CAJ | 학술논문

目的：构建表达猪囊尾蚴cC1抗原的重组耻垢分枝杆菌疫苗。方法提取pET28a-cC1质粒DNA，用xho I和BamH I双酶切，用Klenow酶补平xho I酶切末端，同时提取pMV261穿梭质粒载体，用Hind III和BamH I双酶切，用Klenow酶补平Hind III酶切末端，纯化后的酶切产物通过T4连接酶连接，构建pMV261-cC1穿梭质粒，测序验证正确后，将pMV261cC1穿梭质粒经电穿孔法转化耻垢分枝杆菌，构建重组耻垢分枝杆菌，经热诱导后，以猪囊尾蚴病患者血清为一抗进行Western-blotting鉴定。此外，比较正常耻垢分枝杆菌和重组cC1耻垢分枝杆菌生长状态并绘制生长曲线。结果构建的重组穿梭载体经酶切、测序验证正确。构建的重组耻垢分枝杆菌经热诱导后，SDS-PAGE电泳分析显示，在40 kDa处有外源性蛋白表达，与预期值相符。Western-blotting显示其可被猪囊尾蚴病患者血清识别，表明cC1抗原基因在耻垢分枝杆菌中表达成功。重组耻垢分枝杆菌与正常耻垢分枝杆菌的增殖特性无明显差异。结论成功构建了表达猪囊尾蚴特异性抗原cC1的重组耻垢分枝杆菌疫苗。
목적：구건표체저낭미유cC1항원적중조치구분지간균역묘。방법제취pET28a-cC1질립DNA，용xho I화BamH I쌍매절，용Klenow매보평xho I매절말단，동시제취pMV261천사질립재체，용Hind III화BamH I쌍매절，용Klenow매보평Hind III매절말단，순화후적매절산물통과T4련접매련접，구건pMV261-cC1천사질립，측서험증정학후，장pMV261cC1천사질립경전천공법전화치구분지간균，구건중조치구분지간균，경열유도후，이저낭미유병환자혈청위일항진행Western-blotting감정。차외，비교정상치구분지간균화중조cC1치구분지간균생장상태병회제생장곡선。결과구건적중조천사재체경매절、측서험증정학。구건적중조치구분지간균경열유도후，SDS-PAGE전영분석현시，재40 kDa처유외원성단백표체，여예기치상부。Western-blotting현시기가피저낭미유병환자혈청식별，표명cC1항원기인재치구분지간균중표체성공。중조치구분지간균여정상치구분지간균적증식특성무명현차이。결론성공구건료표체저낭미유특이성항원cC1적중조치구분지간균역묘。
Objective To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 anti-gen. Methods The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction en-zymes,and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both frag-ments were modified by Klenow fragment to form blunt end,then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. Results The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction,a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band,which sug-gested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis,the recombi-nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. Conclusion The recombi-nant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.