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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(OmpU_(AP))为疫苗靶点,构建真核表达重组质粒pcDNA3.1-OmpU_(AP)和基于草鱼Iclp的pcDNA3.1-Iclp-OmpU_(AP)。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-OmpU_(AP)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-OmpU_(AP)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-OmpU_(AP)免疫组与空质粒对照组。攻毒后pcDNA3.1-Iclp-OmpU_(AP)和pcDNA-OmpU_(AP)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。
In order to investigate whether Iclp can serve as an immunological carrier and improve the immunogenicity of gene vaccines, we constructed the eukaryotic expression vector using the OmpU AP (adhesion domain) Recombinant plasmids pcDNA3.1-OmpU_ (AP) and grass carp Iclp-based pcDNA3.1-Iclp-OmpU_ (AP). Two kinds of eukaryotic expression plasmids were used to immunize the goldfish, and the empty plasmid pcDNA3.1 (+) control group and the PBS control group were set at the same time. The expression and distribution of recombinant plasmids in different tissues of fish were detected by PCR and RT-PCR at different time points after immunization (7, 14, 21, 28 and 35 days). Serum antibodies were detected by double antibody sandwich ELISA Level, and in 35 days after immunization protective challenge experiments to detect the rate of immunoprotection. The results showed that recombinant plasmids were distributed in muscle, gill, head kidney, liver and spleen on the 7th day after immunization and continued to be expressed within 35 days. Antibody levels in the pcDNA-OmpU_ (AP) vaccinated grass carp increased slowly. Except for the 21th day, antibody levels at the other time points were not significantly different from those of the control group. Antibody levels were detected at 7 d after immunization with antibodies against pcDNA3.1-Iclp-OmpU_ (AP). The antibody levels increased with the prolongation of immunization and peaked at 28 d, and then the antibody levels began to decline. After 7 days, the levels of serum antibody in other test time points were significantly or extremely significantly higher than those in pcDNA3.1-OmpU AP group and empty plasmid control group. After challenge, the relative immunoprotection rates of pcDNA3.1-Iclp-OmpU AP and pcDNA-OmpU AP were 46.7% and 20% respectively, while those of the empty plasmid control group and the PBS control group All died. Iclp can be used as an immunological carrier to improve the immune effect of fish genetic vaccine.