禽白血病A亚群病毒gp85的单因子血清制备及其特异性鉴定

摘要

[目的]为了研究出一种能够针对A亚群禽白血病的快速特异性诊断试剂.[方法]将A亚群禽白血病病毒(ALV-A)SDAU09E1株接种于DF1细胞上,以感染细胞DNA为模板,通过PCR方法扩增出1023 bp的ALV-A-gp85基因.将其正确阅读框架插入表达载体PET-32a(+)中,实现在BL21(Rosetta)宿主菌中表达.将纯化的融合蛋白常规免疫小鼠,制备得抗血清.[结果]实验成功获得52.8 kDa的重组融合蛋白,且具有良好的免疫原性.间接免疫荧光试验(IFA)表明该血清可与ALV-A和ALV-B反应,但不与ALV-J反应.[结论]该实验首次在国内外研制出能用于鉴别性检测经典的A/B亚群ALV的单因子血清,可与ALV-J特异性单抗互补作用于外源性ALV感染的鉴别性诊断.我国鸡群同时受经典的ALV-A/B和新出现的ALV-J困扰,鉴别诊断非常必要,研究这种试剂具有较高的实用价值.%[Objective]In order to get a rapid specific diagnostic reagent for subgroup A Avian Leukosis Virus detection.[Methods]The Avian Leukosis Virus Subgroup A(ALV-A) SDAU09E1 strain was inoculated into DF1 cells,an ALV-A-gp85 DNA fragment of 1023bp was amplified from infected cells and inserted into PET-32a( + ) plasmid at the location between restriction endonucleases BamH Ⅰ and Not Ⅰ sites.The recombinant plasmid PET-SDAU09E1-gp85 was transformed into E coli.BL21 ( Rosetta) for gp85 gene expression.Then we used the purified recombinant fusion protein to immunize 6 weeks old Kunming white mice, and the antiserum were prepared.[Results]The recombinant ALV-A gp85 fusion protein with a molecular weight of 52.8kDa demonstrated a good antigenecity.Mon-specific serum produced by vaccinated mice came out reactive with subgroups A and B ALV ( ALV-A and ALV-B but not subgroup J ALV) by the indirect immunofluorescence ( IFA ) method.[Conclusion]This was the first time to demonstrate a mono-specific antiserum specific to ALV-A and ALV-B, it could be used for differential diagnosis of exogenous ALV infections in CEF cultures when in complement with ALV-J specific monoclonal antibodies.Chickens in our country are now distressed by both classic ALV-A/B and emerging ALV-J, making differential diagnosis necessary, so studying this reagent has high practical value.