HIV自然感染与疫苗诱导产生抗体鉴别肽的原核表达及纯化

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。

Prokaryotic Expression and Purification of HIV Differential Peptides for Natural Infection-elicited and Vaccine-induced Antibodies

Objective To express HIV differential peptides sk1,sk2,sk3 and their concatemers for natural infection-elicited and vaccine-induced antibodies.Methods By routine PCR and overlap extension PCR,sk1,sk2 and sk3 gene sequences as well as six concatemers of them were amplified from whole HIV-1 B genome plasmid,and cloned into prokaryotic expression vectors pET32a(+) and pEGX4T-2 separately.The constructed recombinant plasmids were transformed to E.coli BL21(DE3) and induced with IPTG.The expressed product was purified by Ni-NAT or glutathione-Sepharose-4B affinity chromatography.The expressed fusion peptides were identified by SDS-PAGE and analyzed for antigenicity by Western blot.Results A total of 18 recombinant plasmids were constructed by using pET-32a(+) and pGEX4T-2 as vectors.The his-sk1,his-sk123,his-sk213 and his-sk231 proteins were expressed by using vector pET-32a(+),while GST-sk3,GST-sk123 and GST-sk213 by using pGEX4T-2,both in soluble form.The expressed products showed specific reaction with HIV-1 positive serum while no reaction with immune serum induced by HIV DNA-Tiantan vaccine.After expression and purification in a large quantity,the expression level of GST-sk3 was the highest in all the proteins,which reached 40 mg / L,while the purity was more than 90%.Conclusion The bioactive differential peptides for natural infection-elicited and vaccine-induced antibodies were successfully expressed,which laid a foundation of developing differentially diagnostic kit for antibodies induced by natural infection and by vaccination.