Bcl-xL是Bcl-2家族中一类重要的抗凋亡因子,其功能主要是抑制细胞凋亡、促进肿瘤发生.为了探知Bcl-xL在生物硫化物应激中的作用,本实验以耐硫生物单环刺螠(Urechis unicinctus)为研究对象,RACE扩增获得一个长度为1 426 bp的单环刺嗌Bcl-xL全长cDNA序列,推导的氨基酸序列包含Bcl-xL4个保守的BH结构域和1个跨膜区域.使用该cDNA的完整开放阅读框序列,构建了原核表达载体pET28a-Bcl-xL,体外诱导表达该重组蛋白;镍柱纯化得到纯度为90％的纯化蛋白,并制备多克隆抗体.Western blotting结果显示:单环刺螠暴露于50 μmol/L硫化物中2 h-24 h内其呼吸肠中Bcl-xL含量显著增加(P＜0.05),然而在150 μmol/L硫化物中48 h-72 h内其呼吸肠Bcl-xL含量显著降低(P<0.05),暗示Bcl-xL抑制细胞凋亡途径在单环刺螠应对低浓度硫化物暴露时发挥作用,而在高浓度硫化物暴露下Bcl-xL不能发挥抑制细胞凋亡的作用.%Bcl-xL,a member of Bcl-2 family is an important anti-apoptosis factor participating in the inhibition of cell apoptosis and promotion of tumour forming.In the present study,we employed Urechis unicinctus,a sulfide-tolerant Echiuran worm to investigate the role of Bcl-xL in the response to sulfide exposure.A full-length cDNA of 1,426 bp from U.unicinctus Bcl-xL was cloned by RACE amplification,and the deduced amino acid sequence included 4 conserved BH domains and a transmembrane domain found in all Bcl-xL.A prokaryotic expression vector,pET28a-Bcl-xL was constructed using the open reading frame sequence of U.unicinctus Bcl-xl,and the recombination protein of Bcl-xL was inductively expressed in vitro.The protein of 90 ％ purity was obtained by nickel column purification and the polyclonal antibody was made.Western blotting showed that content of Bcl-xL increased significantly (P＜0.05) in the hindgut tissue of U.unicinctus exposed to 50 μmol/L sulfide from 2 h to 24 h,while decreased significantly (P＜0.05) in that of 150 μmol/L sulfide from 48 h to 72 h,implying that the Bcl-xL inhibiting apoptosis pathway played a role in U.unicinctus resisting the exposure of lower concentration sulfide,while no apoptosis inhibition of Bcl-xL occurred in the higher concentration sulfide.
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