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1、To generate a biologically pure and homogeneous PCV2 infectious virus stock for the animal inoculation experiment, PK-15 cells free of PCV1 contamination were cultivated in T-25 culture flasks and transfected with the PCV2 molecular DNA clone. 1， Briefly, PK-15 cells were grown to about 85% confluen
2、cy in T-25 flasks. 2， The cells were washed once with sterile phosphate-buffered saline (PBS) buffer before transfection. 3， For each transfection reaction in a T-25 flask, 12 ug of the PCV2 plasmid DNA was mixed with 16 ul of Plus Reagent in 0.35 ml of MEM. A flask of mock-transfected cells with em
3、pty pSK vector was included as the negative control. 4， After incubation at room temperature for 15 min, 50 ul of Lipofectamine Reagent diluted in 0.35 ml of MEM was added to the mixture and was incubated at room temperature for another 15 min. 5， The transfection mixture was then added to a T-25 fl
4、ask of PK-15 cells containing 2.5 ml of fresh MEM.6， After incubation at 37C for 3 h, the media were replaced with fresh MEM containing 2% fetal bovine serum and 1antibiotics. 7， The transfected cells were harvested at 3 days posttransfection and stored at -80C until use. 8， To determine the infecti
5、ous titer of the homogeneous PCV2 virus stock, PK-15 cells were cultivated on eight-well LabTek chamber slides. 9， The virus stock was serially diluted 10-fold in MEM, and each dilution was inoculated onto 10 wells of the monolayers of the PK-15 cells growing on the LabTek chamber slides. Wells of n
6、oninoculated cells were included as controls.10， The infected cells were fixed at 3 days postinoculation with a solution containing 80% acetone and 20% methanol at 4C for 20 min.11， After washing of the cells with PBS buffer, the infected cells were incubated with a 1:1,000-diluted PCV2 specific rab
7、bit polyclonal antibody at 37C for 1 h. 12， The cells were then washed three times with PBS buffer and were incubated with a secondary fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin G at 37C for 45 min. 13， After the slides were washed three times with PBS buffer, they were mount
8、ed with fluoromount-G, coverslipped, and examined under a fluorescence microscope. 14， The 50% tissue culture infective dose (TCID50) per ml was calculated.1 8 重组质粒细胞转染将无PCV1 污染的PK 15 细胞接种于单孔细胞培养板内，待细胞生长至80% 融合度时，按照LipofectamineTM2000转染试剂说明书，用重组质粒pcDNA31( + ) PCV1 转染PK 15 单层细胞，并以转染空载体pcDNA3 1( + ) 的
9、细胞作为阴性对照。细胞转染5 d 后，收取培养物反复冻融3 次，作为种毒继续盲传3 代。1 9 拯救病毒的初步检测病毒盲传3 代的细胞培养液反复冻融 3 次后，以12 000 r /min 离心10 min; 取上清液，经DNase 消化后于37 水浴1 h，参照1 4 中的方法提取DNA，参照1 5 中的条件进行PC 扩增。转染真核细胞参照脂质体产品说明书，将环化后的病毒基因组转染PK-15细胞。转染后的细胞继续培养 48-72h，然后盲传3代。间接免疫荧光试验转染后的细胞经盲传3代后，以丙酮：甲醇（4：1 ）溶液于-20 固定20min 。PH7.4 PBS缓冲液冲洗3 次后，与1：100
10、稀释的PCV2高免血清37温育1h ，PBS洗3次后，再用1 ：300稀释的FITC标记羊抗猪二抗,37 作用45min， PBS缓冲液冲洗 3次，每次 5min。置于荧光显微镜下观察。在试验中，我们曾尝试用300mmol/L D-氨基葡萄糖对转染细胞进行处理以提高病毒滴度，但由于 D-氨基葡萄糖对PK-15细胞毒性太大而告失败。1.8.1 DNA 转染：用QIAprep Spin Miniprep Kit 提取pSK-dPCV1-2 重组质粒。将(2.510)105 个Dulac 细胞及PK-15 细胞移于Dish 中，待细胞长到9095时，进行DNA 转染。按照转染试剂盒（Lipofect
11、amine2000）说明书进行转染。1.8.2 PCR 检测：转染细胞进行传代，经5 次传代后，参照步骤1.5，采用PCR 方法检测细胞中PCV2 ORF2基因。同时，设正常 Dulac 细胞作为阴性对照。1.8.3 间接免疫荧光试验：参照文献18方法进行。1.8.4 病毒 TCID50 的测定：转染后的细胞连续盲传 5 代，收获1 mL 细胞悬液，备用。Dulac 细胞分装到96 孔板，加入连续10 倍稀释（10110 6 稀释）的病毒液10 L （病毒的稀释度为10210 7）；72 h 后弃去培养液，以猪抗PCV2 阳性血清及兔抗猪IgG 荧光抗体作间接免疫荧光试验判定结果。按Reed
12、与Muench 氏法计算病毒的 TCID50。 病毒的制备：Dulac细胞经过脂质体转染PCV1-2 嵌合质粒DNA 后，进行传代培养，每代细胞培养24 h 后用300 mmol/L D-氨基葡萄糖处理 30 min，用10 mmol/L PBS 洗涤后换上新鲜培养液（血清含量为20 mL/L 的维持液）继续培养。测定所收获病毒的TCID50。Transfection of PK-15 cells with pIC-PCV2. 1，To test the infectivityof the molecular DNA clone in vitro, PK-15 cells were seede
13、d in24-well plates (210 4 cells per well). 2，When the PK-15 cells were approx. 60% confluent, the cells were transfected with 200 ng of pIC-PCV2 DNA using the Qiagen Effectene protocol (Qiagen).pUC18-transfected cells were included as a negative control. Cells transfected with 200 ng of PCV2 DNA wer
14、e used as a positive control. 3，Seven days after transfection, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. 4，After washing with PBS, cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature. 5，After washing a further three times, cells were blo
15、cked with 3% fetal calf serum and 0.1% Tween in PBS for 1 h at 37 in a humid atmosphere. 6，Once blocked, cells were washed three times with PBS and incubated with polyclonal sera specific for PCV1 (diluted 1 : 400 in blocking solution) and PCV2 (diluted 1 : 200 in blocking solution), respectively. 7
16、，Subsequently, cells were washed three times with PBS and incubated with speciesspecific FITC-labelled anti-IgG antibodies (Dianova 1 : 200 in blocking solution) for 1 h. Cells were washed again three times in PBS and a fourth time in double distilled water. 8，Afterwards the cells were covered with Immu-mount (Shandon). The samples on microscopic slides were analysed for expression of viral antigen using confocal laserscan microscopy (Zeiss
