K亚群禽白血病病毒gp85单因子血清的制备及其特异性鉴定

为了制备一种快速特异性识别K亚群禽白血病病毒(ALV-K)的单因子血清,本试验将ALV-K接种DF1细胞,提取感染细胞DNA作为模板,PCR扩增1 005 bp ALV-K-gp85和510 bp ALV-K-gp85(fragment)基因.将其正确的阅读框插入表达载体pET-28a(+)中,转化大肠杆菌BL21(Rosetta)菌株中表达.将表达蛋白常规免疫昆明白小鼠制备抗血清.结果表明成功获得39.85和22.01 ku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)结果表明两血清均可与ALV-K反应,而不与ALV-A、ALV-B和ALV-J反应.本试验首次研制获得能特异性识别ALV-K的单因子血清,为K亚群禽白血病的快速检测奠定了基础.

Preparation and Specific Identification of Single Factor Serum of Subgroup K Avian Leukosis Virus gp85

This study was to prepare a diagnostic serum to rapidly diagnose subgroup K avian leukemia virus (ALV-K).The ALV-K were inoculated in DF1 cells and extracted DNA from infected cells to amplify ALV-K-gp85 (1 005 bp) and ALV-K-gp85 (fragment) (510 bp) genes by PCR.Their correct reading frames were inserted into pET-28a(+) expression vector,then transformed into BL21 (Rosetta) strain.The expressed proteins were used to immune Kunming White mice to prepare antiserum.Recombinant fusion proteins of 39.85 and 22.01 ku were successfully obtained and showed good immunogenicity.Indirect immunofluorescence assay (IFA) showed that the two sera could react with ALV-K,but could not react with ALV-A,ALV-B and ALV-J.The experiment prepared the single factor serum that could be used to specifically diagnosis subgroup K avian leukemia for the first time,at the same time,the single factor serum laid the foundation for rapid diagnoses of subgroup K avian leukemia.