저자 rWSN-PB2-Strep 바이러스 나다 Naffakh과 마리 앤 Rameix-Welti (Institut 파스퇴르)을 감사드립니다.

<html> 프로토콜의 개략도 순서도는 그림에 표시됩니다. 1과 시약의 테이블이 아래에 제공됩니다. 1. 감염 (16-24 H) <ol><li> Dulbecco의 수정된 이글 배지 (DMEM) - 높은들은 다음날 (약 5 X 10 8 셀) 약 80 %가 confluency 도달할 것이라는 등의 포도당 중간, 5 150mm 플라스틱 세포 배양 접시에 HELA 세포 밖으로 종자. 그것은 세포가 100 % confluency 도달하지 않는 것이 중요합니다. </li><li> 다음 날에는로 Strep-태그가 인플루엔자 바이러스 주식을 희석 인산 버퍼 식염수 (PBS)가 포함된 0.3 % 소 (BSA) 3 감염의 다수에 대한 혈청 알부민. </li><li> 세포 성장 매체를 제거 PBS로 한번 씻어, 10 ML PBS는 세포에 바이러스를 포함하는 추가합니다. 상온에서 1 H 동안 품어. </li><li> DMEM 높은 포도당으로 감염 매체를 교체, 37에서 부화를 계속 ° C. </li></ol>&quot;&gt; 부화의 길이는 (토론 참조) 공부되는 감염의 단계에 따라 달라집니다. 2. Subcellular 분별화 (3 H) <ol><li> 차가운 PBS로 한 번 세포를 씻어하고 고무 스크레이퍼와 50 ML 원뿔 튜브로 전송 차가운 PBS로 세포를 일시 중지합니다. </li><li> 1,000 rpm으로 5 분 용 테이블 탑 원심 분리기에서 다음 펠렛 세포는 PBS를 기음. </li><li> 10 ML 차가운 자당 버퍼의 Resuspend 세포 펠렛 (10 밀리미터 HEPES의 산도 7.9, 10 MM KCl, 2 밀리미터 MG 아세트산 (MgOAc), 3 MM CaCl 2, 340 밀리미터 자당, 1 ㎜ dithiothreitol (DTT), 1 % 테아제 억제제 믹스) . 정지 후, 세포 대단히 짧은 시간을 일부러 망치려고 10 ML 플라스틱 혈청의 피펫에 부착된 200 μl 피펫 팁을 통해 세포를 전달합니다. </li><li> 얼음에 10 분 알을 품다. 부드럽게 세포를 resuspend하기 위해 주기적으로 바꾸란. </li><li> 15 초위한 고속으로 0.5 %와 와동의 최종 농도로 Nonidet P-40 (NP-40)를 추가합니다. 즉시 테이블 상단 centrifu에서 4 ° C에서 3,500 XG에 10 분 동안 원심 분리기GE. </li><li> 4에서 뜨는 및 저장소를 제거 ° C. 이것은 세포질 분획이다. 펠렛은 원래 세포 펠렛보다 작고 하얀이 있어야합니다. 모든 분수는 4 ° C.에 최소 4 H 위해 저장할 수 있습니다 </li><li> 단계 2.5에서와 같이 5 ML 차가운 자당 버퍼, 다시 원심 분리기에 펠릿 씻고 뜨는 버리고. </li><li> 1.5 ML 차가운 nucleoplasmic 추출 완충액 (50 밀리미터 HEPES 산도 7.9, 150 MM KOAc, 1.5 밀리미터 MgCl 2, 0.1 % NP-40, 1 밀리미터 DTT, 1 % 프로 테아제 억제제 믹스)에 펠렛을 (핵 포함) Resuspend. </li><li> 꽉 끼는 유봉과 냉장, 전체가 유리로 4 ML 다운스 호모 지 나이저 편입 및 20 스트로크로 핵 homogenize. 균질화 동안 거품 마십시오. 저항은 약 10 스트로크 후에 증가한다. 효율적인 균질은 위상 대비 현미경으로 확인할 수 있습니다. </li><li> 1.5 ML의 microcentrifuge 튜브로 균질 핵 전송과 4에서 회전 바퀴에서 20 분 품어 °C. </li><li> 10 분 4 ° C에서 microcentrifuge에 16,000 X g에서 원심 분리기. </li><li> 4에서 뜨는 및 저장소를 제거 ° C. 이것은 nucleoplasmic 분수입니다. </li><li> 1.5 ML의 소화 버퍼 (50 밀리미터 HEPES 산도 7.9, 10 MM NaCl, 1.5 밀리미터 MgCl 2, 1 밀리미터 DTT, 1 % 프로 테아제 억제제 믹스, 100 U / ML Benzonase (RNA 분석을위한, 20 U / ML RNase와 대용품에 Resuspend 펠릿 무료 DNase I)) 37에 미리 예열 ° C, 37시 10 분 품어 ° C. </li><li> 5 M NaCl 42 μl를 (150 MM NaCl의 최종 농도)을 추가, 4시 20 분 추가 품어 ° C. </li><li> 10 분 4 ° C에서 microcentrifuge에 16,000 X g에서 원심 분리기. </li><li> 4에서 뜨는 및 저장소를 제거 ° C. 이것은 ch150 분수입니다. </li><li> Resuspend 펠렛 1.5 ML 추위 높은 소금 버퍼 (50 밀리미터 HEPES의 산도 7.9, 500 MM NaCl, 1.5 밀리미터 MgCl 2, 0.1 % NP-40, 1 밀리미터 DTT, 1 % 프로 테아제 억제제 믹스)와 얼음 속에서 20 분 알을 품다. </li><li> 16,000에서 X G &lt;을 원심 분리기/ em&gt;는 10 분, 4 ° C에서 microcentrifuge 인치 </li><li> 4에서 뜨는 및 저장소를 제거 ° C. 이것은 ch500 분수입니다. </li></ol> 3. Strep-태그 정화 (2 H) <ol><li> 세포질 분획 (150 MM NaCl의 최종 농도의 경우)에 5 M NaCl 300 μl를 추가합니다. </li><li> 15 ML 원뿔 튜브 분율 당 포장 Strep-Tactin 세파 로스 비즈의 나누어지는 100 μl. </li><li> 믹스, 그리고 탁상 1,000 X g에서 5 분 원심 분리기하도록 튜브 반전, 구슬로 Strep 세척 버퍼 10 볼륨 (20 밀리미터 HEPES의 산도 7.9, 150 MM NaCl (ch500 샘플 500 MM NaCl), 1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산)) 추가 원심 분리기. </li><li> , 뜨는을 삭제하시겠습니까 두번 단계 3.3 반복하고 Strep 세척 버퍼의 동일한 볼륨에 비드 펠렛을 resuspend. </li><li> 각각의 분율 (n​​uceloplasmic위한 1.5 ML의 microcentrifuge 튜브, ch150, 그리고 ch500 분수와 세포질 분획을위한 15 ML 원뿔 튜브)로 형편없는 Strep-Tactin 비드 슬러리 200 μl를 추가합니다. </li><li> 회전4에 튜브 1 시간 ° 회전 바퀴에서 C #. </li><li> 튜브에게 4에 1000 X g에서 1 분 ° C를 원심 분리기 뜨는 폐기하십시오. </li><li> 각 관 1 ML 추운 Strep 세척 버퍼를 추가합니다. 15 ML 튜브 (세포질 분획)에서 1.5 ML 관에 구슬을 전송합니다. 4 시에 튜브 1 분 ° C 단계 3.7에서와 같이 이후 centrifuging 씻으십시오. 두번 세차 단계를 반복합니다. </li><li> 마지막 세척 단계 후, 플랫 피펫 팁을 사용하여 세척 버퍼의 모든 흔적을 제거합니다. 100 μl 용출 버퍼 (Strep 세척 버퍼 2.5 MM desthiobiotin를 포함)을 추가하고 가볍게 섞는다. </li><li> 얼음에 15 분 알을 품다. 부드럽게 튜브의 바닥을 도청하여 가끔 구슬을 섞는다. </li><li> 원심 분리기 튜브 4에 1000 X g에서 1 분 ° C microcentrifuge 인치 뜨는 포함된 eluted Strep-태그를 vRNPs를 수집합니다. </li></ol> 4. 대표 결과 분별화의 효율성을 최고의 antib을 사용 fractionated lysates의 서양 얼룩 분석에 의해 규정되는subcellular 마커 (그림 2) 특정 오디. 특히, 성공적인 subnuclear 분별화는 nucleoplasmic에서 거의 없거나 전혀없는 세포 RNA 중합 효소 II (폴 II) 또는 용해 핵 분획 4, 대부분의 150 MM NaCl 5 추출해야 표시해야합니다. 폴 II의 저하도 reproducibly 문학 6 일관성 감염되지 않은 세포에 비해 인플루엔자 바이러스에 감염된 세포에서 관찰된다. 그림에서 세포질 eluate 위해 그림과 같이 vRNPs의 정화는 최고, 은색 또는 Coomassie 염색법에 의해 판단된다. 3. 우리의 경험에서, 대부분의 Strep-PB2는 완전하게 구성된 vRNP의 일환으로, 즉 약간 용해 효소 포로가되어로 정화된다. 은색 또는 Coomassie의 염색법으로 관찰 PA/PB1/PB2 비율 : 이것은 높은 NP에 반영됩니다. 여러 유비 쿼터스 RNases (예 : RNase 등) dissoci에 같은 방식으로 분열 바이러스성 RNA로 알려져있다으로 낮은 비율은 RNases와 버퍼 오염을 제안합니다NP multimers 7 효소를 먹었어요. 흥미롭게도, 혼자 Benzonase 치료는 바이러스 RNA를 소화하기에 충분하면서 vRNP 단백질 stochiometric 비율에 아무런 영향을 미치지 않습니다 것처럼 보인다. 우리가이 현상을 설명할 수는 없지만, 우리는 그 Benzonase 제안 기타 RNases (데이터가 표시되지 않음)과 소화 후에 vRNPs의 장애가 특정 구조적으로 중요한 RNA 영역을 그대로두고 있습니다 관찰했다. 세포질과 nucleoplasm (nuclease의 소화하지 않고 수행)에서 vRNPs 정화 후, 고분자 중량 8 희미하지만 이산 밴드는 8 인플루엔자 게놈 분야의 예측 분자 무게에 해당하는 실버 염색법, (심판을 참조하십시오. 3 관찰할 수있다 ). 9 H 포스트 감염에서 각 분획에서 정화 vRNPs의 상대적인 양의 그림에 표시됩니다. 4. vRNPs의 분포는 초기 시간 지점에서 ch150 분수의 강한 축적과 함께, 감염의 과정을 통해 다양하고,늦은 시간 지점에서 nucleoplasm 및 세포질에서 증가 축적. <img src="/files/ftp_upload/4028/4028fig1.jpg" alt="그림 1"/> subcellular 분별화의 그림 1. 플로우 차트. 심판에서 적응. 3. <img src="/files/ftp_upload/4028/4028fig2.jpg" alt="그림 2"/> 그림 2. 인플루엔자 바이러스에 감염된 세포에서 subcellular 분수의 서양 얼룩 분석. 10 9 HELA 세포는 subcellular 분별화 이전 9 H에 대한 인플루엔자 바이러스 스트레인 WSN에 감염되었다. 총 단백질 균등 금액이 각 레인에로드하고 표시된 항체를 사용하여 분석되었다. RNA 중합 효소 II는 (폴 II) C-말단 도메인 (hypophosphorylated 밴드가 가장 두드러진입니다) 모든 형태를 인식 클론 8WG16를 사용하여 감지되었습니다. 심판에서 적응. 3. <img src="/files/ftp_upload/4028/4028fig3.jpg" alt="그림 3"/> 그림 3.vRNPs 투석의 실버 얼룩 분석. HELA 세포는 rWSN (마이너스 컨트롤) 또는 세포질 분획에서 Strep 정화 이어 9 H 용 rWSN-PB2-Strep, 감염되었다. 별표는 RNase의 소화 후 보이지 않는 밴드를 나타냅니다. 심판에서 적응. 3. <img src="/files/ftp_upload/4028/4028fig4.jpg" alt="그림 4"/> 그림 4. 다른 세포 분수에서 정화 vRNPs의 실버 얼룩 분석. 10 9 HELA 세포가 9 rWSN-PB2-Strep 또는 rWSN 감염된 H는 subcellular 분별화 및 Strep 정화로 따라갔다. 각 분획에서 eluate의 균등 금액이로드되었습니다. 심판에서 적응. 3.

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관심의 어떠한 충돌 선언 없습니다.

많은 연구가 최근에 각각의 단백질이나 인플루엔자 바이러스 감염 8 개입 셀룰러 네트워크를 확인하고 있지만, 이러한 상호 작용의 대부분의 기능 의미는 불분명 남아 있습니다. 독감 바이러스는 RNA 합성과 핵 9 복잡 biophysical 및 생화 학적 자연을위한 염색질 기반 기능의 절대적인 의존성을 감안할 때, 새로운 기술은 이러한 기능을 명료하게하다하셔야합니다. vRNPs의 친화력 정화...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> DMEM 높은 포도당 </td><td> Gibco </td><td> 11965-092 </td><td></td></tr><tr><td> BSA </td><td> 시그마 </td><td> A9418 </td><td></td></tr><tr><td> 단백 분해 효소 억제제 믹스 G </td><td> Serva </td><td> 39,101 </td><td></td></tr><tr><td> Benzonase Nuclease </td><td> Novagen </td><td> 71,206 </td><td> 25 U / μl </td></tr><tr><td> DNase 나 RNase 부담 </td><td> ThermoScientific </td><td> EN0523 </td><td> 50 U / μl </td></tr><tr><td> 균 질기를 Dounce </td><td> Wheaton의 </td><td> 432-1271 </td><td> &quot;B&quot;유봉을 입력하십시오 </td></tr><tr><td> Strep-Tactin 세파 로스 </td><td> IBA 게엠베하 </td><td> 2-1201-025 </td><td> 50 % 서스펜션 칼럼 형식도 사용할 수 있습니다 </td></tr><tr><td> Desthiobiotin </td><td> IBA게엠베하 </td><td> 2-1000-002 </td><td></td></tr></tbody></table>

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

모든 부정적인 - 가닥 RNA 바이러스와 마찬가지로 인플루엔자 바이러스의 게놈은 단일 좌초된 게놈은 핵 단백질 (NP)에 의해 encapsidated, 그리고 구성된 trimeric 효소 복합과 연관된되는 바이러스성 ribonucleoprotein 컴플렉스 (vRNP)의 형태로 패키지되어 PA, PB1, 그리고 PB2 subunits의. 그러나 대부분의 RNA 바이러스와는 달리, 인플루엔자 바이러스는 감염된 세포의 핵에 바이러스 RNA 합성을 수행합니다. 흥미롭게도, 바이러스성 mRNA 합성이 primers로 세포 사전 mRNAs를 사용하고,이 과정은 염색질 1 이루어지는 것이 제안되었습니다. 바이러스 중합 효소와 RNA 중합 효소 II 호스트뿐만 아니라, NP 및 호스트 nucleosomes 간의 상호 작용은 또한 1,2를 묘사하고있다. 최근 원 - Strep-태그를 인코딩 재조합 독감 바이러스의 세대 유전자가 바이러스 중합 효소의 PB2 subunit의 C-말단 (rWSN-PB2-Strep 3)로했습니다 융합실내는 설명했다. 이러한 재조합 바이러스가 감염된 세포에서 vRNPs 포함한 PB2 함유 단지,의 정화를 허용합니다. vRNPs 투석을 받기 위해서는, 세포 배양은 감염 및 vRNPs이 세포에서 파생된 lysates에서 정화 친화력 있습니다. 그러나 탁아소를 운영하는 용해 절차는 일반 nuclease의 존재에도 불​​구하고, 주로 inefficiently 염색질 바인딩된 자료를 추출 한 단계 세제 용해에 기반하고있다. 우리의 예비 작업은 핵 vRNPs의 많은 부분은 전통적인 세포 용해 동안 추출되지 않은 것을 제안, 따라서 친화력 정화 될 수 없었습니다. 이 추출 효율을 향상시키기 위해, 비 염색질 바인딩된 핵 vRNPs에서 염색질 표지인 분리, 우리는 인플루엔자 바이러스에 감염된 세포 단계 현명한 subcellular 추출 프로토콜을 적응. 간단히,이 절차는 먼저 세포에서 핵을 분리하고 용해 핵 단백질 (여기서 &quot;nucleoplasmic&quot;분수 되나) 추출합니다.남은 불용성 핵 자료는 다음 Benzonase, 두 소금 추출 단계 뒤에 unspecific의 DNA / RNA nuclease로 소화된다 : 먼저 150 MM NaCl (이하 &quot;ch150 &#39;되나)를 사용하여 다음 500 MM NaCl (&quot;ch500 &quot;) (그림 1 ). 이러한 소금 추출 단계 500 밀리미터 NaCl 아직 여전히 친화 행렬에 태그를 vRNPs의 바인딩을 허용 핵 vRNPs의 85 % 이상 solubilize하기에 충분하다고 우리의 관찰을 바탕으로 선정되었습니다. 감염된 세포의 subcellular 분별화 후, 그것은 각각의 분획의 친화력 정화 PB2-태그를 vRNPs하는 것이 가능하고 단백질과 각각 서양 얼룩 및 프라이머 확장자를 사용하여 RNA 구성 요소를 분석합니다. 최근, 우리는 500 MM NaCl (ch500) 3 추출한 염색질 분획에서 감염 후 늦은 점 중 그 vRNP 수출 단지 양식을 발견하는이 방법을 활용.

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

인플루엔자 바이러스는 호스트 세포 염색질과 협회들의 RNA 게놈을 복제. 여기서는 감염된 세포의 염색질에서 그대로 바이러스성 ribonucleoprotein 컴플렉스를 정화하는 방법을 제시한다. 바이러스 단지 투석을하는 것은 각각 서양 얼룩 및 단백질과 RNA 내용의 프라이머 확장명을 모두에 의해 분석할 수 있습니다.

감염된 세포의 염색질에서 인플루엔자 바이러스 Ribonucleoprotein 컴플렉스의 동질 정제

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

affinity-purification-influenza-virus-ribonucleoprotein-complexes

Research

JoVE Journal

Immunology and Infection

13.0K 조회수.  Universitätsklinikum Freiburg. 인플루엔자 바이러스는 호스트 세포 염색질과 협회들의 RNA 게놈을 복제. 여기서는 감염된 세포의 염색질에서 그대로 바이러스성 ribonucleoprotein 컴플렉스를 정화하는 방법을 제시한다. 바이러스 단지 투석을하는 것은 각각 서양 얼룩 및 단백질과 RNA 내용의 프라이머 확장명을 모두에 의해 분석할 수 있습니다.

비디오: 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.

플라스미드 DNA의 재조합 독감 바이러스의 생성

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

연구

면역학 및 감염병학

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.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

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.

병원체 Vacuoles의 정제에서<em&gt; Legionella</em&gt; - 감염된 Phagocytes

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.

배큘로 바이러스 발현 시스템을 사용하여 소설 H7N9 인플루엔자 바이러스의 기능 재조합 적혈구 응집소와 뉴 라미니다 아제 단백질의 발현

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.

인간 백신 닭 계란에 인플루엔자 바이러스 전파

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.

인플루엔자 A 바이러스의 핵 단백질과 마우스 MX1 단백질의 공동 면역 침전

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.

돼지에서 인플루엔자 바이러스 탐지 및 격리를위한 코 닦음

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.

표현형 및 만성 림프 구성 맥락 수막염 바이러스에 감염된 마우스에서 고립 활성화 규제 T 세포의 기능 분석

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.

박테리아와 균류에 의해 바이러스에 감염된 세포에 바이오 필름 개시의 결정

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.

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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 ...