猪圆环病毒与口蹄疫病毒二联重组鸡痘病毒的鉴定和BALB/c小鼠的免疫试验

Identification of recombinant fowlpox viruses coexpressing ORF2/ORF2ORF1 of PCV2 and P12A of FMDV and experimental study on injecting BALB/c Mice

以鸡痘病毒(FPV282E4)为活载体,用猪2型圆环病毒内蒙株的衣壳蛋白、与复制相关蛋白基因及O型口蹄疫结构蛋白基因作为目的基因,经同源重组的方法获得2个重组鸡痘毒,分别命名为:vUTALP1-ORF2-ORF1、vUTALP1-ORF2。在复制、转录和翻译水平对这2个重组毒进行鉴定,结果表明这2个重组毒均能表达目的蛋白。将它们免疫6～8周龄BALB/c小鼠,每隔19d免疫1次,共3次;每10d尾跟采血1次,三免后10d杀鼠取脾。用ELISA方法检测小鼠血清抗体效价,流式细胞仪检测脾T淋巴细胞亚类CD4+、CD8+数量。结果发现2个重组毒试验免疫组均是从最后一次采集的小鼠血清能获得最高的抗体效价,与对照组FPV相比都能产生一定的体液免疫反应,且以vUTALP1-ORF2-ORF1重组毒免疫小鼠产生的血清抗体效价最高;脾T淋巴细胞亚类数量测定发现,免疫试验组的CD4+与CD8+的T细胞亚类总数显著高于对照组(P<0.05),并以vUTALP1-ORF2-ORF1数值较高。结果表明,这2个重组鸡痘毒既能较好的刺激细胞免疫又能产生一定的体液免疫,将作为候选活载体重组鸡痘毒进行本体动物免疫试验研究。

Using fowlpox virus（wt 282E4） as live virus vector,PCV2（Inner Mongolis strain） capsid or replicon genes and FMDV capsid polypeptide genes of O/NY00 were interest genes.The two recombinant viruses which were named as vUTALP1-ORF2 and vUTALP1-ORF2ORF1 were gained by homologous recombination.The two recombinant viruses were identified from replication,transcript and translation level,and tested that they were capable of express interest proteins.Six/eight week-old BALB/c mices（12 mices each）were divided into three groups,one of which was control for inoculating by FPV（wt 282E4 strain） and the other two groups were injected by vUTALP1-ORF2,vUTALP1-ORF2ORF1,respectively.It lasted one week until mices received the first intramuscular injection.Mices were inoculated three times every 20 days.Mice sera gained before inoculation were used as negative control.Then,during the immunization,mice sera were from the mice＇s tails every 10 days.On the 10th day after the third inoculation,mices were killed by excising eyeballs ane removing spleens with sterility.Detecting sera antibody using ELISA and calculating mice spleen T lymphocytes subgroups（CD4＋,CD8＋） by flow cytometry.And find that the last collected sera has the highest titre among experimental groups;They all produced certain degree of humoral immunity compared with those from control group,and the vUTALP1-ORF2ORF1 is superior.In addition,the numbers of experimental groups＇CD4＋,CD8＋ T lymphocytes subgroups are higher than the control group,the total number of T subgroups in vUTALP1-ORF2ORF1 group is also much higher.The results show vUTALP1-ORF2ORF1 can efficiently stimulate both cellular immunity and humoral immunity.It will be tested continually as a specific PCV2 vaccine candidate.