杆状病毒表达系统中NLS序列对鸭圆环病毒Cap基因表达的影响及其免疫原性研究  

Study on the effects of NLS sequence on the expression of Duck circovirus cap gene in baculovirus expression system and its immunogenicity

作　　者：栾明朱 马跃宇 刘立 孙莉 李明 费东亮 马鸣潇 LUAN Mingzhu;MA Yueyu;LIU Li;SUN Li;LI Ming;FEI Dongliang;MA Mingxiao(College of Animal Science and Veterinary Medicine,Jinzhou Medical University,Jinzhou 121001,China;Inner Mongolia Autonomous Region Chifeng City Keshiketeng Banner Agriculture and Animal Husbandry Bureau,Chifeng 024000,China)

机构地区：[1]锦州医科大学畜牧兽医学院,辽宁锦州121001 [2]内蒙古赤峰市克什克腾旗农牧局,内蒙古赤峰024000

出　　处：《黑龙江畜牧兽医》2024年第3期66-72,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：辽宁省教育厅2021年度科学研究经费项目“鸭圆环病毒(DuCV)二价病毒样颗粒疫苗研制”(LJKZ0799)。

摘　　要：为了研究核定位信号肽(Nuclear localization signal,NLS)序列对鸭圆环病毒(duck Circovirus,DuCV)Cap蛋白表达的影响,并探究不同免疫策略下重组蛋白的免疫原性,试验将去除和未去除NLS序列的DuCV Cap基因分别克隆至杆状病毒载体pFastBacⅠ中,通过杆状病毒表达系统进行表达,分析NLS序列对Cap蛋白表达的影响。采用不同的免疫策略,分别以原核表达系统构建的重组蛋白rCap(rCap组)、杆状病毒表达系统构建的rvAc-Cap(rvAc-Cap组)及rCap与rvAc-Cap联合(Prime-Boost组)作为免疫源免疫Balb/c小鼠,并设置PBS对照组,定期采血,检测DuCV IgG抗体水平、淋巴细胞增殖指数(SI)和γ干扰素(IFN-γ)、白细胞介素-2(IL-2)、白细胞介素-4(IL-4)等细胞因子水平,评价其免疫效果。结果表明:利用杆状病毒表达系统成功表达出重组杆状病毒rvAc-Cap和rvAc-NLS-Cap,且去除NLS序列的Cap基因表达水平更高;小鼠免疫重组蛋白rCap后,重组蛋白rCap与重组杆状病毒rvAc-Cap均能够刺激小鼠产生特异性IgG抗体,三免后Prime-Boost组的IgG抗体水平最高,而与其他免疫组差异不显著(P>0.05);三免后各免疫组淋巴细胞增殖指数显著高于PBS对照组,差异显著(P<0.05);各免疫组IFN-γ、IL-2和IL-4水平与PBS对照组比较差异显著(P<0.05),且Prime-Boost组水平最高。说明在杆状病毒表达系统中去除NLS序列可以提高Cap基因的表达;重组杆状病毒rvAc-Cap能够活化小鼠的免疫系统,刺激小鼠机体产生体液免疫与细胞免疫,而Prime-Boost免疫策略有效提高了免疫效果。In order to study the effects of nuclear localization signal(NLS) on the expression of Cap protein of Duck circovirus(DuCV),and to explore the immunogenicity of recombinant protein under different immune strategies,in this experiment,the DuCV Cap gene with or without NLS deletion was expressed by baculovirus expression system;the DuCV Cap gene with or without NLS deletion was cloned into the baculovirus vector pFastBacI,and expressed by baculovirus expression system to analyze the effects of NLS sequence on Cap protein expression.Different immunization strategies were used to immunize Balb/c mice with recombinant protein rCap constructed by prokaryotic expression system(rCap group),rvAc-Cap constructed by baculovirus expression system(rvAc-Cap group),and rCap combined with rvAc-Cap(Prime-Boost group) as immunogens,and PBS control group was set up.Blood was collected regularly to measure the level of DuCV IgG antibody,lymphocyte proliferation indexes(SI),and the levels of cytokines γ interferon(IFN-γ),interleukin-2(IL-2),and interleukin-4(IL-4) to evaluate its immune effects.The results showed that the recombinant baculovirus rvAc-Cap and rvAc-NLS-Cap were successfully expressed by baculovirus expression system,and the expression level of Cap gene with NLS sequence removal was higher.After mice were immunized with recombinant protein rCap,both recombinant protein rCap and recombinant baculovirus rvAc-Cap could stimulate mice to produce specific IgG antibodies,and the Prime-Boost group had the highest level of IgG antibody after 3 immunization,but there was a significant difference between the other immunization groups(P>0.05).The lymphocyte proliferation index of each immune group was significantly higher than that of the PBS control group after the third immunization,and the difference was significant(P<0.05).The levels of IFN-γ,IL-2 and IL-4 in each immunization group were significantly different from those in the PBS control group(P<0.05),and the levels in the Prime-Boost group were the highest.These results

关 键 词：鸭圆环病毒 CAP蛋白 核定位信号肽 杆状病毒表达系统 免疫原性 

分 类 号：S852.659.2[农业科学—基础兽医学]