HIV-1型gp41抗原蛋白在大肠杆菌无细胞体系中的表达和免疫反应性检测

HIV-1 Antigenic Protein Gp41: Expression in E. coli Cell-Free System and Immunoreactivity Analysis

HIV-1型gp41抗原蛋白在HIV初筛诊断中有重要应用。该研究合成了gp41全长基因序列,将其与p IVEX2.4c酶切连接构建表达载体,在大肠杆菌无细胞体系中实现了目标蛋白的全长可溶表达。考察了添加不同浓度非离子型去污剂Brij78对蛋白可溶性的影响,在0.2%Brij78条件下,可溶性gp41重组蛋白的表达水平达到650μg/m L。Ni2+亲和层析纯化目标蛋白,纯度达到95.6%;超滤浓缩后,蛋白浓度为0.9 mg/m L,总回收率为70.8%。免疫反应性检测结果显示该gp41重组抗原制备的乳胶HIV抗体诊断试纸能与不同滴度的HIV-1型阳性标本反应,在检测线位置出现红色条带,而与HIV-1型阴性标本无反应。该研究制备了具有完整氨基酸序列的HIV-1型gp41重组抗原,为进一步提高HIV抗体诊断试剂的质量打下了基础。

HIV-1 antigenic protein gp41 is one vitally important raw material with wide application in the initial screening and diagnosis of HIV. Nevertheless,as a transmembrane protein,gp41 is difficult to be expressed in vivo of E. coli because of its hydrophobic toxicity to E. coli cells and the current commercial gp41 protein is expensive to import from abroad. In order to fulfill the production of HIV diagnostic reagents,the source problem of gp41 antigenic material with favorable immunoreactivity must be solved. This research synthesized full-length gene sequence of gp41,which was ligated to p IVEX2. 4c after enzyme digestion to construct the expression vector,achieving full-length and soluble expression of target protein in cell-free system in vitro of E. coli. Then,a study was presented to make sure the effect on the soluble expression of gp41 when different concentrations of non-ionic detergent Brij78 were added into cell-free system. Under the optimal conditions of non-ionic detergent brij78（ a concentration of 0. 2%）,the maximum soluble expression level of gp41 recombinant protein could reach up to 650 μg / m L. By using Ni2 ＋affinity chromatography and then concentrated by ultrafiltration,the purified gp41 protein showed a purity of 95. 6%,a concentration of 0. 9 mg / m L and a total recovery rate of 70. 8%. On this basis,the purified gp41 recombinant antigen was used to prepare latex test strips to assess its immunoreactivity by detecting 4 HIV-1 positive samples with different antibody titres and 4 HIV-1 negative samples. The results of immunoreactivity analysis proved that,a red test line emerged on the test strips when HIV-1 positive samples with different antibody titres were loaded,while there was no red test line appearing when HIV-1 negative samples were dropped on the test strips. In conclusion,this work provides a promising HIV-1 gp41 recombinant antigen which has integral structure and favorable immunoreactivity,laying the foundation for further improving the quality of existing HIV diagnostic kit.