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当前位置: 首页 > 文章 > 基于分子佐剂EDA的弓形虫DNA疫苗研究 中国兽医科学 2018 (11) 1374-1380
Position: Home > Articles > Study on Toxoplasma DNA vaccine based on molecular adjuvant EDA and SAG1 Chinese Veterinary Science 2018 (11) 1374-1380

基于分子佐剂EDA的弓形虫DNA疫苗研究

作  者：
龚丽贞;黄春芳;陈欢;黄潇航;林霞琳;宋肇程;黄志坚;殷光文
关键词：
弓形虫;EDA;SAG1;分子佐剂;小鼠免疫保护
摘  要：
为构建分子佐剂纤维连接蛋白外域A(EDA)与弓形虫表面膜蛋白1(SAG1)基因融合的真核表达载体并探讨其对小鼠的免疫原性,以本实验室保存的质粒为模板,进行PCR扩增,获得EDA-SAG1与SAG1目的片段,再分别连接到真核表达载体pVAX1上,构建出重组质粒pVAX1-EDA-SAG1和pVAX1-SAG1。将构建的质粒pVAX1-EDA-SAG1、pVAX1-SAG1同空载体pVAX1通过肌肉注射的方式免疫小鼠,通过ELISA、CCK-8法和小鼠攻虫试验对该DNA疫苗的免疫效果进行评价。经PCR扩增获得的SAG1片段长为855 bp,EDA-SAG1片段长为1 131 bp。双酶切和测序结果显示成功构建了上述重组质粒。在ELISA检测中,实验组血清中抗体Ig G、Ig G1、Ig G2a水平和细胞因子IL-4、IL-10、IL-12p70及IFN-γ的质量浓度较高,与对照组相比差异显著(P<0.05)。在脾淋巴细胞增殖试验中,分子佐剂EDA-SAG1组的增殖效果最明显(P<0.05)。攻虫后,pVAX1-EDA-SAG1实验组小鼠存活时间最长,较空白对照组多存活4 d。实验组pVAX1-EDA-SAG1相对于对照组pVAX1-SAG1具有较好的免疫效果,分子佐剂EDA可以显著地增强弓形虫SAG1的免疫原性。
译  名：
Study on Toxoplasma DNA vaccine based on molecular adjuvant EDA and SAG1
作  者：
GONG Li-zhen;HUANG Chun-fang;CHEN Huan;HUANG Xiao-hang;LIN Xia-lin;SONG Zhao-cheng;HUANG Zhi-jian;YIN Guang-wen;Fujian Animal Medicine Engineering Laboratory/College of Animal Sciences,Fujian Agriculture and Forestry University;
单  位：
GONG Li-zhen%HUANG Chun-fang%CHEN Huan%HUANG Xiao-hang%LIN Xia-lin%SONG Zhao-cheng%HUANG Zhi-jian%YIN Guang-wen%Fujian Animal Medicine Engineering Laboratory/College of Animal Sciences,Fujian Agriculture and Forestry University
关键词：
Toxoplasma gondii;;EDA;;SAG1;;molecular adjuvant;;mouse immune protection
摘  要：
To construct a DNA vaccine expressing the molecular adjuvant fibronectin extra domain A(EDA) and the surface antigen 1(SAG1) gene of Toxoplasma gondii and to investigate immunogenicity of this vaccine in mice,the EDA-SAG1 and SAG1 fragments were amplified by PCR and then ligated into the eukaryotic expression vector p VAX1 to construct the recombinant plasmids p VAX1-EDA-SAG1 and p VAX1-SAG1. The plasmids p VAX1-EDA-SAG1,p VAX1-SAG1 and p VAX1 were respectively immunized into mice by intramuscular injection,and the immune effect of the DNA vaccines were evaluated by ELISA,CCK-8 method.The results showed that the fragment of SAG1 was 855 bp in length,and EDA-SAG1 was 1 131 bp in length.The plasmids p VAX1-EDA-SAG1 and p VAX1-SAG1 were successfully constructed after confirmed by double enzyme digestion and sequencing.The levels of Ig G,Ig G1,and Ig G2 a antibodies in the serum of the experimental group and cytokines IL-4, IL-10,IL-12 p70 and IFN-γ in the serum were higher than that inthe control groups(P<0.05).The proliferation effect of the molecular adjuvant EDA was the most obvious(P<0.05). After the mice were infected with T.gondii RH strain, the mice in the p VAX1-EDA-SAG1 group survived the longest time,and they survived 4 days more than the blank control group.The molecular adjuvant EDA could significantly enhance the immunogenicity of the SAG1.

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