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目的 构建HIV-1 Tat核心碱性区多肽Tat38-61重组原核表达质粒,在大肠杆菌中表达融合蛋白并进行纯化及免疫反应性检测。方法采用PCR法从HIV-1 HXB2株Tat1-101基因中扩增编码Tat38-61的基因序列,克隆至原核表达载体pET32a(+)中,构建重组原核表达质粒pET32a(+)-Tat38-61。转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经Ni2+-NTA柱亲和层析法纯化后,ELISA法鉴定其免疫反应性。结果重组原核表达质粒pET32a(+)-Tat38-61经双酶切及测序表明构建正确;SDS-PAGE分析显示,在相对分子质量约21 300处可见目的 蛋白条带,表达量占菌体总蛋白的67.4%,主要以可溶形式表达;纯化后融合蛋白的纯度可达97%以上;ELISA结果显示,该融合蛋白与兔抗PEPTIDE-Tat1-101血清及HIV阳性血清均呈特异性反应。结论已成功构建了HIV-1 Tat核心碱性区多肽Tat38-61的重组原核表达质粒,表达并纯化了PET32a(+)-Tat38-61融合蛋白,该融合蛋白碱性区表位得到较好的保留,为Tat38-61噬菌体突变文库的构建及亲和筛选奠定了基础。
Objective To construct the recombinant plasmid pET38-61 of HIV-1 Tat, and express the fusion protein in Escherichia coli for purification and immunoreactivity detection. Methods The gene encoding Tat38-61 was amplified from Tat-101 gene of HIV-1 HXB2 strain by PCR and cloned into prokaryotic expression vector pET32a (+) to construct recombinant prokaryotic expression plasmid pET32a (+) - Tat38-61. Escherichia coli BL21 (DE3) was transformed into E.coli BL21 and induced with IPTG. The expressed product was purified by Ni2 + -NTA column affinity chromatography and its immunoreactivity was identified by ELISA. Results The recombinant prokaryotic expression plasmid pET32a (+) - Tat38-61 was confirmed by double enzyme digestion and sequencing. SDS-PAGE analysis showed that the total protein was expressed in about 21 300 molecular weight , Which was mainly expressed in soluble form. The purity of the fusion protein was over 97% after purification. ELISA showed that the fusion protein reacted specifically with rabbit anti-PEPTIDE-Tat1-101 serum and HIV-positive serum. Conclusion The recombinant prokaryotic expression plasmid Tat38-61 of HIV-1 Tat core region has been successfully constructed, and the PET32a (+) - Tat38-61 fusion protein was expressed and purified. The basic epitopes of the fusion protein were well Reserved, which laid the foundation for the construction of Tat38-61 phage mutation library and affinity screening.