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VP28是对虾白斑综合征病毒(White Spot Syndrome Virus,WSSV)的囊膜蛋白。将VP28基因序列密码子优化后合成,经限制性内切酶NdeⅠ、XhoⅠ酶切后,按正确的阅读框顺序插入到p ET-28a(+)表达载体上。重组质粒转化大肠杆菌BL21(DE3),经质粒双酶切、测序鉴定后成功地构建了VP28基因原核表达载体p ET-28a-VP28。转化菌经IPTG诱导,SDS-PAGE显示含有与预期大小一致的约30 ku蛋白带,主要以包涵体形式表达。采用Ni-IDA-Sepharose CL-6B亲和层析柱对重组蛋白进行纯化,获得了纯度为95%的大肠杆菌重组蛋白VP28。该纯化蛋白作为绝对定量Western的标准品,梯度稀释建立标准曲线,以定量检测转基因鱼腥藻7120中的VP28绝对表达量。实验结果表明,大肠杆菌BL21(DE3)表达的重组蛋白VP28与鱼腥藻7120中表达的VP28分子量基本相同,纯化后的大肠杆菌重组蛋白梯度稀释作为绝对定量Western标准曲线,计算出转基因鱼腥藻7120在培养第17天时,VP28的表达量最大,为5.14μg/m L,占转基因鱼腥藻7120总蛋白浓度的1.45%。这不仅对转基因鱼腥藻的高效培养具有重要意义,也为日后确定投喂对虾口服疫苗有效剂量防治白斑综合征奠定了基础。
VP28 is an envelope protein of White Spot Syndrome Virus (WSSV). The codon 28 of VP28 gene was optimized and synthesized. The fragment was digested with restriction endonucleases Nde I and Xho I and inserted into p ET-28a (+) expression vector in the correct reading frame. The recombinant plasmids were transformed into E. coli BL21 (DE3). The prokaryotic expression vector pET-28a-VP28 of VP28 gene was successfully constructed by double enzyme digestion and sequencing. Transformants were induced by IPTG. SDS-PAGE showed about 30 ku protein bands consistent with the expected size, mainly expressed as inclusion bodies. The recombinant protein was purified by Ni-IDA-Sepharose CL-6B affinity chromatography to obtain E. coli recombinant protein VP28 with a purity of 95%. The purified protein serves as a standard for absolute quantification of Western, and a standard curve is established by gradient dilution to quantitatively detect the absolute amount of VP28 in the transgenic Anabaena 7120. The experimental results showed that the recombinant protein VP28 expressed in E. coli BL21 (DE3) was basically the same as the VP28 expressed in Anabaena 7120, and the gradient of purified E. coli recombinant protein was used as an absolute quantitative Western standard curve to calculate transgenic Anabaena 7120 had the highest expression of VP28 at day 17, which was 5.14μg / m L, which accounted for 1.45% of the total protein concentration of transgenic Anabaena 7120. This is of great significance not only for the efficient cultivation of the transgenic Anabaena, but also for the future determination of the effective dose of the oral vaccine for shrimp feeding to prevent white spot syndrome.