摘要
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 a prokaryotic expression vector for HIV-1 Tat core and basic region polypeptide Tat38-61,express fusion protein in E.coli,identify the expressed product and analyze its immunoreactivity.Methods The gene encoding Tat38-61 was amplified by PCR from HIV-1 HXB2 Tat1-101 gene and cloned into prokaryotic expression vector pET32a（＋）.The constructed recombinant plasmid pET32a（＋）-Tat38-61 was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was purified by Ni2＋-NTA column affinity chromatography and determined for immunoreactivity by ELISA.Results Both restriction analysis and sequencing proved that recombinant plasmid pET32a（＋）-Tat38-61 was constructed correctly.SDS-PAGE showed a target protein band with relative molecular mass of about 21 300.The expressed product,mainly in a soluble form,contained 67.4% of total somatic protein and reached a purity of more than 97% after purification.ELISA showed specific reactions of the fusion protein with both rabbit anti-PEPTIDE-Tat1-101 serum and HIV-positive serum.Conclusion The prokaryotic expression vector for HIV-1 Tat core and basic region polypeptide Tat38-61 was successfully constructed,and Tat38-61 fusion protein was expressed and purified.The basic area epitope of the fusion protein was remained well,which laid a foundation of construction and screening of Tat38-61 mutant phage display library.
出处
《中国生物制品学杂志》
CAS
CSCD
2011年第1期10-13,24,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金资助项目(30872246
30972799)
上海市基础研究重点项目(08JC1405200)
