新型胰腺癌MUC1 DNA疫苗的优化构建及体外转染研究

The optimized construction and cell transfection assay of new MUC1 DNA vaccine for pancreatic cancer

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（HBV）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2×10^6个293T细胞和Panc-02细胞，观察转染后48h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1100bp大小片段，pIRES2-EGFP—A—C和pIRES2-EGFP—A—B—C重组质粒都插入了1200bp大小片段，分别与目的基因大小一致。在转染转染后48h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。

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 tbrm epitope. The HBcAg gene （C1-144） and interleukin （1L）-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 transfeeted into Panc-02 and 293T （2 × 106 ） ceils by using the Lipeffectamine 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 I restriction 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 EeoR 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 recombinant plasmids was examined in 293T ceils, and HBV C1-144 and mouse IL-18 （raiL-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.