CAJ | 학술논문

构建褶纹冠蚌热休克蛋白70基因的原核表达质粒pET30a-cpHSP70，并经酶切和DAN测序鉴定。将其转入到表达宿主BL21（DE3）菌中，用 IPTG诱导表达，最后用 SDS-PAGE和 Western-blot检测。结果显示：新表达的重组蛋白分子量约为74 kD。在 IPTG的诱导下，其表达量随时间的增加而增加，最适表达条件是37℃诱导8 h。表达蛋白可溶性分析发现，一部分蛋白以不溶性的包涵体形式存在，一部分蛋白以可溶性蛋白形式存在。Western-blot检测显示蚌在35℃热激诱导1 h后，其血、鳃、肌肉、肝胰腺和外套膜组织 cpHSP70蛋白表达水平相对于蚌在20℃暂养3 h后的表达量明显升高。
구건습문관방열휴극단백70기인적원핵표체질립pET30a-cpHSP70，병경매절화DAN측서감정。장기전입도표체숙주BL21（DE3）균중，용 IPTG유도표체，최후용 SDS-PAGE화 Western-blot검측。결과현시：신표체적중조단백분자량약위74 kD。재 IPTG적유도하，기표체량수시간적증가이증가，최괄표체조건시37℃유도8 h。표체단백가용성분석발현，일부분단백이불용성적포함체형식존재，일부분단백이가용성단백형식존재。Western-blot검측현시방재35℃열격유도1 h후，기혈、새、기육、간이선화외투막조직 cpHSP70단백표체수평상대우방재20℃잠양3 h후적표체량명현승고。
Prokaryotic expression plasmid,pET30a-cpHSP70 of C.plicata was constructed in this study.The construct was identified by endonuclease digestion and DNA sequencing and then transformed into the host bacteria BL21 (DE3).1 mmol·L-1 IPTG was used to induce the expression of the fusion protein.The ex-pression product was verified by SDS-PAGE and Western blot.It was found that the molecular weight of new expressed protein was 74 kD,which was similar to the expected molecular weight.The expression products increased with the increasing of induction time by IPTG.The optimal condition for protein expres-sion was induction with 1 mmol·L-1 IPTG at 37 ℃ for 8 h.Part of the expressed protein was soluble in supernatant fluid,while there was still some insoluble protein appeared in the inclusion bodies.The results of western-blot indicated that the protein levels of HSP70 at 35 ℃ were increased rapidly in bloods,gills, muscles,hepatopancreas and mantles from C.plicata,compared to that of HSP70 at 20 ℃.