细粒棘球绦虫EGR-03347基因的原核表达及其对小鼠免疫效果的初步研究  

Prokaryotic expression of 3-hydroxyacyl-Co A dehydrogenase gene from Echinococcus granulosus(EGR-03347) and preliminary study on its immune effect on mice

作　　者：县锦雯 王炜烨 王芸菲 张艳艳[2] 马勋[1] 孟季蒙[2] 王正荣[2] 薄新文[2] XIAN Jin-wen;WANG Wei-ye;WANG Yun-fei;ZHANG Yan-yan;MA-Xun;MENG Ji-meng;WANG Zheng-rong;BO Xin-wen(College of Animal Science and Technology,Shihezi Univeristy,Shihezi 832000,China;State Key Laboratory of sheep Genetic Improvement and Healthy Production/Institute of Animal Husbandry and Veterinary,Xinjiang Academy of Agricultural and Reclamation Sciences,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所,新疆石河子832000

出　　处：《中国预防兽医学报》2021年第8期873-879,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金项目(31860701、31360608);国家重点基础研究发展计划(973计划)项目(2015CB150300);新疆生产建设兵团国际科技合作项目(2017BC003、2020BC007)。

摘　　要：为了阐明细粒棘球绦虫3-羟基酰基-CoA脱氢酶基因(EGR-03347)生物学功能及其免疫原性,本研究通过分析已发表细粒棘球绦虫原头蚴高通量mRNA测序数据,从中筛选出细粒棘球绦虫脂肪酸代谢主要的调控基因之一—EGR-03347,将其克隆于原核表达载体pET-32a中,构建的重组质粒经诱导、纯化后通过SDS-PAGE及western blot检测。结果显示,重组EGR-03347蛋白(rEGR-03347)以包涵体的形式存在,分子量约47 ku;western blot结果显示,纯化的蛋白可与犬棘球蚴阳性血清特异性结合,具有较好的反应原性。将纯化的rEGR-03347与弗氏佐剂等体积混合并乳化后对小鼠经皮下多点注射(50μg/只),共免疫4次,分别于首免后不同时间采血分离血清,经ELISA检测小鼠血清抗体水平;同时采用qPCR方法检测首免后不同时间小鼠脾细胞因子(IL-4、IL-10、TNF-α、IFN-γ)的转录水平。结果显示,随着免疫次数的增加,免疫组小鼠血清抗体水平逐渐升高,且在首免后56 d,即第4次免疫后抗体水平达到峰值,与对照组(PBS及佐剂组)相比差异极显著(p<0.01)。qPCR结果显示,rEGR-03347可诱导小鼠产生IFN-γ、IL-4和IL-10细胞因子,其中免疫后14 d~56 d rEGR-03347可致Th1型细胞因子IFN-γ的表达上调,免疫后56 d,可诱导Th 2型细胞因子IL-4和IL-10的表达上调,表明rEGR-03347可诱导小鼠产生Th1和Th2两种类型的免疫反应。综上,本研究首次初步证明细粒棘球绦虫EGR-03347具有作为诊断抗原及疫苗候选抗原的潜力,为后期其诊断方法及疫苗研发提供了参考依据。To study the function and immunogenicity of the 3-hydroxyacyl-CoA dehydrogenase of Echinococcus granulosus,the coding sequence of this gene(EGR-03347) was subcloned into prokaryotic expression vector p ET-32 a and transformed into E. coli. After induction and purification, the expression of the recombinant protein(rEGR-03347) was detected by SDS-PAGE and western blot. The results showed that the rEGR-03347 exists in the form of inclusion bodies with a molecular weight of about47 ku. The r EGR-03347 could react with a positive canine serum against Echinococcus granulosus as indicated by western blot. The purified rEGR-03347 was mixed with Freund’s adjuvant and then used to immunize mice(50μg/mouse). Antibody levels were detected by ELISA and the transcription levels of mouse spleen cytokines(IL-4, IL-10, TNF-α, IFN-γ) were examined by qPCR.The results indicated that the serum antibody level of the mice gradually increased after the rEGR-03347 immunization, and were significantly higher than that in the control groups(p<0.01). The qPCR results showed that rEGR-03347 could stimulate the expression of IFN-γ, IL-4 and IL-10 cytokines in mice after immunization with the rEGR-03347. The expression of IFN-γ started to increased at 14 d after immunization and the expression of IL-4 and IL-10 were up-regulated 56 d after immunization. These results indicated that the rEGR-03347 could induce two types of immune responses(Th1 and Th2) in mice. In conclusion, this study preliminarily proved that Echinococcus granulosus EGR-03347 has the potential to be used as a diagnostic antigen and candidate vaccine antigen, which provides a reference basis for later development of diagnostic methods and vaccines.

关 键 词：细粒棘球绦虫 3-羟基酰基-CoA脱氢酶 原核表达 蛋白纯化 细胞因子 

分 类 号：S852.4[农业科学—基础兽医学]