HIV-1 HXB2株Tat半胱氨酸富集区N端移位突变体的构建及在大肠杆菌中的高效表达

Effective expression of HIV-1 HXB2 subtype Tat protein shifted the cysteine-rich region to N terminal in E. coli

目的构建HIV-1HXB2株Tat半胱氨酸富集区N端移位突变体,并在大肠杆菌E.coliBL21(DE3)中进行高效表达和纯化。方法应用PCR拼接的方法将首轮合成Tat基因的半胱氨酸富集区(64～111位核苷酸,22～37位氨基酸)移位至缺失半胱氨酸富集区的Tat(△C)的N末端,获得其突变体序列(Tat(CN)DNA),构建其原核表达质粒pET32a-Tat(CN),并转入大肠杆菌E.coli BL21(DE3)中进行诱导表达及纯化,经SDS-PAGE鉴定表达及纯化产物,纯化后的产物用间接ELISA进行鉴定。结果构建了pET32a-Tat(CN)突变体,pET32a-Tat(CN)融合蛋白在大肠杆菌中表达水平为5.17mg/ml,相对分子质量为31ku。间接的ELISA结果表明,pET32a-Tat(CN)能与Tat兔抗血清呈特异反应。结论成功构建了HIV-1 HXB2株Tat半胱氨酸富集区移位至TatN端的突变体,并能在大肠杆菌中高效表达,其纯化的蛋白pET32a-Tat(CN)很好地保留了与HIV-1Tat兔抗血清的免疫反应性,为HIV-1Tat的疫苗基础研究提供了一有价值的研究结论。

Objective To construct mutant of HIV-1 HXB2 subtype Tat which shifted the cysteine-rich region to N terminal, and to effectively express and purify the mutant protein in E. coli BL21 （DE3）. Methods Tat mutant DNA shifted the eysteine-rich region （64-111 nucleotides, 22-37 amino acids） to N-terminal of Tat deleting the cysteine-rich region Tat（/XC）, named as Tat （CN） DNA, was obtained by PCR and cloned into pET32a vector. pET32a-Tat （CN） plasmid was established and transformed into E. coli BL21 （DE3） strains. The expressed and purified pET32a-Tat （CN） protein was analyzed by SDS-PAGE, then testified the reactivity of the purified protein by indirect ELISA. Results The mutant of pET32a-Tat （CN） was established, the dense of the purified fusion protein pET32a-Tat （CN） was 5.17 mg/ml expressed in E. coli BL21 （DE3） , and the relative molecular mass of the protein was 31 ku. Indirect ELISA showed that anti-sera of pET32a-Tat protein from rabbit could react specifically with pET32a-Tat （CN） protein and pET32a-Tat protein, and titers of the sera were 1 ： 128 000 and 1：64 000 respectively. The reaction of the sera with pET32a protein was rather weak. Conclusion The recombinant mutant of HIV-1 HXB2 subtype Tat which shift the cysteine-rich region to N terminal is successfully established, and expressed efficiently in E. coli. The purified pET32a-Tat （CN） protein remains good antigenicity with the anti-sera of pET32a-Tat protein from rabbit. The results provide a valuable conclusion for further development of HIV-1 Tat vaccine.