1个新的白介素-32剪接异构体的克隆与表达

Cloning and expression of a novel splicing variant of interleukin-32

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。

Interleukin-32 is a recently-described proinflammatory cytokine,and six splice variants of IL-32 have been reported previously.In this study we aimed to clone and express a novel isoform of IL-32,termed IL-32η.The ORF of IL-32η contains 336 bp nucleoside acids,which encode a 111-amino acid protein with a theoretical molecular weight of 12.56 KDa.A recombinant plasmid of pET28-IL32η was constructed and then introduced into BL21（DE3）.SDS-PAGE analysis showed that the recombinant IL-32η was mainly expressed in the periplasm of host bacteria as a soluble protein.The recombinant IL-32η（rIL-32η） was purified by using His-bind resin,and a rabbit anti-IL32η polyclonal antibody was obtained from rabbit immunized with purified rIL-32η.Western blot analysis revealed that the rabbit anti-IL-32η antibody could recognize the whole isoforms of IL-32（α,β,γ,δ,ε,ζ,η）.In conclusion,the identification of IL-32η will provide a new insight for fully understanding the function of IL-32.