Kumar Manoj, Saravanan P, Jalali S K
Foot-and-Mouth Disease Vaccine Centre, Indian Veterinary Research Institute, Bangalore Campus, Hebbal, Bangalore, Karnataka 560024 India.
National Bureau of Agricultural Insect Resources, H A Farm Post, Bellary Road, Hebbal, Bangalore, Karnataka 560024 India.
Virusdisease. 2016 Mar;27(1):84-90. doi: 10.1007/s13337-015-0290-8. Epub 2015 Nov 24.
Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7 days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47 kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.
口蹄疫（FMD）是一种高度传染性的病毒性疾病，会给家畜造成严重的经济损失。通过重组DNA技术生产的病毒样颗粒（VLPs）因其免疫原性和安全性，在开发口蹄疫新疫苗方面越来越受到重视。在本研究中，描述了一种在蓖麻蚕（Samia cynthia ricini）幼虫中表达、纯化和鉴定口蹄疫病毒（FMDV）VLPs的实用且经济可行的方法。尽管对三种鳞翅目昆虫幼虫（棉铃虫、斜纹夜蛾和蓖麻蚕）进行了VLPs生产测试，但仅在蓖麻蚕幼虫中获得了表达。在Sf9细胞中制备了编码FMDV多聚蛋白P1-2A-3C的高滴度重组杆状病毒。与口服喂养导致的低水平表达相比，将重组杆状病毒注射到蓖麻蚕幼虫的血腔中，感染后3至7天（dpi）血淋巴中VLPs的表达水平有所增加。VLPs在夹心ELISA中与针对O/IND/R2/75型FMDV全病毒颗粒产生的血清发生反应，并且在蛋白质印迹中26、37和47 kDa的蛋白条带模式表明它们与天然病毒具有抗原相似性。蔗糖密度梯度纯化的VLPs用于免疫兔子和豚鼠以评估免疫原性。此外，兔子和豚鼠血清样本在间接ELISA中的反应性（滴度为1.30 - 2.81 Log10）表明VLPs在本质上具有抗原性和免疫原性。我们首次证明蓖麻蚕幼虫可用于生产O/IND/R2/75型FMDV的VLPs。这种方法对于大规模生产用于疫苗或口蹄疫控制计划中诊断用途的重组VLPs可能有用。
