抗猪流行性腹泻病毒卵黄抗体制剂的研究及其临床应用
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摘要
为开发抗猪流行性腹泻病毒(PEDV)卵黄抗体制剂,应用RT-PCR技术从患猪流行性腹泻(PED)仔猪肠组织中扩增出523 bp的基因片段,构建了重组表达质粒pET32a-S1-523,转化至感受态细胞BL21 (DE3)中,经0.4 mmoL/L IPTG诱导表达7 h后成功表达出32.8 kDa大小的可溶性重组蛋白。该重组蛋白经Ni-NTA蛋白纯化柱纯化后,进行SDS-PAGE鉴定。Western Blot检测结果显示特异性良好。以重组蛋白作为抗原免疫50只初产蛋鸡,收集高免卵黄液,并采用喷雾干燥技术和冷冻干燥技术研制卵黄抗体粉和卵黄抗体冻干粉,利用间接ELISA方法检测其抗体效价。应用高效价高剂量的卵黄抗体制剂对规模化猪场中患病仔猪进行治疗试验,结果显示,卵黄抗体粉组的治愈率为74.67%,卵黄抗体液组的治愈率为78.26%,卵黄抗体冻干粉组的治愈率为75.93%。表明卵黄抗体制剂对患PED仔猪具有一定的治疗效果。
In this study, the 523 bp gene was amplified from diseased piglets tissues by RT-PCR technology, and was then cloned into the pET-32 a expression vector to obtain the recombinant expression plasmid pET-32 a-S1-523.The recombinant fusion protein was abouts 32.8 kDa,which was induced for 6 h by 0.4 mmoL/L IPTG. The recombinant protein was purified by NTA agarose gel and canfirmed by Western blot. 50 primiparous laying hens were immunized by recombinant protein and high-immunized egg yolk was collected to develop yolk antibody powder and freeze-dried yolk antibody by the spray drying and freeze drying technology. Indirect ELISA method was applied to detect the antibody titer. High titer and high dose yolk antibody preparations were applied to sick piglets in large-scale pig farms. The results showed that the cure rate was 74.67% in the yolk antibody powder group, the cure rate was 78.26% in the yolk antibody solution group and the cure rate was 75.93% in egg yolk antibody freeze-dried powder group. The results show that egg yolk antibody preparations have a certain therapeutic effect on PED piglets.
In this study, the 523 bp gene was amplified from diseased piglets tissues by RT-PCR technology, and was then cloned into the pET-32 a expression vector to obtain the recombinant expression plasmid pET-32 a-S1-523.The recombinant fusion protein was abouts 32.8 kDa,which was induced for 6 h by 0.4 mmoL/L IPTG. The recombinant protein was purified by NTA agarose gel and canfirmed by Western blot. 50 primiparous laying hens were immunized by recombinant protein and high-immunized egg yolk was collected to develop yolk antibody powder and freeze-dried yolk antibody by the spray drying and freeze drying technology. Indirect ELISA method was applied to detect the antibody titer. High titer and high dose yolk antibody preparations were applied to sick piglets in large-scale pig farms. The results showed that the cure rate was 74.67% in the yolk antibody powder group, the cure rate was 78.26% in the yolk antibody solution group and the cure rate was 75.93% in egg yolk antibody freeze-dried powder group. The results show that egg yolk antibody preparations have a certain therapeutic effect on PED piglets.
引文
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[14] 崔焕忠,姜海龙,张加力,等. 抗猪传染性胃肠炎病毒与猪流行性腹泻病毒卵黄抗体制备及其临床应用研究[J]. 中国畜牧兽医杂志,2012, 39(6): 173- 175.
[2] Pensaert M B, De Bouck P. A new coronavirus-like particle associated with diarrhea in swine [J]. Arch Virol, 1978, 58(3): 243- 247.
[3] Ojkic D, Hazlett M, Fairles J, et al. The first case of porcine epidemic diarrhea in Canada [J]. Canadian Veterinary Journal-Revue Veterinaire Canadienne, 2015, 56(2): 149- 152.
[4] Vlasova A N, Marthaler D, Wang Q, et al. Distinct characteristics and complex evolution of PEDV strains, North America, May 2013-February 2014 [J]. Emerging Infectious Diseases, 2014, 20(10): 1 620- 1 628.
[5] Lee D K, Park C K, Kim S H,et al. Heterogeneity in spike protein genes of porcine epidemic diarrhea viruses isolated in Korea [J]. Virus Research, 2010, 149(2): 175- 182.
[6] Chen J, Liu X, Shi D,et al. Detection and Molecular Diversity of Spike Gene of Porcine Epidemic Diarrhea Virus in China [J]. Viruses, 2013, 5(10): 2 601- 2 613.
[7] 黄冬妮,黄良宗,马春全,等. 广东省猪流行性腹泻病毒ORF3和M基因的克隆与序列分析[J]. 黑龙江畜牧兽医,2015(5): 136- 139.
[8] 罗六龙,黄冬妮,黄良宗,等. 广东省猪流行性腹泻病毒S1基因的克隆与序列分析[J]. 中国兽医杂志,2016, 52(3): 19- 21.
[9] 李井春,王姗姗,姚伟,等. 猪流行性腹泻病毒辽宁株序列测定及分析[J]. 中国兽医杂志,2016, 52(3): 41- 43.
[10] Chen F, Ku X, Li Z, et al. Genetic characteristics of porcine epidemic diarrhea virus in Chinese mainland, revealing genetic markers of classical and variant virulent parental/attenuated strains [J]. Gene, 2016, 588(1): 95- 102.
[11] Su Y, Liu Y, Chen Y,et al. Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3 gene and the S1 gene [J]. Virology Journal, 2016, 13 (1): 192.
[12] Yang X, Huo J Y, Chen L,et al. Genetic variation analysis of reemerging porcine epidemic diarrhea virus prevailing in central China from 2010 to 2011 [J]. Virus Genes, 2013, 46 (2): 337- 344.
[13] 秦文. 抗猪PED、TGE、RV高免卵黄抗体的研制与应用[D].杨凌:西北农林科技大学,2014.
[14] 崔焕忠,姜海龙,张加力,等. 抗猪传染性胃肠炎病毒与猪流行性腹泻病毒卵黄抗体制备及其临床应用研究[J]. 中国畜牧兽医杂志,2012, 39(6): 173- 175.