传染性法氏囊病vp2基因原核表达及免疫原性研究.pdfVIP

摘 要 本研究以RT-PCR技术从鸡传染性法氏囊病毒(IBDV)超强野毒株HB （河北）株基 因组中克隆出该病毒VP2基因，显示其长度大约为1536bp，并将VP2基因连接pMDl9-T 载体后转化感受态DH5 α大肠杆菌。经测序后，与GenBank中己报道的B87株疫苗株 IBDV毒株基因组比对，发现其和B87疫苗株同源性达到90%。利用胶回收试剂盒将其 回收之后进行双酶切鉴定，将VP2基因连接pGEx-4T-1表达载体，再将其转入BL21受 体菌里进行诱导表达。通过SDS及Western- Blotting分析所表达的蛋白约 66kDa。 经GST琼脂糖凝胶亲和层析柱对重组蛋白进行纯化，用白油佐剂乳化制备亚单位 疫苗。并与不同厂家IBDV灭活疫苗免疫比较，检验重组表达pGEx-4T-1-VP2蛋白免疫 原性。经口服和滴眼对鸡进行攻毒，其结果表明商品化疫苗组保护率为100%，重组 表达pGEx-4T-1-VP2蛋白组保护率为50%。 关键词：传染性法氏囊病；VP2；重组蛋白；基因；同源性 Prokaryotic expression of infectious bursal disease virus VP2 gene and Immunogenic detection Abstract In this study,VP2 gene of IBDV was cloned from super wild stra in HB(hebei) of IBDV of chicken by means of RT-PCR technique,and a fragement of 1536bp was amplified from it. And it connects the pMD19-T transformed into competent DH5a E.coli.Comparing with IBDV strain reported in GenBank has been found that B87 vaccine strain homology of 90% or more after sequencing of IBDV super wild strain (HB).Doing the identification of double enzyme digest after recycling the agarose gel,then VP2 has been transfered to the pGEx-4T-1 vector and expressed and transferred to the BL21 recipient strain that has been induced expression.The results of SDS and Western blot indicated that the expressed VP2 protein of molecular weight was about 66 kDa. The recombinant protein was purified by GST agarose gel affinity chromatography and was made into subunit vaccine by white oil adjuvant that has been emulsionized.Compared with the inactive vaccine of IBDV from different manufacture in order to test the immunogenicity of recombinant expression pGEx-4T-1-VP2 protein.Chicken were challenged with a v