Wang Aiping, Yin Jiajia, Zhou Jingming, Ma Hongfang, Chen Yumei, Liu Hongliang, Qi Yanhua, Liang Chao, Liu Yankai, Li Jinge, Zhang Gaiping
Zhengzhou University, School of Life Sciences, Zhengzhou University, Henan, PR China.
PeerJ. 2021 Jan 4;9:e10543. doi: 10.7717/peerj.10543. eCollection 2021.
The VP2 on the surface of the virus particle is the main structural protein of BTV, which can induce the host to produce neutralizing antibodies and play an important role in the antiviral immunity process. This study aimed to obtain the soluble VP2 and analyze its immunogenicity.
The gene encoding the full-length VP2 of BTV1 was amplified by PCR. The products from restriction enzyme digestion and ligase reaction between VP2 and vector pET-28a were transformed into DH5α. After PCR and sequencing detection, the positive plasmid PET28a-VP2 was transformed into BL21(DE3) and Rosetta(DE3) competent cells, expression induced by IPTG. The fusion protein was expressed in the optimized conditions with the induction of IPTG, purified by affinity chromatography and identified by SDS-PAGE and Western blotting. A total of 5 Balb/c mice aged 6-8 weeks were immunized with the fusion protein at a dose of 30 µg per mouse. Each mouse was immunized three times at an interval of 3 weeks.
The recombinant plasmid PET28a-VP2 was successfully constructed. The expression strains were induced by 0.4 mmol/L IPTG at 16 °C for 10 h, and BTV1 VP2 was expressed in a soluble form. The purity of the recombinant VP2 protein (∼109 kDa) was about 90% in the concentration at 0.2 mg/ml afterpurification. The purified VP2 had good immunoreactivity with BTV1 positive serum. Taken together, thisstudy offered a route for producing soluble BTV VP2, which retains activity and immunogenicity, to bebeneficial to the research on developing BTV vaccine, and lay the foundation for further research on BTV.
病毒粒子表面的VP2是蓝舌病毒（BTV）的主要结构蛋白，可诱导宿主产生中和抗体，在抗病毒免疫过程中发挥重要作用。本研究旨在获得可溶性VP2并分析其免疫原性。
通过PCR扩增编码BTV1全长VP2的基因。将VP2与载体pET-28a经限制性内切酶消化和连接反应后的产物转化至DH5α。经PCR和测序检测后，将阳性质粒PET28a-VP2转化至BL21(DE3)和Rosetta(DE3)感受态细胞中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。在IPTG诱导的优化条件下表达融合蛋白，通过亲和层析进行纯化，并经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹法（Western blotting）鉴定。将5只6-8周龄的Balb/c小鼠用融合蛋白以每只小鼠30μg的剂量进行免疫。每只小鼠每隔3周免疫3次。
成功构建重组质粒PET28a-VP2。表达菌株在16℃用0.4mmol/L IPTG诱导10小时，BTV1 VP2以可溶性形式表达。纯化后浓度为0.2mg/ml的重组VP2蛋白（~109kDa）纯度约为90%。纯化的VP2与BTV1阳性血清具有良好的免疫反应性。综上所述，本研究提供了一条生产具有活性和免疫原性的可溶性BTV VP2的途径，有利于BTV疫苗研发的研究，并为BTV的进一步研究奠定了基础。
