摘要
将猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)核衣壳蛋白(nucleocapsid protein,NP)的基因片段克隆至载体pET-30a,转化重组质粒至Escherichia coli BL21(DE3)表达融合蛋白6×His-N,并以其为抗原免疫蛋鸡,制备特异性抗PRRSV-N卵黄抗体(egg yolk immunoglobulin,IgY).结果显示:6×His-N在BL21中的表达量为60.2mg·L-1;将纯化浓缩的6×His-N与弗氏佐剂乳化,连续3次免疫海兰蛋鸡后,经ELISA检测,首免后第36天抗6×His-NIgY抗体为阳性(P/N=2.44);第83天的P/N值达到最高,为5.63,抗体经1:640稀释后,P/N值为2.29;经SDS-PAGE和Western blot分析,提取的卵黄抗体的纯度高且具有良好的免疫反应性.研究表明,抗PRRSV-N蛋白的IgY制备具有简便、成本低廉等特点,可用于PRRSV检测试剂盒的开发.
The gene encoding nucleocapsid protein （N） of porcine reproductive and respiratory syndrome virus （PRRSV） was cloned into prokaryotic expression vector pET-30a（＋）,and the recombinant plasmid was transformed into Escherichia coli BL21（DE3） pLysS for expression of the fusion protein 6×His-N.The results showed that the yield of fusion protein 6×His-N in BL21 was 60.2 mg·L^-1.The purified fusion protein 6×His-N was emulsified with an equal volume of Freund’s complete adjuvant and used as antigen to immunize Hylan hen for three times to produce chicken egg yolk immunoglobulins （IgY） against N protein.ELISA results revealed that the specific antibody was firstly detected in the egg yolk at day 36 post-immunization （P/N=2.44）.The peak was reached at day 83 post-immunization （P/N=5.63） and still had P/N=2.29 by a 1：640 dilution of yolk.SDS-PAGE and Western blot analysis showed that the purified egg yolks possessed high purity and good immunoreactivity.These results indicate that specific IgY antibodies can be easily produced by immunization of hens with the recombinant fusion protein 6×His-N and is a novel alternative antibody source as a diagnostic kit reagent of PRRSV.
出处
《浙江大学学报（农业与生命科学版）》
CAS
CSCD
北大核心
2010年第3期269-274,共6页
Journal of Zhejiang University:Agriculture and Life Sciences
基金
浙江省科技厅重大专项资助项目(2007C12072)
关键词
猪呼吸与繁殖综合征病毒
核衣壳蛋白
卵黄抗体
ELISA
porcine reproductive and respiratory syndrome virus
nucleocapsid protein
egg yolk immunoglobulins
ELISA
