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目的原核表达并纯化HIV自然感染与疫苗诱导产生抗体鉴别肽sk1、sk2、sk3及3条肽段串联序列。方法用普通PCR及重叠延伸PCR法从HIV-1 B亚型质粒扩增得到sk1、sk2、sk3基因序列及3条肽段基因片段的6种连接序列,并分别克隆入原核表达载体pET-32a(+)及pGEX4T-2中,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经Ni-NAT或谷胱甘肽-Sepharose-4B层析柱亲和层析纯化。SDS-PAGE分析融合肽的表达,Western blot分析融合肽的抗原性。结果分别以pET-32a(+)和pGEX4T-2为载体,共构建了18个重组表达质粒;his-sk1、his-sk123、his-sk213、his-sk231及GST-sk3、GST-sk123、GST-sk213分别在pET-32a(+)及pGEX4T-2表达载体中以可溶形式表达,且均能与HIV-1阳性血清反应,而不能与HIV DNA-天坛疫苗免疫血清反应;经大量诱导表达纯化后,GST-sk3表达量最高,可达40 mg/L,纯度超过90%。结论已成功表达并纯化了具有生物学活性的HIV自然感染与疫苗诱导产生抗体鉴别肽,为研制HIV自然感染与疫苗诱导产生抗体鉴别诊断试剂盒奠定了基础。
Objective To express and purify the natural infection of HIV and to induce the production of antibody-identifying peptides sk1, sk2, sk3 and three peptide tandem sequences by prokaryotic expression. Methods The sk1, sk2 and sk3 genes were amplified from the subtype HIV-1 B subtype plasmid by PCR and overlap extension PCR, and 6 kinds of junction sequences of 3 peptide fragments were cloned into prokaryotic expression vector pET-32a (+) And pGEX4T-2, transformed into E.coli BL21 (DE3) and induced by IPTG. The expressed product was purified by Ni-NAT or glutathione-Sepharose 4B chromatography. The expression of the fusion peptide was analyzed by SDS-PAGE and the antigenicity of the fusion peptide was analyzed by Western blot. Results A total of 18 recombinant plasmids were constructed using pET-32a (+) and pGEX4T-2 as vectors respectively. His-sk1, his-sk123, his-sk213, his-sk231 and GST-sk3, GST- -sk213 were expressed in soluble form in pET-32a (+) and pGEX4T-2 expression vectors, respectively, and all of them were able to react with HIV-1 positive sera without reacting with HIV DNA-Tiantan Vaccine serum. After purification, the highest expression of GST-sk3, up to 40 mg / L, more than 90% purity. CONCLUSION: The natural HIV infection with biological activity and the identification of antibody-producing peptides by the vaccine have been successfully expressed and purified, which lays the foundation for the development of a kit for differential diagnosis between HIV natural infection and vaccine-induced antibody.