摘要
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)感染所致的一种急性高度传染性疾病,该病主要引起妊娠母猪早产、流产、死胎、木乃伊胎等繁殖障碍以及各年龄段猪的呼吸道症状和高死亡率。该病于1987年首先发现于美国,随后波及多个国家和地区。我国1991年在台湾发现该病,郭宝清在1996年首次从流产胎儿中分离到PRRSV,证实了PRRSV在我国的存在。2006年下半年的“无名高热”疫情在我国南方大面积暴发,最后确诊为高致病性PRRS。该病造成较高的发病率和死亡率,而且临床症状逐渐复杂,流行范围逐渐扩大。目前PRRS已经遍及全球主要养猪的国家和地区,给养猪业带来了极大的经济损失,也成为了世界各国科学家研究的热点。
我们前期的研究发现感染PRRSV猪的血清中存在两种不同的抗GP5和M蛋白的自身抗独特型抗体(Auto-anti-idiotypic antibodies),它们对PRRSV感染后猪的免疫反应网络可能起到正向或负向调控作用。为了探索抗独特型抗体对PRRSV免疫反应的调控作用,本试验用GP5的抗独特型抗体Mab2-5G2及其单链抗体免疫小鼠后再免疫纯化的PRRSV病毒,然后通过间接ELISA检测各种抗体水平和流式细胞术检测外周血中CD4+/CD8+淋巴细胞的数量比例变化及血象分析来说明问题。因为有报道称不同剂量的抗独特型抗体有着不同的调控作用,所以我们设计了不同的免疫剂量下的试验,并且同时设置了无关抗体对照组。
通过对实验结果统计分析发现,抗独特型抗体Mab2-5G2及其scFv都可以增强小鼠免疫系统对PRRSV的免疫反应,反映在可以提高体液免疫中GP5的抗体水平并且能显著性的提高外周血中CD4+和CD8+T细胞的比例,从而能够增强小鼠对PRRSV的免疫能力。另外我们发现,不同剂量的抗独特型抗体及其scFv有不同的调控作用,在我们所分的3个剂量梯度中,只有0.25uM/鼠的剂量才可以增强小鼠外周血中CD4+和Cd8+T淋巴细胞的比例及总淋巴细胞占外周血白细胞的比例,而0.025uM/鼠和1.0uM/鼠的免疫剂量都可以显著性的降低CD4+和CD8+T细胞的比例,从而可能消弱小鼠免疫系统对PRRSV的免疫能力。该研究证明了抗独特型抗体及其单链抗体对机体免疫系统的调控受免疫剂量的影响,不同剂量可能产生正向或反向的调控。该研究结果给用抗独特型抗体或其单链抗体作为免疫调节剂来提高猪对PRRSV的免疫力提供了依据。
Porcine reproductive and respiratory syndrome (PRRS) is an acute infectious disease caused by Porcine reproductive and respiratory syndrome virus (PRRSV). PRRS mainly caused reproductive disorder, including of premature, abortion, fetal death, mummy and porcine respiratory syndrome at variety of ages and high mortality. The disease was first discovered in American in 1987 and then prevailed in many countries. In 1991, this disease was discovered in Taiwan. In 1996, Guo Baoqing first isolated a PRRSV strain from aborted fetuses and verified the evidence of PRRSV in our country. In 2006, there was a large outbreak in our South districts and this disease was diagnosed as highly pathogenic PRRS. The disease caused high morbidity and mortality and the clinical symptom became more complicated and the prevalence also was gradually expanded. At the present, PRRS has prevailed in the main worldwide pig-raising country and districts and caused pig-raising industry large economic losses. So it becomes one of the hot research areas.
Our previous studies demonstrated that pigs infected with PRRSV produced auto-anti-idiotypic antibodies (auto-Ab2s) against idiotypic antibodies to M and GP5 protein, which may up- or down-regulate the immune responses against PRRSV infection. To explore the regulating effect of anti-idiotypic antibodies to PRRSV infection, we use the anti-idiotypic antibody specific to GP5 and its scFv to immunize mice and then challenge the mice with purified PRRSV, and at last we detect the antibody levels of the immunized mice by I-ELISA and the percentage of CD4+/CD8+ T cell in PBL by FCM, and do hemogram analysis. And because some reports indicate that different dosage of anti-idiotypic antibody have different regulating effect, we set three different dosage groups, with every group its own irrelevant control.
After analysis of the statistics, we found that Mab2-5G2 and its scFv can enhance the immune response of PRRSV. It shows that they can increase the anti-GP5 antibody levels and the percentage of CD4+/CD8+ T cell in PBL, which may improve the mice immunity. In addition, we found that different dosage of immunogens have different effect, and only the mice immunized in the dosage of 0.025uM/mouse increase the percentage of CD4+/CD8+ T cell in PBL and the percentage of total lymphocyte in peripheral blood WBC, whereas the dosage of 0.025uM and 1.0uM/mouse immunogen can significantly reduce the percentage of CD4+/CD8+ T cell in PBL, which may weaken the mice immunity. In conclusion, our study demonstrates that the effect of anti-idiotypic antibody and its scFv can be influenced by different dosage, and this result pave the way for anti-idiotypic antibody and its scFv to be the immune regulator against PRRSV in pigs.
引文
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