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目的对白介素-32(interleukin-32,IL-32)的不同剪接异构体进行克隆及表达。方法设计保守引物,以Jurkat细胞的cDNA为模板进行PCR扩增,克隆IL-32不同剪接异构体;DNA测序扩增产物,计算机辅助分析IL-32基因序列特征;构建IL-32原核及真核表达重组质粒,并利用SDS-PAGE和Western blot检测其表达情况。结果克隆得到已知的IL-32的6个剪接异构体(α、β、γ、δ、ε、ζ),还克隆得到1个新的IL-32剪接异构体,将其命名为IL-32η,Genbank登录号JN546102。IL-32η编码区长度为336 bp,编码的蛋白含111个氨基酸残基,其理论相对分子质量为12 560。IL-32η原核表达产物主要以可溶形式位于宿主菌的周质腔。亲和纯化了重组IL-32η,利用重组IL-32η制备了兔多克隆抗体,该抗体能够识别IL-32全部的7个剪接异构体。结论克隆得到1个新的IL-32剪接异构体IL-32η,它的发现将为全面理解IL-32的功能提供新思路。
Objective To clone and express different splicing isoforms of interleukin-32 (IL-32). Methods The conservative primers were designed and used to amplify the IL-32 splicing isoforms based on the cDNA of Jurkat cells. The products of DNA sequencing were amplified by PCR. The IL-32 gene sequence was characterized by computer. The recombinant plasmids were expressed by nuclear translocation and detected by SDS-PAGE and Western blot. Results Six known splice variants (α, β, γ, δ, ε, ζ) of IL-32 were cloned and one new IL-32 splicing isoform was cloned and named as IL -32η, Genbank accession number JN546102. The coding region of IL-32n was 336 bp in length, encoding a protein of 111 amino acid residues with a theoretical relative molecular mass of 12 560. The prokaryotic expression product of IL-32n is mainly located in the periplasm of the host bacteria in a soluble form. Recombinant IL-32n was affinity-purified and a rabbit polyclonal antibody was prepared using recombinant IL-32n, which recognizes all seven splice isoforms of IL-32. Conclusion One new IL-32 splicing isoform IL-32n was cloned, and its discovery will provide a new idea to fully understand the function of IL-32.