缺失半胱氨酸富集区的HIV-1 HXB2株Tat蛋白在大肠杆菌中的高效表达及免疫原性分析

摘要

目的 删除人免疫缺陷病毒Ⅰ型(HIV-1) HXB2株Tat蛋白(长度101个氨基酸)的半胱氨酸富集区(22～37位氨基酸)以提高其在大肠杆菌中的表达量和分子稳定性,并分析缺失半胱氨酸富集区后的Tat蛋白[Tat(△C)蛋白]免疫原性的改变.方法 应用PCR方法对Tat基因的半胱氨酸富集区(64～111位核苷酸)进行缺失突变,获得其突变体序列[Tat(△C)DNA] ,并构建其重组表达质粒pET-32a-Tat(△C).相同的条件下将pET-32a-Tat(△C)和pET-32a-Tat质粒转入大肠杆菌BL21(DE3)中进行诱导表达及纯化.用pET-32a-Tat(△C)融合蛋白免疫家兔制备抗血清,ELISA和Western blot方法对抗血清进行免疫原性分析.结果 pET-32a-Tat(△C)蛋白在大肠杆菌中的表达水平(7.12 mg/ml)明显高于pET-32a-Tat蛋白(1.50 mg/ml);pET-32a-Tat蛋白在表达纯化后及25℃和4℃放置7 d后均有明显二聚体的产生,而pET-32a-Tat(△C)蛋白则未形成二聚体;pET-32a-Tat(△C)蛋白免疫家兔可诱导产生高滴度的抗体(1∶320 000),该抗体与Tat(△C)蛋白、Tat蛋白(1～101 AA)及化学合成Tat(1～86 AA)蛋白均呈特异性反应.结论 缺失HIV-1 Tat蛋白的半胱氨酸富集区可明显提高其原核表达水平和蛋白的稳定性并较好地保留其免疫原性,为HIV-1 Tat疫苗研究提供了一种新型免疫原.%Objective Deleting the cysteine-rich region (22-37 amino acids)of HIV-1 HXB2 Tat protein(whole length is 101 amino acids) to improve its stability and expression level in E.coli and to analyze the immunogenicity of Tat protein without the cystein-rich region [Tat(△C)protein]. Methods Tat DNA deleted the cysteine-rich region (64-111 nucleotides), named as Tat(△C)DNA, was obtained in vitro by PCR and cloned into pET-32a vector. pET-32a-Tat(△C)plasmid and the pET-32a-Tat plasmid were established and transformed into E.coli BL21(DE3) strains respectively to express and purify the protein. Three rabbits were vaccinated with pET-32a-Tat(△C)protein, then testify the reactivity of sera from rabbits by ELISA and Western blot. Results The dense of the purified pET-32a-Tat(△C)protein was 7.12 mg/ml,which was greatly more than pET-32a-Tat protein(1.50 mg/ml). Dimer of pET-32a-Tat protein can be observed just after the protein purification and stored at 25℃ and 4℃ for 7 days, but dimer of pET-32a-Tat(△C)protein was not formed at the same condition. Experimental rabbits were immunized with pET-32a-Tat(△C)protein and produced high titre of anti-pET-32a-Tat(△C)serum(1∶320 000), the antibody can react specifically with Tat(△C)protein, Tat protein (1-101 AA)and synthetic Tat(1-86 AA) protein. Deletion mutation of the cysteine-rich region of Tat protein was first performed in the study. Conclusion The expression level in E.coli and the stability of Tat protein deleted the cysteine-rich region can be increased greatly, and the protein remains good immunogenicity. The results may provide a novel antigen for further development of HIV-1 Tat vaccine.