质粒DNA重组流感病毒的一代

Summary

质粒DNA的A型流感病毒的救援是一个基本的和必要的实验技术，使流感的研究人员，以产生重组病毒在流感病毒的生物学特性研究的多个方面，被视为潜在的载体或疫苗使用。

Abstract

多项流感研究小组的努力已在A型流感病毒反向遗传学的发展和改善的关键。成立于1999年^1,2质粒为基础的反向遗传技术，以产生重组病毒已经彻底改变了流感研究领域，因为具体问题，通过基因工程，传染病，重组流感病毒已回答。这些研究包括病毒的复制，病毒蛋白的功能，特定的突变病毒蛋白在病毒复制和/或发病机制中的贡献，此外，使用病毒载体表达外源蛋白的重组流感病毒³。

Protocol

1。流感病毒救援转 A型流感病毒属于负链RNA包膜病毒的 Orthomyxoviridae家庭。 A型流感病毒的基因组由8个不同的RNA负极的基因编码，至少11病毒蛋白（图1） 4。在这份报告中，我们将重点抢救其中一个最常见的实验室菌株，使用含8流感病毒A/PR/8/34段ambisense质粒（PDZ）流感A/PR/8/34，5 （图2）。 对于抢救质粒DNA的重组流感病毒，我们建议3?…

Discussion

质粒DNA的重组流感病毒的救援是一个简单明了的过程，一旦该协议是经常在实验室中进行，但在开始的时候，多个事情都可能出错。生成的病毒，它必须有良好的质粒准备。正确保养的细胞株（293T和MDCK）是一个成功的病毒救援的关键。传统上，一个遗传标记是插入基因编码质粒流感，沉默突变。这种沉默突变（S）的简介和创作，例如，一个新的限制性内切酶站点是用来区分野生型和经酶切重组?…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
DMEM		Invitrogen	11995-065	Store at 4°C
OptiMEM		Invitrogen	51985-034	Store at 4°C
Lipofectamine 2000 (LPF2000)		Invitrogen	11668-019	Store at 4°C
TPCK-trypsin		Sigma	T-8802	Store at -20°C
Bovine Albumin (BA)		Sigma	A7979	Store at 4°C
Trypsin-EDTA		Invitrogen	25300-054	Store at -20°C
Penicillin/Streptomycin (PS) 100X		Invitrogen	15140-122	Store at -20°C
Fetal Bovine Serum (FBS)		Hyclone	SH30070.03	Store at -20°C
V-bottom 96-weel plates		Nunc	249570

Cell lines

293T (catalogue number CRL-11268) and MDCK (catalogue number CCL-34) cell lines are maintained in a 37°C incubator with 5% CO₂ in DMEM 10% FBS, 1% PS. Cells are available form the American Type Culture Collection (ATCC, 10801 University Boulevard, Manassas, VA. 20110-2209 USA).

Embryonated chicken eggs

Embryonated 10-day-old 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. Eggs are incubated at 37°C preceding and after viral infection. Before and after viral infection, eggs are candled to determine viability of the embryos. It is very important to look for dead eggs before and after viral infection. Before infection a dead egg can be easily spotted by the absence of blood vessels as well as the absence of embryo mobility. When candled, live embryos move. After viral infection a dead egg (probably related to influenza virus infection) will be easily spotted by the bad appearance of the egg as seen by the smaller and bloody volume of allantoic fluid. Infected-eggs are discarded in double autoclavable bags and autoclaved following standard procedures.

Chicken red blood cells (RBC)

Chicken RBC can be purchased from Truslow Farms, 201 Valley Road, Chestertown, Md 21620. Store at 4°C. For HA assays, wash 5 ml of the chicken RBC with 45 ml of PBS 1X in a 50 ml centrifuge tube. Centrifuge for 5 minutes at 1000 rpms, RT. Discard carefully the supernatant and use a 1:1000 dilution of the pelleted RBC in PBS 1X (final concentration of 0.5-1.0% RBC).

Tissue culture supernatants and allantoic fluids

Both, tissue culture supernatants and allantoic fluids can be stored at 4°C for a short period of time. After confirming virus rescue, viruses from cell supernatants or allantoic fluid are stored at -80°C.

Plasmids

All plasmids are prepared using a plasmid maxi kit following manufacturer’s recommendations. All plasmids are aliquot at concentrations of 1 μg/ml in ddH₂O and stored at -20°C. For short-term storage, the plasmid can be keep at 4°C. The concentration of the purified DNA plasmid is determined by spectrophotometry at 260 nm, with purity being estimated using the 260:280 nm ratio. Preparations with 1.8-2.0 260:280 nm ratios are considered appropriated for virus rescue purposes. Additionally, plasmid concentration and purity should be confirmed with agarose gel chromatography. Ambisense pDZ plasmids (6) containing the eight influenza A/PR/8/34 viral genes (7) are illustrated in Figure 2.

Viruses

The described protocol for rescuing influenza A/PR/8/34 can be performed under biosafety level (BSL) 2 conditions. Contaminated material, including tissue culture supernatants and embryonated eggs, should be sterilized before disposal. Rescue of other influenza virus may require higher BSL conditions and, therefore, special conditions/security measurements will need to be followed.

Tissue culture media and solutions

DMEM 10%FBS 1%PS: 445 ml Dulbecco’s modified Eagle’s medium (DMEM), 50 ml of Fetal Bovine Serum (FBS), and 5 ml of 100X Penicillin/Streptomycin (PS). Store at 4°C. This media will be used to maintain 293T and MDCK cells as well as for the transfections. DMEM 0.3%BA 1%PS: 495.7 ml of DMEM, 4.3 ml of 35% Bovine Albumin (BA). Store at 4°C. Just before use, add TPCK treated trypsin to a final concentration of 1 μg/ml. Infectious media.

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 liter. 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.