Zhang Dandan, Li Hongmei, Zhang Zhongsheng, Sun Shuhong, Cheng Ziqiang, Liu Jianzhu, Zhao Peng, Ren Qingya, Guo Huijun
College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai'an 271018, China.
College of Veterinary Medicine, Research Center for Animal Disease Control Engineering Shandong Province, Shandong Agricultural University, Tai'an 271018, China.
Antiviral Res. 2015 Apr;116:20-6. doi: 10.1016/j.antiviral.2015.01.007. Epub 2015 Jan 28.
To observe the antibody responses induced by recombinant A subgroup avian leukosis virus (ALV-A) gp85 protein vaccine plus CpG-ODN adjuvant and the protection of maternal antibodies (MAbs) for the hatched chickens against early infection, the gp85 gene was amplified from the proviral cDNA of ALV-A-SDAU09C1 strain using PCR and the recombinant plasmid containing target gene was constructed and expressed in EscherichiaColi. The expressed product was confirmed using SDS-PAGE and western blot that it is about 46KD of recombinant protein. The purified recombinant proteins combining with CpG-ODN adjuvant or Freund's adjuvant were inoculated into the breeder hens, the ALV-A antibodies in serum and in egg-yolk were detected; the fertilized eggs from the vaccinated hens with different titers of egg-yolk antibody were hatched and then challenged with 10(4.2)/0.1mL TCID50 of ALV-A-SDAU09C1 strain, all the hatched chickens were weekly detected for the viremias and the cloacal swab P27 antigen and pathological lesions; the neutralizing test of antisera in vitro was conducted. The results showed that the recombinant gp85 proteins combining with CpG-ODN adjuvant could induce the breeder hens to produce better antibody responses than gp85 protein with Freund's adjuvant or without adjuvant; the MAbs with higher titers induced by CpG-ODN+gp85 proteins could obviously decrease the ratios of viremias (13% vs 33%), cloacal detoxification (20% vs 67%) and death (0% vs 22%) caused by ALV-A infection than those by gp85 protein without adjuvant. The results of the neutralizing test indicated that the antisera from the hatched chickens could neutralize the ALV-A-SDAU09C1 strain in vitro, but which depends on the antibody titers. The results of IFA confirmed that the serum antibody could combine with the ALV in DF1 cells. It can be concluded that the prepared ALV-A gp85 subunit vaccine combining with CpG-ODN adjuvant could induce the breeder hens to produce better neutralizing antibody responses and protect 80% of their offspring chickens against early infection.
为观察重组A亚群禽白血病病毒（ALV-A）gp85蛋白疫苗加CpG-ODN佐剂诱导的抗体反应以及母源抗体（MAbs）对出壳雏鸡早期感染的保护作用，采用PCR从ALV-A-SDAU09C1株前病毒cDNA中扩增gp85基因，构建含目的基因的重组质粒并在大肠杆菌中表达。用SDS-PAGE和western blot证实表达产物为约46KD的重组蛋白。将纯化的重组蛋白与CpG-ODN佐剂或弗氏佐剂混合接种于种母鸡，检测血清和蛋黄中的ALV-A抗体；将不同蛋黄抗体效价的接种母鸡所产的受精卵孵化，然后用10(4.2)/0.1mL TCID50的ALV-A-SDAU09C1株进行攻毒，对所有出壳雏鸡每周检测病毒血症、泄殖腔拭子P27抗原及病理损伤；进行抗血清体外中和试验。结果表明，重组gp85蛋白与CpG-ODN佐剂联合使用比gp85蛋白单独使用或与弗氏佐剂联合使用能诱导种母鸡产生更好的抗体反应；CpG-ODN+gp85蛋白诱导产生的高效价母源抗体比无佐剂的gp85蛋白能明显降低ALV-A感染引起的病毒血症比例（13%对33%）、泄殖腔排毒比例（20%对67%）和死亡比例（0%对22%）。中和试验结果表明，出壳雏鸡的抗血清能在体外中和ALV-A-SDAU09C1株，但这取决于抗体效价。IFA结果证实血清抗体能与DF1细胞中的ALV结合。可以得出结论，制备的ALV-A gp85亚单位疫苗与CpG-ODN佐剂联合使用能诱导种母鸡产生更好的中和抗体反应，并保护80%的后代雏鸡免受早期感染。
