盘尾丝虫ASP1蛋白佐剂活性区不同标签融合表达与佐剂活性比较

为了探明纯化标签对盘尾丝虫活化相关分泌蛋白1(activation-associated secreted protein 1,ASP1)佐剂活性的影响,将其佐剂活性区PR-1编码序列分别与类弹性蛋白多肽(elastin-like polypeptide,ELP)、ELK16自聚肽或His标签进行融合表达,用相变循环、离心洗涤和镍亲和层析进行融合蛋白纯化;将ELP与传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)VP2基因片段进行融合表达,用蛋白酶切除ELP标签后与不同标签融合PR-1免疫小鼠,两次免疫后不同时间采血分离血清,采用ELISA检测VP2特异IgG、IgG1和IgG2c滴度,用试剂盒检测免疫小鼠血清的IFN-γ、TNF-α、IL-6、IL-10浓度。结果显示,ELP-PR1、ELK-PR1、His-PR1和ELP-VP2融合蛋白在重组大肠杆菌中均获得正确表达,纯化蛋白纯度大于90%;3种标签融合PR-1均能增强免疫小鼠的抗原特异IgG、IgG1和IgG2应答,并能刺激小鼠产生IFN-γ、TNF-α、IL-6或IL-10细胞因子。在3种PR-1融合蛋白中,ELP-PR1的佐剂活性最强,ELK-PR1与不完全弗氏佐剂相当。本研究探索了不同纯化标签对PR-1免疫佐剂活性的影响,为传染性法氏囊病新型亚单位疫苗佐剂研制提供思路。

EFFECT OF FUSION TAGS ON ADJUVANT ACTIVITY OF ONCHOCERCA VOLVULUS ASSOCIATED SECRETED PROTEIN-1

To investigate the influence of fusion tags on the adjuvant activity of Onchocerca volvulus activation associated secreted protein-1(ASP-1), the PR-1 functional domain of ASP-1 was expressed as an elastin-like polypeptide(ELP), self-aggregating peptide ELK16 or His-tagged protein and then purified by inverse transition cycling, centrifugation and washing or nickel affinity chromatography.The VP2 segment of infectious bursal disease virus was used as an ELP fusion protein and to immunize mice after cleavage of ELP tag.The VP2-specific IgG, IgG1 and IgG2 c were detected using indirect ELISA, and concentrations of IFN-γ, TNF-α, IL-6 and IL-10 were detected using ELISA kit. The results showed that ELP-PR1, ELK-PR1, His-PR1 and ELP-VP2 fusion proteins were correctly expressed in recombinant E. coli and purified to more than 90% purity. All of three PR-1 fusion proteins enhanced the VP2-specific IgG, IgG1 and IgG2 c responses and IFN-γ, TNF-α, IL-6 and/or IL-10 production. Among the three PR-1 fusion proteins, ELP-PR1 showed the strongest humeral and cellular adjuvant effects, followed by ELK-PR1 and His-PR1.