对虾白斑综合征病毒囊膜蛋白VP53B原核表达及抗血清的制备

Prokaryotic expression and serum preparation of envelope protein VP53B of white spot syndrome virus (WSSV)

摘要:

【目的】研究对虾白斑综合征病毒(White spot syndrome virus，WSSV)囊膜蛋白sVP53B克隆、表达、纯化及抗血清制备。【方法】根据WSSV囊膜蛋白基因序列，设计引物，PCR扩增出功能序列(Svp53B)，构建到pET-16b载体后，转化至大肠杆菌Rosetta 2诱导表达，用SDS-PAGE、Western blotting检测优化表达。表达产物采用Ni-NTA琼脂糖磁珠进行纯化、割胶回收融合蛋白，以纯化的Svp53B-his为抗原，免疫兔子获得多克隆抗体，通过间接ELISA检测抗体的效价。【结果】构建重组质粒pET-16b-Svp53B，在大肠杆菌Rosetta 2中以1 mmol/L IPTG诱导表达量最高，主要以包涵体形式表达。纯化包涵体蛋白免疫兔子，获得多克隆血清，效价达到1:150 000。【结论】原核表达并纯化得到高纯度的WSSV囊膜蛋白sVP53B，制备的兔源多克隆血清亲和力高、特异性好，这对后期进一步研究VP53B与经口侵染相关功能奠定了基础。

Abstract:

[Objective] To clone, express and purify envelope protein sVP53B of WSSV (white spot syndrome virus) for polyclonal serum preparation. [Methods] Based on the GenBank sequence of WSSV vp53B, primers were designed, and PCR was done to amplify functional sequence of Svp53B. Svp53B was inserted into prokaryotic expression vector, pET-16b. Recombinant plasmid was transformed into Escherichia coli Rosetta 2. The expression of sVP53B was detected by SDS-PAGE and Western blotting. The protein was purified with Ni-NTA agarose beads, then applied to immune rabbits for polyclonal serum detecting it’s titer by indirect ELISA (enzyme-linked immuno sorbent assay). [Results] The sVP53B was expressed most by 1 m mol/L IPTG (isopropyl-β-D-thiogalactoside) inducing and mainly expressed as inclusion bodies. Polyclonal serum, prepared by immunizing rabbit, revealed a titer as high as 1:150 000. [Conclusion] Recombinant protein sVP53B was highly expressed in E. coli and showed high purity after purification. The prepared polyclonal serum had high affinity and specificity against sVP53B. The results pave the way for functional study of VP53B during WSSV oral infection.