乌鳢水泡病毒P蛋白异构体的鉴定及其功能初探

为了鉴定乌鳢水泡病毒(SHVV)磷蛋白(P)的异构体并研究其在病毒增殖中的作用,本研究扩增了SHVV的P基因,构建了原核表达质粒pET32a-P,通过Ni-NTA亲和层析柱纯化His-P蛋白,免疫新西兰大白兔制备了多克隆抗体,利用该抗体对SHVV的P蛋白异构体进行鉴定。进一步利用增强性绿色荧光蛋白(EGFP)研究了P蛋白异构体的亚细胞定位,并通过定量PCR(qRT-PCR)、Western blot及TCID_(50)研究了过表达P蛋白异构体对SHVV增殖的影响。结果显示,SHVV感染斑点叉尾鮰卵巢细胞(CCO)出现3条P蛋白带,进一步验证表明,其中2条带分别是P蛋白(又称P1)及其异构体P2。亚细胞定位实验发现P1和P2主要定位在细胞质,但是可以在核质中穿梭。在CCO细胞中过表达P1、P2均能促进SHVV增殖。因此,SHVV在感染过程中能产生P蛋白(P1)及其异构体P2,且P1和P2对SHVV增殖发挥重要作用。研究结果有助于阐明SHVV的致病机理以及弹状病毒P蛋白异构体的功能。

Identification of snakehead vesiculovirus P protein isomers and primary study of their function

In order to identify the snakehead vesiculovirus (SHVV) phosphoprotein (P) isoforms and study their role in virus proliferation, the P gene of SHVV was amplified, and a prokaryotic expression plasmid pET32a-P was constructed. The recombinant His-P protein was purified by Ni-NTA affinity chromatography column and polyclonal antibody against the His-P protein was acquired through immunization of New Zealand white rabbits. Using the P protein polyclonal antibody, the SHVV P isoforms were determined. Furthermore, the subcellular localization of P isoforms was assessed using enhanced green fluorescent protein (EGFP), and the effect of overexpression of P isoforms on SHVV proliferation was determined using qRT-PCR, Western Blot, and TCID50. The results showed that three P protein bands were detected during SHVV-infected channel catfish ovary (CCO) cells. Further verification determined that two of the three bands were P protein (also called P1) and its isoform P2. Subcellular localization experiment showed that both P1 and P2 localized mainly in cytoplasm, and P1 and P2 could shuttle between nucleus and cytoplasm. Overexpression of P1 or P2 in CCO cells promoted the proliferation of SHVV. Thereby, SHVV can produce P protein isoforms P1 and P2, and P1 and P2 play an important role in SHVV replication. Our results will be helpful to illustrate SHVV pathogenicity.