HIV-1跨膜蛋白gp41重组抗原的表达及其免疫反应性

HIV-1 transmembrane glycoprotein 41:Expression of recombinant antigen and antigenicity analysis

目的为开发和建立敏感、特异的抗HIV-1抗体的检测方法研制重组gp41抗原。方法根据HIV-1的基因序列,设计并合成了1对PCR扩增引物,应用PCR技术从HIV-1外膜基因组中扩增出HIV-1的gp41截短体,将扩增的基因片段插入质粒pET-28a中构建成重组表达质粒pET-gp41。诱导表达并纯化gp41重组抗原,对gp41进行了初步应用分析。结果gp41在大肠埃希菌BL21(DE3)中获得高表达,表达量占菌体总蛋白量的26.08%。纯化后截短体gp41的纯度为97.94%。经间接ELISA和免疫印迹检测,纯化后的表达产物gp41具有很高的抗原特异性和免疫反应性。结论研制的重组抗原gp41有较强的抗原性和潜在的应用价值。

Objective To develop and establish a new sensitive, specific method for serologic detection of HIV-1. Methods A pair of primer for PCR was designed and synthesized according to the sequence of human immunodefieieney virus type-1 （ HIV-1 ） gene. By using the synthesized primers, the traneated fragment of transmembrane glyeoprotein 41 （gp41） gene was amplified from the envelop-en- coding region in HIV genome by PCR. The amplified DNA fragment was cloned into a plasmid pET-28a and i＇eeombinant expression plasmid pET-gp41 was constructed. The recombinant gp41was indueibly expressed and purified. Preliminary applicable analysis for the antigenieity of the recombinant gp41 was conducted. Results The gp41 was highly expressed in E. eoli BL21 （ DE3 ）. The amount of gp41 accounted for 26.08% of the total lytie protein. The purity of the purified gp41 was 97.94%. By using ELISA and Western blot, high specificity and strong antigenieity were shown. Conclusion The recombinant gp41 exhibited strong antigenieity, thus its potential availability for HIV diagnosis may be expected.