HEPC1和HEPC2真核表达载体的构建及其在NIH3T3细胞中的表达

Construction of eukaryotic expression vectors of mouse HEPC1 and HEPC2 genes and expression in NIH3T3 cells

目的 构建小鼠HEPC1和HEPC2基因的真核表达载体,并观察其分别稳定转染NIH 3T3细胞后的表达情况。方法 从小鼠肝组织中分离总RNA ,采用RT PCR获得小鼠HEPC1和HEPC2基因全长cDNA ,分别将其克隆至真核表达载体pcDNA3上,酶切和测序鉴定后,用脂质体将重组子分别转染至NIH3T3细胞中,G418进行长期筛选,然后用RT PCR和dot ELISA检测HEPC1和HEPC2的表达。结果 RT PCR获得预期大小的片段,重组子pcDNA3 HEPC1和pcDNA3 HEPC2酶切正确,测序表明HEPC1有1个碱基与GenBank中报告的不同,但其位于密码子的第3位在此不改变编码的氨基酸,HEPC2则完全与GenBank中报道的一致,经RT PCR和dot ELISA检测HEPC1和HEPC2在其稳定转染的NIH3T3细胞中均有表达。结论 成功构建了小鼠HEPC1和HEPC2基因的真核表达载体,HEPC1和HEPC2在其稳定转染的NIH 3T3细胞中获得了表达,为下一步探讨HEPC1和HEPC2的功能及作用机制奠定了基础。

Objective To construct the eukaryotic vectors of mouse HEPC1 and HEPC2 genes and observe their expressions in NIH3T3 cells lines. Methods To obtain HEPC1 and HEPC2 complete cDNAs, the total RNA was isolated from liver of mouse and cDNAs were synthesized by RT-PCR. The specific amplification products were cloned into pcDNA3 and then were identified by restriction enzymes digestion and sequencing analysis. The recombinant plasmids pcDNA3-HEPC1 and pcDNA3-HEPC2 were respectively transfected into NIH3T3 cells by lipofectin. The positive clones of the transfected cells were obtained by G418 screening, and their expressions were analyzed by RT-PCR and dot-ELISA. Results The expected gene fragments were obtained by RT-PCR. The recombinant plasmids were identified to contain target fragments by restriction enzyme digestion analysis. DNA sequencing showed the sequence of HEPC2 gene had no difference with the GenBank reports while the sequence of HEPC1 gene had a base difference from the GenBank reports, but it did not affect the coded amino acid. The HEPC1 and HEPC2 were successfully expressed in transfected NIH3T3 cells. Conclusion Eukaryotic expression vectors pcDNA3-HEPC1 and pcDNA3-HEPC2 were constructed successfully. HEPC1 and HEPC2 were successfully expressed in the transfected NIH3T3 cells, which laid the foundation for further studies on the biological functions of HEPC1 and HEPC2 and their mechanisms.