CAJ | 학술논문

目的优化构建新型胰腺癌MUC1 DNA疫苗.方法目的基因增加重复序列可变数目串联重复序列(VNTR),并分别插入乙型肝炎病毒(HRV)核心抗原基因(C1-144)和/或小鼠白细胞介素(IL)-18基因序列,插入真核表达载体pIRES2-EGFP,构建成优化重组质粒,酶切及测序鉴定.4种优化重组质粒分别体外转染2×106个293T细胞和Panc-02细胞,观察转染后48 h后绿色荧光表达,评估转染效率收集转染后的细胞,裂解后Western blot检测目的基因的表达,验证各优化重组质粒在真核细胞中的表达.结果双酶切鉴定证实了pIRES2-EGFP-A-B插入了1100 bp大小片段,pIRES2-EGFP-A-C和pIRES2-EGFP-A-B-C重组质粒都插入了1200 bp大小片段,分别与目的基因大小一致.在转染转染后48 h后,293T和Panc-02均可见绿色荧光.Western blot检测可见在Panc-02细胞中pIRES2-EGFP-A-B质粒、pIRES2-EGFP-A-B-C质粒均可表达乙肝病毒核心抗原C1-144,pIRES2-EGFP-A-C质粒、pIRES2-EGFP-A-B-C质粒均可表达小鼠IL-18 (mIL-18).在293T细胞中,pIRES2-EGFP-A质粒、pIRES2-EGFP-A-B质粒、pIRES2-EGFP-A-C质粒、pIRES2-EGFP-A-B-C质粒均表达VNTR.结论优化构建的4个重组质粒均可在真核细胞中表达插入的目的抗原.
목적 우화구건신형이선암MUC1 DNA역묘.방법 목적기인증가중복서렬가변수목천련중복서렬(VNTR),병분별삽입을형간염병독(HRV)핵심항원기인(C1-144)화/혹소서백세포개소(IL)-18기인서렬,삽입진핵표체재체pIRES2-EGFP,구건성우화중조질립,매절급측서감정.4충우화중조질립분별체외전염2×106개293T세포화Panc-02세포,관찰전염후48 h후록색형광표체,평고전염효솔수집전염후적세포,렬해후Western blot검측목적기인적표체,험증각우화중조질립재진핵세포중적표체.결과 쌍매절감정증실료pIRES2-EGFP-A-B삽입료1100 bp대소편단,pIRES2-EGFP-A-C화pIRES2-EGFP-A-B-C중조질립도삽입료1200 bp대소편단,분별여목적기인대소일치.재전염전염후48 h후,293T화Panc-02균가견록색형광.Western blot검측가견재Panc-02세포중pIRES2-EGFP-A-B질립、pIRES2-EGFP-A-B-C질립균가표체을간병독핵심항원C1-144,pIRES2-EGFP-A-C질립、pIRES2-EGFP-A-B-C질립균가표체소서IL-18 (mIL-18).재293T세포중,pIRES2-EGFP-A질립、pIRES2-EGFP-A-B질립、pIRES2-EGFP-A-C질립、pIRES2-EGFP-A-B-C질립균표체VNTR.결론 우화구건적4개중조질립균가재진핵세포중표체삽입적목적항원.
Objective To construct new MUC1 DNA vaccine for pancreatic cancer and investigate whether the target genes are expressed in eukaryotic cells.Methods Three strategies were combined to optimize new MUC1 DNA vaccine.Variable number of tandem repeat (VNTR) number was increased to facilitate target antigen to form epitope.The HBeAg gene (C1-144) and interleukin (IL)-18 gene were also inserted alone or in combination.All the recombinant plasmids were successfully constructed and further confirmed by restricting enzyme digestion and DNA sequencing.Four recombinant plasmids were all transfected into Panc-02 and 293T (2 × 106) cells by using the Liperfectamine 2000.After incubation for 48 h at 37℃ in 5％ CO2,cells were observed under fluorescent microscope and harvested in 100 μL of lysis buffer for Western blotting assay.Results The pIRES2-EGFP-A-B clone was digested with Sac I and EcoR Irestriction enzymes and a 1100 bp.fragment could be seen in the 1％ argrose gel.The pIRES2-EGFP-A-C and pIRES2-EGFP-A-B-C clones were both digested with EcoR I and Sal I restriction enzymes and 1200 bp fragments were generated in the argrose gel.Green fluoresces could be seen under the fluorescent microscope 48 h after transfection.The expression of VNTR from the four recomhinant plasmids was examined in 293T cells,and HBV C1-144 and mouse IL-18 (mIL-18) were detectable in panc-02 cells.Conclusion The new MUC1 DNA vaccine for pancreatic cancer were successfully constructed to express the target genes in eukaryotic cells.