猪圆环病毒2型的分离鉴定及高免卵黄抗体的研制

Isolation and identification of porcine circovirus type 2 and development of the specific yolk immunoglobulin

为制备猪圆环病毒2型(PCV2)特异性高免卵黄抗体(IgY),本研究首先对2017年~2018年河南部分地区规模化养猪场采集的19份疑似感染PCV2猪的病料样品进行PCR检测,对阳性样品进行PCV2全基因组测序分析、病毒分离鉴定、制备灭活疫苗免疫蛋鸡并制备其IgY,对制备的IgY经中和试验检测其中和活性。比较氯仿法与水稀释-硫酸铵沉淀法制备的IgY的纯度差异。结果显示,河南省部分地区的PCV2阳性率为63.2%(12/19)。PCV2全基因组序列分析显示,12株PCV2之间的同源性为96.9%~100%,与其它PCV2代表株之间的同源性为95.1%~99.9%。遗传进化分析显示,8株PCV2属于PCV2d亚型,4株属于PCV2b亚型。病毒分离结果显示,7份阳性样品可以分离出病毒,其中一株病毒效价达106.5 TCID50/mL。用该株病毒制备的PCV2灭活疫苗免疫蛋鸡能够诱导其产生较高水平的特异性IgY。中和试验结果显示,该IgY对PCV2具有较高的中和活性,中和效价达1:181。此外,通过比较氯仿法和水稀释-硫酸铵沉淀法制备IgY的效果显示,两种方法均较为理想,但前者获得的样品纯度更高。本研究为进一步开展IgY抗PCV2研究奠定了基础。

In order to prepare chicken egg yolk immunoglobulin(IgY)specific for porcine circovirus type 2(PCV2),19 suspected PCV2-positive pig samples were collected from large-scale pig farms in Henan province and detected by PCR.Virus isolation was performed on PCV2 positive samples,and the whole-genome sequence was analyzed.Inactivated virus was prepared to immunize layer hens and IgY was prepared from egg yolk and detected by neutralization test.In addition,the purity of the IgY prepared by chloro form method and water dilution-ammonium sulfate precipitation method was compared.The result showed that the positive rate of PCV2 in some areas of Henan province was 63.2%(12/19).Whole genomic sequence analysis showed that the homology rates were 96.9-100% between the newly emerged PCV2 strains,and 95.1-99.9%between these new strains and other PCV2 representative isolates.Among the 12 newly emerged PCV2 isolates,8 strains belonged to PCV2d subtype and 4 strains belonged to PCV2b subtype.In addition,7 strains were isolated and one of the virus isolates titer was 106.5 TCID50/mL.The hens immunized with inactivated PCV2 produced high levels of specific IgY.The neutralization test results showed that the IgY had higher neutralizing activity against PCV2.And the neutralization titer had reached 1∶181.In addition,the comparison of the preparation of IgY by chloroform method and water dilution-ammonium sulfate precipitation method showed that both methods were ideal,but the former method was better in IgY purity.This study laid the foundation for further research on IgY against PCV2.