Name: using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy
Uploaded: 2011-11-01T12:00:00
Description: Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

이 작품은 부여 5 번 우수 서양 지역 센터 (WRCE)를 통해 U54 AI057156 - 07, 알레르기 및 전염병 국립 연구소에서 1 R01 AI08764301 - A1, 그리고 대학에서 백신 개발을위한 Sealy 센터에서 내부 자금으로 투자되었다 텍사스 의료 지점.

1. 플라스미드 DNAs 2 재조합 MP - 12 인코딩 NSS 변이 (들)의 복구 <ol><li> 보급 아기 햄스터 신장 (BHK) / T7 - 9 세포 15, 안정적으로 표현 T7 RNA 효소, 최소 필수 매체 6 cm 요리로 (가상) 알파 10% 태아 소 혈청 (FBS를 포함하는 (Invitrogen, 고양이 # 32561037) ), 페니실린 - 스트렙토 마이신 (페니실린 : 100 U / ML, 스트렙토 마이신 : 100 μg / ML) (Invitrogen, 고양이 # 15140122), 600 μg / hygromycin B의 ML (Cellgro, 고...

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RVFV에 대한 유전학 시스템을 리버스는 T7 프로 모터 2,4,5 드나 마우스 3 인간 4 POL - I 프로 모터를 이용하여 여러 그룹에 의해 개발되었습니다. 이 원고에서는, 우리는 BHK/T7-9 세포 15되는 안정 표현 T7 RNA 효소를 사용하여 재조합 RVFV MP - 12 계통을 생성하는 프로토콜을 설명합니다. 바이러스 복구의 효율 BHK/T7-9 세포의 상태에 따라 다양, plasmids의 양을, transfected 세포 ?...

<table border="1"><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 최소 필수 중간 (가상) - 알파 </td><td> Invitrogen </td><td> 32561037 </td><td></td></tr><tr><td> Dulbecco의 수정 최소 필수 배지 </td><td> Invitrogen </td><td> 11965092 </td><td></td></tr><tr><td> 수정 독수리 중간 (가상 2X) </td><td> Invitrogen </td><td> 11935046 </td><td></td></tr><tr><td> 페니실린 - 스트렙토 마이신 </td><td> Invitrogen </td><td> 15140122 </td><td></td></tr><tr><td> Hygromycin B </td><td> Cellgro </td><td> 30-240 - CR </td><td></td></tr><tr><td> Tryptose 인산 국물 </td><td> MP의 biomedicals </td><td> 1,682,149 </td><td></td></tr><tr><td> 귀족 한천 </td><td> VWR </td><td> 101170-362 </td><td></td></tr><tr><td> 대중 교통 - LT1 </td><td> Mirus </td><td> MIR2300</td><td></td></tr><tr><td> Opti - 가상 </td><td> Invitrogen </td><td> 31985070 </td><td></td></tr><tr><td> 에어로졸 꽉 뚜껑 </td><td> Eppendorf </td><td> C - 2223-25 </td><td></td></tr><tr><td> 0.33 % 중성 빨간색 솔루션 </td><td> 시그마 알드리치 </td><td> N2889 - 100ML </td><td></td></tr><tr><td> C57/WT MEF 세포 </td><td> InvivoGen </td><td> mef - c57wt </td><td></td></tr><tr><td> Blasticidin S </td><td> InvivoGen </td><td> 개미 - BL - 1 </td><td></td></tr><tr><td> Zeocin </td><td> InvivoGen </td><td> 개미 - zn - 1 </td><td></td></tr><tr><td> QUANTI - 블루 </td><td> InvivoGen </td><td> 담당자 - qb1 </td><td></td></tr><tr><td> BHK/T7-9 세포 15 </td><td> 기후 대학, 일본 </td><td></td><td></td></tr><tr><td> Vero E6 세포 </td><td> ATCC </td><td> CRL - 1586 </td><td></td></tr></table>

using reverse genetics to manipulate the nss gene of the rift valley fever virus mp-12 strain to improve vaccine safety and efficacy

출혈성 발열, 신경 장애 또는 인간의 실명 및 고속 낙태와 ruminants 1 태아의보기 흉한 것의 원인이 리프트 밸리 열병 바이러스 (RVFV)는, 선택한 에이전트 및 위험 그룹 3 병원체를 중복 HHS / USDA으로 분류되었습니다. 그것은 가족 Bunyaviridae에 속 Phlebovirus에 속한이 가족의 가장 악성 멤버 중 하나입니다. RVFV MP - 12 백신 스트레인 2,3뿐만 아니라 ZH548 및 ZH501를 포함한 야생 형 RVFV의 변종 4-6에 대한 몇 가지 역방향 유전 시스템은 2006 년부터 개발되었습니다. MP - 12 변형은 (위험 그룹 2 병원체가 아닌 선택 대리인되는) 몇몇은의 돌연변이 M과 L - 세그먼트로 높은 감쇠 있지만, 여전히 기능 독기를 인코딩 악성 S - 세그먼트 RNA 3, 운반 요소, NSS. 그것으로 effic 복제하면서 rMP12 - C13type (C13type)는 NSS ORF의 삭제 - 프레임의 69 % 나르는 것은, 모든 알려진 NSS 기능을 결여ient는 VeroE6 세포의 MP - 12 타입 - I IFN에 부족 않습니다. NSS는 인터페론 (IFN) - 베타 mRNA 7,8를 포함하여 호스트 전사의 종료 - 오프를 유도하고 사후 translational 수준에서 두 번 좌초 RNA - 의존 단백질 키나제 (PKR)의 저하를 추진하고 있습니다. 9,10 IFN - 베타 transcriptionally입니다 인터페론 규제 요인 3 (IRF - 3), NF - KB 및 활성 단백질 1 (AP - 1) 및 IFN-alpha/beta 수용체에 IFN - 베타의 바인딩 (IFNAR)에 의해 upregulated는 IFN - 알파의 전사를 촉진 NSS에 의한 IFN - 베타 유전자를 포함하여 호스트 전사 억제 바이러스는 복제에 대한 응답으로 그 ISGs의 유전자 upregulations을 방지 반면, 호스트 항바이러스 활동을 유도 유전자 또는 다른 인터페론 자극 유전자 (ISGs) 11, 비록 IRF - 3, NF - KB와 활성제 단백질 1 (AP - 1) RVFV7에 의해 활성화될 수 있습니다. . 따라서, NSS 추가 MP - 12을 자제하고, IFN - 베타 억제 기능을 폐지하여 호스트 타고난 면역 반응을 향상시킬 수있는 좋은 대상입니다. 여기에우리는 재조합 MP - 12 인코딩 변이된 NSS 창출을위한 프로토콜을 설명하고, IFN - 베타 mRNA 합성을 억제하는 기능을 부족한 NSS 돌연변이를 확인하기 위해 검사 방법의 예를 제공합니다. 타고난 면역에 그 본질적인 역할뿐만 아니라, 입력 - I IFN이 돌기 세포 및 적응 면역 반응의 유도 12-14의 성숙을 위해 중요합니다. 따라서, NSS의 돌연변이 타입 - I IFN을 유도하는 것이 더 감쇠 있지만, 동시에 접종 방식에 적합하게 후보자 야생 형 MP - 12보다 자극 호스트 면역 반응에 더 효율적입니다.

Watch this Scientific Journal Video about Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy at JoVE.com

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

리프트 밸리 열병 바이러스 MP - 12 백신 스트레인에 대한 역방향 유전 시스템은 증가 감쇄 및 immunogenicity로 추가 MP - 12 돌연변를 만들기위한 유용한 도구입니다. 우리는 NSS 돌연변이 변종을 생성하고 특성화 프로토콜을 설명합니다.

리프트 밸리 열병 바이러스 백신 안전 및 효능을 향상하기 위해 MP - 12 스트레인의 NSS 진을 조작하는 역방향 유전자를 사용하여

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

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