不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究

doi:10.16431/j.cnki.1671-7236.2016.10.030

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2716-2723.doi: 10.16431/j.cnki.1671-7236.2016.10.030

不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究

董瑞凯, 郭晓宇, 袁维峰, 贾红, 鑫婷, 侯绍华, 朱鸿飞

中国农业科学院北京畜牧兽医研究所, 北京 100193

收稿日期:2016-03-01 出版日期:2016-10-20 发布日期:2016-10-28
通讯作者: 朱鸿飞 E-mail:bioclub@vip.sina.com
作者简介:董瑞凯(1989-),男,河北石家庄人,硕士,研究方向:动物疫苗与分子免疫学,E-mail:dongruikai1989@126.com
基金资助:
国家自然科学基金（31402232）

Immune Enhancement Activity of Different Types of CpG Motifs on the Recombinant Eg95 Antigen of Echinococus granulosus

DONG Rui-kai, GUO Xiao-yu, YUAN Wei-feng, JIA Hong, XIN Ting, HOU Shao-hua, ZHU Hong-fei

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received:2016-03-01 Online:2016-10-20 Published:2016-10-28

摘要/Abstract

摘要：

为探究不同类型CpG基序对细粒棘球蚴（Echinococus granulosus）Eg95抗原的免疫增强作用，将pET-32a-3Eg95重组大肠杆菌，经IPTG诱导表达，亲和层析纯化重组蛋白pET-32a-3Eg95。将重组蛋白分别与常规佐剂Quli-A、pUC18-CpG和CpG ODN混合制备基因工程亚单位疫苗样品，免疫小鼠。通过间接ELISA方法检测抗体水平，通过实时荧光定量PCR方法测定体外刺激试验后细胞因子的相对表达量，检测不同佐剂在体液免疫和细胞免疫方面对Eg95抗原免疫效果的增强作用。结果显示，诱导表达重组蛋白的大小约为56 ku，与理论值相符，用羊细粒棘球蚴阳性血清检测有特异性条带；pUC18-CpG组和CpG ODN组小鼠免疫后14、28、42 d的抗体平均水平（D_{450 nm}）分别为3.10、3.03、3.22和2.98、3.12、3.27，与Quli-A组抗体平均水平相比均差异显著（P<0.05）；CpG ODN组血清中抗体平均水平略高于pUC18-CpG组，但差异不显著（P>0.05）。重组蛋白刺激后pUC18-CpG组与CpG ODN组IFN-β、IFN-γ和TNF-α的相对表达量分别为6.88、2.35、6.28和5.03、2.85、7.07，与Quli-A组相比差异均显著（P<0.05）。因此相对于常规佐剂Quli-A，pUC18-CpG和CpG ODN在体液免疫和细胞免疫方面对Eg95抗原都有显著的免疫增强作用，CpG ODN的免疫增强作用略优于pUC18-CpG，二者均可作为免疫佐剂，增强现有包虫病基因工程疫苗抗原的免疫效果。

关键词: 细粒棘球蚴; pUC18-CpG; CpG ODN; 免疫刺激活性

Abstract:

To study the immune enhancement activity of CpG motifs on the recombinant Eg95 antigen of Echinococcus granulosus,Escherichia coli BL21(DE3) containing pET-32a-3Eg95 was grown in LB medium,and IPTG was then added for induction.The recombinant Eg95 protein was expressed and later harvested by affinity chromatography.The obtained antigen was mixed either with the conventional adjuvant Quli-A or with different types of CpG motifs (pUC18-CpG or CpG ODN),to prepare vaccine candidates for mice immunization.The average levels of serum IgG antibody were tested by indirect ELISA and the quantification of cytokine expression were determined by Real-time quantitative PCR after in vitro stimulation.The results identified that the size of the recombinant protein was 56 ku as predicted,and the recombinant protein could be recognized by serum from Echinococcus granulosus-infected sheep.The average levels of serum IgG antibody (D_{450 nm}) of the pUC18-CpG and CpG ODN groups on 14,28 and 42 d were 3.10,3.03,3.22 and 2.98,3.12, 3.27,respectively.Antibody levels from both CpG groups were significantly higher compared to the conventional adjuvant Quli-A group (P<0.05) The average level of serum IgG antibody in CpG ODN group was a little higher than that in pUC18-CpG group(P>0.05).After stimulation by recombinant protein 3Eg95,the relative expression levels of IFN-β,IFN-γ and TNF-α of pUC18-CpG and CpG ODN groups were 6.88,2.35,6.28 and 5.03,2.85,7.07,respectively,and both were higher than that of the Quli-A group (P<0.05).The results indicated that pUC18-CpG and CpG ODN had significant immune enhancement property on Eg95 antigen in both humoral and cellular immune response,and the immune enhancement activity of CpG ODN was slightly better than that of the pUC18-CpG.Hence,pUC18-CpG and CpG ODN could be used as efficient immune adjuvants enhancer in future vaccines against Echinococcus granulosus infection.

Key words: Echinococus granulosus; pUC18-CpG; CpG ODN; immune enhancer

中图分类号:

S855.9

董瑞凯, 郭晓宇, 袁维峰, 贾红, 鑫婷, 侯绍华, 朱鸿飞. 不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究[J]. 《中国畜牧兽医》, 2016, 43(10): 2716-2723.

DONG Rui-kai, GUO Xiao-yu, YUAN Wei-feng, JIA Hong, XIN Ting, HOU Shao-hua, ZHU Hong-fei. Immune Enhancement Activity of Different Types of CpG Motifs on the Recombinant Eg95 Antigen of Echinococus granulosus[J]. , 2016, 43(10): 2716-2723.

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不同类型CpG基序对细粒棘球蚴Eg95抗原的免疫增强作用研究

Immune Enhancement Activity of Different Types of CpG Motifs on the Recombinant Eg95 Antigen of Echinococus granulosus

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