Coll Julio
Instituto Nacional de Investigación y Tecnología Agrarias y Alimentarias, Dpto. Biotecnología, INIA, crt.Coruña km 7, 20040 Madrid, Spain.
Fish Shellfish Immunol. 2017 Aug;67:393-401. doi: 10.1016/j.fsi.2017.06.038. Epub 2017 Jun 15.
Bacterins obtained from recombinant bacteria displaying heterologous antigens in its surface coded by prokaryotic rather than eukaryotic expression plasmids (here called "spiny" bacterins or spinycterins), have been used to increase fish immunogenicity of recombinant viral protein fragments. To explore their immunogenicity, five bacterial-specific membrane anchor-motifs characterized in the literature (Nmistic, Mistic, NTD, YAIN and YBEL) were genetically fused to the immunorelevant cystein-free 117 amino acid fragment II from the ORF149 of cyprinid herpes virus 3 (frgII). The fusion of anchor-motifs to the N-terminus of frgII enriched expression in E.coli outer membranes as demonstrated by ELISA, immunofluorescence and flow cytometry of formaldehyde-fixed recombinant bacteria (spinycterins). Unconventional low-intensity ultrasound inducing mucosal micropores in a reversible non-harmful manner was used before carp or zebrafish immersion on spinycterin suspensions as a practical delivery alternative to fish-to-fish injection. After ELISA screening for anti-frgII-specific antibodies of spinycterin-immunized fish plasma, the YBEL constructs were identified as the most immunogenic in both carp and zebrafish, correlating with one of the best expressed recombinant proteins as demonstrated by Western blot and surface enriched as demonstrated by ELISA and flow cytometry. The use of prokaryotic expression plasmids to express viral immunorelevant protein fragments in traditionally used fish vaccination bacterins should reduce the environmental concerns raised by DNA vaccination based on eukaryotic expression plasmids. Therefore, spinycterins may be a useful alternative to develop safer fish viral vaccines and mass vaccination methods.
从原核而非真核表达质粒编码的、在其表面展示异源抗原的重组细菌中获得的菌苗（这里称为“多刺”菌苗或多刺菌素），已被用于提高重组病毒蛋白片段在鱼类中的免疫原性。为了探究它们的免疫原性，将文献中描述的五个细菌特异性膜锚定基序（Nmistic、Mistic、NTD、YAIN和YBEL）与鲤疱疹病毒3（CyHV-3）开放阅读框149的无半胱氨酸的117个氨基酸免疫相关片段II（frgII）进行基因融合。通过对甲醛固定的重组细菌（多刺菌素）进行ELISA、免疫荧光和流式细胞术检测表明，将锚定基序融合到frgII的N端可使大肠杆菌外膜中的表达增加。在鲤鱼或斑马鱼浸泡于多刺菌素悬浮液之前，采用以可逆无害方式诱导黏膜微孔的非常规低强度超声，作为鱼类间注射的一种实际递送替代方法。在对多刺菌素免疫的鱼血浆进行抗frgII特异性抗体的ELISA筛选后，YBEL构建体被确定为在鲤鱼和斑马鱼中免疫原性最强的，这与通过蛋白质印迹法证明的最佳表达重组蛋白之一以及通过ELISA和流式细胞术证明的表面富集相关。使用原核表达质粒在传统使用的鱼类疫苗菌苗中表达病毒免疫相关蛋白片段，应可减少基于真核表达质粒的DNA疫苗接种所引发的环境问题。因此，多刺菌素可能是开发更安全的鱼类病毒疫苗和大规模疫苗接种方法的一种有用替代物。
