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基于Iclp的拟态弧菌OmpUAP真核表达重组质粒的构建 及其免疫效果初步评价
刘艳,丁淑荃,陶会竹,曹际,周昊,李槿年
0
(安徽农业大学动物科技学院,合肥 230036)
摘要:
为了探明鱼类恒定链类似蛋白(Iclp)能否作为免疫载体,提高基因疫苗的免疫效果,以拟态弧菌OmpU蛋白的黏附功能域(OmpUAP)为疫苗靶点,构建真核表达重组质粒pcDNA3.1-OmpUAP和基于草鱼Iclp的pcDNA3.1-Iclp-OmpUAP。用2种真核表达重组质粒分别免疫草金鱼,同时设置空质粒pcDNA3.1(+)对照组与PBS对照组。于免疫后不同时间(7、14、21、28和35 d)采用PCR和RT-PCR 方法检测重组质粒在鱼体不同组织中的分布与转录水平的表达情况,使用双抗体夹心ELISA检测血清抗体水平,并于免疫后第35天进行攻毒保护性实验,检测其免疫保护率。结果显示,免疫后第7 天在肌肉、鳃、头肾、肝脏和脾脏中均有重组质粒分布,并在35 d内持续表达。pcDNA-OmpUAP免疫组草金鱼的血清抗体水平缓慢上升,除第21天外,其余检测时间点的抗体水平与空质粒对照组无显著差异。pcDNA3.1-Iclp-OmpUAP 免疫组草金鱼在免疫后7 d即有血清抗体产生,随着免疫时间延长,抗体水平不断上升,至28 d达到峰值,随后抗体水平开始下降;除第7 天外,其余各检测时间点的血清抗体水平均显著或极显著高于pcDNA3.1-OmpUAP免疫组与空质粒对照组。攻毒后pcDNA3.1-Iclp-OmpUAP和pcDNA-OmpUAP免疫组草金鱼的相对免疫保护率为别为46.7%和20%,而空质粒对照组和PBS对照组的实验鱼全部发生死亡。表明Iclp可以作为免疫载体,提高鱼类基因疫苗的免疫效果。
关键词:  拟态弧菌  OmpU蛋白  Iclp蛋白  真核重组质粒  免疫效果
DOI:10.13610/j.cnki.1672-352x.20170524.019
投稿时间:2016-11-09
基金项目:国家自然科学基金 (31672698)资助。
Construction of Iclp-based recombinant eukaryotic expression plasmid containing the OmpUAP gene from vibrio mimicus and preliminary evaluation on its immune efficacy
LIU Yan,DING Shuquan,TAO Huizhu,CAO Ji,ZHOU Hao,LI Jinnian
(College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036)
Abstract:
In order to investigate if invariant chain-like protein (Iclp) of fish can be used as immune carrier for improving immune efficacy of gene vaccine, the recombinant eukaryotic expression plasmid pcDNA3.1-OmpUAP, which carried the OmpU adhesion function domain gene from Vibrio mimicus, and the plasmid Iclp-based pcDNA3.1-Iclp-OmpUAP were constructed and identified. Healthy Carassius auratus were divided into 4 groups and immunized with pcDNA3.1-OmpUAP, pcDNA3.1-Iclp-OmpUAP, pcDNA3.1 (+) and PBS control. On Day 7, 14, 21, 28 and 35 post-vaccination, the distribution and transcription of the recombinant plasmids in different tissues of Carassius auratus were detected by PCR and RT-PCR, respectively. The specific antibody against OmpUAP and Iclp-OmpUAP in the sera was detected with the triple antibody sandwich ELISA, and then the immuneprotection efficacy was evaluated by challenging V. mimicus at the 35th day of post-vaccination. The results from PCR and RT-PCR indicated that both recombinant plasmids were distributed in the muscle, gill, head kidney, liver and spleen and expressed continuously in different tissues of vaccinated fish within 35 days. Fish immunized with pcDNA3.1-OmpUAP slowly produced low level antibody against OmpUAP, and the antibody level at the other detection time points except the 21th day had no significant difference with the empty plasmid control group. Carassius auratus immunized with pcDNA3.1-Iclp-OmpUAP produced specific antibody against Iclp-OmpUAP on the 7th day of
Key words:  Vibrio mimicus  OmpU protein  invariant chain-like protein  recombinant eukaryotic expression plasmid  immune efficacy

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