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目的:运用基因工程方法制备HIV-1 Nef重组蛋白及其抗体。方法:用PCR技术从HIV-1H×B2质粒中扩增HIV-1 Nef基因,将测序鉴定过的HIV-1 Nef基因克隆到原核表达载体pET28a(+)上,应用酶切鉴定、测序、SDS-PAGE及Western blot等方法鉴定基因片段的正确性及表达蛋白的特异性。纯化的重组蛋白免疫兔3次后,ELISA法测定兔多克隆抗体滴度。用其制备的多克隆抗体通过免疫组织化学方法测定表达Nef基因的内皮细胞。结果:成功地构建了Nef基因的原核表达质粒,测序证明HIV-1Nef基因全长为621bp,编码206个氨基酸。在大肠杆菌BL21(DE3)中高效表达的重组蛋白为包涵体的形式。HIV-1 Nef蛋白N端融合6×His标签便于纯化及鉴定。获得的高纯度HIV-1 Nef融合蛋白,免疫动物后,可产生特异的高滴度抗体(ELISA滴度达1∶10000)。将转染Nef基因的内皮细胞,应用免疫组化的方法证明其抗体有高度的特异性。结论:构建了高表达HIV-1 Nef蛋白的原核表达系统,制备的Nef重组蛋白免疫动物,获得了特异、高效价的抗体,为研究Nef基因的生物学活性提供了实验材料和依据。
OBJECTIVE: To prepare HIV-1 Nef recombinant protein and its antibody by genetic engineering. Methods: The HIV-1 Nef gene was amplified by PCR from HIV-1 × B2 plasmid. The HIV-1 Nef gene was cloned into the prokaryotic expression vector pET28a (+) and identified by restriction enzyme digestion, sequencing, SDS -PAGE and Western blot to identify the correctness of the gene fragment and the specificity of the expressed protein. Rabbit polyclonal antibody titers were measured by ELISA after the rabbits were immunized with purified recombinant protein three times. Polyclonal antibodies prepared therewith were used to determine the Nef gene-expressing endothelial cells by immunohistochemistry. Results: The prokaryotic expression plasmid of Nef gene was successfully constructed. Sequencing proved that the HIV-1 Nef gene was 621bp in length and encoded 206 amino acids. The recombinant protein that is highly expressed in E. coli BL21 (DE3) is in the form of inclusion bodies. HIV-1 Nef protein N-terminal fusion 6 × His tag for ease of purification and identification. High-purity HIV-1 Nef fusion protein was obtained and the animals were immunized to produce specific high-titer antibodies (ELISA titer 1: 10,000). Nef gene will be transfected endothelial cells, the use of immunohistochemical methods to prove that the antibodies have a high degree of specificity. CONCLUSION: The prokaryotic expression system with high expression of HIV-1 Nef protein was constructed. The prepared Nef recombinant protein was used to immunize animals to obtain specific and high titer antibodies, which provided the experimental materials and basis for studying the biological activity of Nef gene.