CAJ | 학술논문

目的：在大肠杆菌中原核表达、纯化日本血吸虫果糖二磷酸醛缩酶（rSjFBPA），观察其在血吸虫生活史各阶段的表达。方法以日本血吸虫成虫cDNA为模板扩增rSjFBPA基因，克隆至pET28a（+）质粒后，再转化入E. coli BL21。含重组质粒的菌株经IPTG诱导后，采用SDS?PAGE和Western blotting分析鉴定重组蛋白rSjFBPA是否表达，用层析法纯化rSjFBPA并用SDS?PAGE鉴定其纯度。同时，用RT?PCR方法分析SjFBPA在血吸虫尾蚴、童虫、成虫和虫卵各阶段的表达情况。结果经PCR扩增出目的基因，含目的基因的TA克隆质粒经双酶切和测序鉴定，证明插入片段与预期目的基因序列相符。Western blotting结果显示，表达后的重组蛋白可与His?tag单克隆抗体发生特异性反应。经镍亲和层析法制备了纯化的重组SjFBPA蛋白，纯化重组蛋白浓度达4 mg/ml。RT?PCR结果显示，SjFBPA在日本血吸虫尾蚴、童虫、成虫和虫卵阶段均有表达。结论 SjFBPA基因被成功克隆和表达，其在日本血吸虫尾蚴、童虫、成虫和虫卵各阶段均有表达。
목적：재대장간균중원핵표체、순화일본혈흡충과당이린산철축매（rSjFBPA），관찰기재혈흡충생활사각계단적표체。방법이일본혈흡충성충cDNA위모판확증rSjFBPA기인，극륭지pET28a（+）질립후，재전화입E. coli BL21。함중조질립적균주경IPTG유도후，채용SDS?PAGE화Western blotting분석감정중조단백rSjFBPA시부표체，용층석법순화rSjFBPA병용SDS?PAGE감정기순도。동시，용RT?PCR방법분석SjFBPA재혈흡충미유、동충、성충화충란각계단적표체정황。결과경PCR확증출목적기인，함목적기인적TA극륭질립경쌍매절화측서감정，증명삽입편단여예기목적기인서렬상부。Western blotting결과현시，표체후적중조단백가여His?tag단극륭항체발생특이성반응。경얼친화층석법제비료순화적중조SjFBPA단백，순화중조단백농도체4 mg/ml。RT?PCR결과현시，SjFBPA재일본혈흡충미유、동충、성충화충란계단균유표체。결론 SjFBPA기인피성공극륭화표체，기재일본혈흡충미유、동충、성충화충란각계단균유표체。
Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.