含分子内佐剂的SARS-CoV-2 RBD域重组蛋白制备及免疫效果评价

Preparation and immungenicity of recombinant protein containing intramolecular adjuvant in SARS-CoV-2 RBD domain

摘要:

制备含破伤风毒素肽(tetanus toxin,TT)、促吞噬肽(tuftsin)和新型冠状病毒刺突蛋白(spike,S蛋白)受体结合域(receptor-binding domain,RBD)的融合蛋白，探讨分子内佐剂对RBD蛋白体液免疫和细胞免疫效果的影响。将破伤风毒素肽、促吞噬肽与S蛋白RBD区域通过柔性多肽串联，密码子优化后构建重组载体，原核表达纯化制备重组S-TT-tuftsin蛋白，与铝佐剂混合后免疫BALB/c小鼠，对其体液及细胞免疫效果进行评价。重组S-TT-tuftsin蛋白以包涵体形式表达，离子交换层析纯化后采用梯度透析进行复性，复性蛋白经Dot blotting鉴定，可与新冠亚单位疫苗(安徽智飞公司)免疫后人血清发生反应。小鼠免疫实验结果表明，免疫35 d时抗体水平到达平台期，含分子内佐剂重组蛋白(铝佐剂)免疫小鼠后血清ELISA抗体效价高达1︰66 240，显著高于S-RBD蛋白(铝佐剂)免疫小鼠抗体效价(P<0.05)。同时，含分子内佐剂重组蛋白刺激小鼠产生更强的淋巴细胞增殖能力，刺激指数可达4.71±0.15，相较于S-RBD蛋白的刺激指数1.83±0.09具有显著性差异(P<0.000 1)。分子内佐剂破伤风毒素肽和促吞噬肽可显著增强新冠S蛋白RBD域的体液免疫和细胞免疫效果，可为新冠亚单位疫苗和其他病毒亚单位疫苗的研制提供理论基础和参考。

Abstract:

A fusion protein containing a tetanus toxin peptide, a tuftsin peptide and a SARS-CoV-2S protein receptor-binding domain (RBD) was prepared to investigate the effect of intramolecular adjuvant on humoral and cellular immunity of RBD protein. The tetanus toxin peptide, tuftsin peptide and S protein RBD region were connected by a flexible polypeptide, and a recombinant vector was constructed after codon optimization. The recombinant S-TT-tuftsin protein was prepared by prokaryotic expression and purification. BALB/c mice were immunized after mixed with aluminum adjuvant, and the humoral and cellular immune effects were evaluated. The recombinant S-TT-tuftsin protein was expressed as an inclusion body, and was purified by ion exchange chromatography and renaturated by gradient dialysis. The renaturated protein was identified by Dot blotting and reacted with serum of descendants immunized with SARS-CoV-2 subunit vaccine. The results showed that the antibody level reached a plateau after 35 days of immunization, and the serum antibody ELISA titer of mice immunized with recombinant protein containing intramolecular adjuvant was up to 1:66 240, which was significantly higher than that of mice immunized with S-RBD protein (P<0.05). At the same time, the recombinant protein containing intramolecular adjuvant stimulated mice to produce a stronger lymphocyte proliferation ability. The stimulation index was 4.71±0.15, which was significantly different from that of the S-RBD protein (1.83±0.09) (P<0.000 1). Intramolecular adjuvant tetanus toxin peptide and tuftsin peptide significantly enhanced the humoral and cellular immune effect of the SARS-CoV-2 S protein RBD domain, which provideda theoretical basis for the development of subunit vaccines for SARS-CoV-2 and other viruses.