 &#176;C. DNA concentration and purity are assessed by spectrophotometry at 260 and 280 nm. Preparations with a 260/280 ratio higher than 1.8 are considered of acceptable quality for the rescue. Plasmid DNA quality is also routinely double-checked by agarose gel chromatography. 
			Viruses 
			The described protocol for rescue of the lentogenic NDV strain Hitchner B1 can be performed under biosafety level (BSL) 2 conditions. The Modified Vaccinia Ankara expressing the T7 RNA polymerase (MVA-T7) was described34 and obtained from Dr. Bernard Moss. This virus is growth in confluent monolayers of chicken embryo fibroblasts and titrated in mammalian (A549 or HEp-2) cells. NDV stocks are grown in embryonated chicken eggs and titrated by IFA using polyclonal serum raised against purified virions. All contaminated material should be safely sterilized and disposed according to standard biosafety procedures. 
			Tissue culture media and solutions: 
			DMEM 10% FBS 1% PS: Dulbecco&#39;s modified Eagle&#39;s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and Penicillin/Streptomycin (P/S): 445 ml of DMEM, 50 ml of heat-inactivated FBS, 5 ml of 100x commercial P/S solution. Store at 4 &#176;C. 
			10x Phosphate buffered saline (PBS): 80 g of NaCl, 2 g of KCl, 11.5 g of Na2HPO4.7H2O, 2 g of KH2PO4. Add ddH2O up to 1 L. Adjust pH to 7.3. Sterilize by autoclave. Store at room temperature. 
			1x PBS: Dilute 10x PBS 1:10 with ddH2O. Sterilize by autoclave and store at room temperature. 
			100x Ca/Mg : 1.327 g CaCl2.2H2O, 2.133 g MgCl2&#8226;6H2O and add ddH2O up to 100 ml. Autoclave and store at room temperature. 
			1x PBS/BA/PS: 50 ml of 10x Phosphate buffered saline (PBS) in 437 ml ddH2O. Autoclave and when cooled down to room temperature, add 5 ml 100x Penicillin/Streptomycin 3 ml 35% Bovine and 5 ml of 100x Ca/Mg. Store at 4&#176;C.
			</td>
		</tr>
	</tbody>
</table>

rescue of recombinant newcastle disease virus from cdna

न्यूकासल रोग वायरस (NDV), परिवार Paramyxoviridae 1 की Avulavirus जीनस का प्रोटोटाइप सदस्य, एक गैर खंडों, नकारात्मक भावना, एकल असहाय, टीकाकरण के लिए एक वेक्टर के रूप में संभावित अनुप्रयोगों के साथ शाही सेना वायरस (चित्रा 1) छा और है मानव रोगों का इलाज. इन आवेदनों की गहराई अन्वेषण केवल सीडीएनए 2-5 के रूप में उनकी पूर्ण जीनोम एन्कोडिंग plasmids से पुनः संयोजक वायरस बचाव के लिए रिवर्स आनुवंशिकी तकनीक की स्थापना के बाद संभव हो गया है. वायरल सीडीएनए सुविधा वायरस के जीनोटाइप को बदलने के लिए और / या नए ट्रांसक्रिप्शनल इकाइयों में शामिल करने के लिए मानक क्लोनिंग प्रक्रियाओं का उपयोग करके इन विट्रो में संशोधित किया जा सकता है. ऐसे आनुवंशिक रूप से संशोधित वायरस से बचाव कारकों संक्रमण के कई चरणों को प्रभावित करने, और साथ ही एंटीजन की अभिव्यक्ति और वितरण के लिए वैक्टर के विकास और सुधार के लिए अनुमति देता है समझने के लिए एक महत्वपूर्ण उपकरण प्रदान करता हैटीकाकरण और उपचार के लिए. यहाँ हम पुनः संयोजक NDVs के बचाव के लिए एक प्रोटोकॉल का वर्णन.

NDV का बचाव नियमित वायरस का पूरा सीडीएनए लिए उपयोग किया है कि प्रयोगशालाओं में प्रदर्शन एक सुस्थापित प्रक्रिया है. हालांकि, विधि के आंतरिक स्टोकेस्टिक प्रकृति यह कठिन 100% बचाव दक्षता हासिल करने के लिए बन?...

Watch this Scientific Journal Video about Rescue of Recombinant Newcastle Disease Virus from cDNA at JoVE.com

Rescue of Recombinant Newcastle Disease Virus from cDNA

न्यूकासल रोग वायरस (NDV) बड़े पैमाने पर अन्य लोगों के अलावा, टीकाकरण और उपचार के लिए नए वैक्टर विकसित करने के लिए पिछले कुछ वर्षों में अध्ययन किया गया है. इन अध्ययनों के कारण इस तरह हम यहाँ वर्णन उन के रूप में सीडीएनए से पुनः संयोजक वायरस बचाव के लिए तकनीक, संभव हो गया है.

सीडीएनए से संयोजक न्यूकासल रोग वायरस का बचाव

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

rescue-of-recombinant-newcastle-disease-virus-from-cdna

Research

JoVE Journal

Immunology and Infection

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.

पुनः संयोजक इन्फ्लुएंजा वायरस के डीएनए प्लाज्मिड से पीढ़ी

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

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.

Baculovirus अभिव्यक्ति प्रणाली का उपयोग उपन्यास H7N9 इन्फ्लूएंजा वायरस से कार्यात्मक संयोजक Hemagglutinin और neuraminidase प्रोटीन की अभिव्यक्ति

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

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

फेज-प्रदर्शित सिंथेटिक एंटीबॉडी पुस्तकालयों से स्केलेबल उच्च Throughput चुनाव

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

अमर छाँटे बी कोशिकाओं से पुनः संयोजक मानव आईजीजी मोनोक्लोनल एंटीबॉडी का सृजन

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

न्युट्रोफिल अलगाव और विश्लेषण करने के लिए चिकित्सीय एजेंट लिंफोमा सेल संवेदनशीलता में उनकी भूमिका का निर्धारण करने के लिए

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

एक नई दुनिया Zika वायरस संक्रमित क्लोन से पुनः संयोजक वायरस के बचाव और विशेषता

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

पता लगाने और संयोजक प्रोटोटाइप फोम वायरस Integrase के शोधन के दौरान Nuclease संदूषण को हटाने

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

तेजी से, सुरक्षित, और सरल मैनुअल बेडसाइड न्यूक्लिक एसिड निष्कर्षण पूरे रक्त के नमूनों में वायरस का पता लगाने के लिए

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

संक्रमित Ifnar1 के Immunoprivileged अंगों में Zika वायरस-विशिष्ट टी-सेल प्रतिक्रियाओं का मूल्यांकन-चूहों/

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

स्वाइन पूरे रक्त में व्यक्त कोडिंग और गैर कोडिंग आरएनए कक्षाओं की पहचान

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...