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目的:重组人鳞状细胞癌抗原(SCCA)的基因及表达融合蛋白,为下一步建立新的肝癌诊断方法奠定基础。方法:提取子宫颈癌细胞株(hela)中的总RNA,应用RT-PCR、PCR等技术扩增出SCCA基因,将其分别与PET-32a及PGEX-4T-1载体连接,转化入DH5α菌中进行克隆,并测序鉴定。异丙基β-D-硫代半乳糖苷(IPTG)诱导转入工程质粒的BL21菌中表达SCCA融合蛋白(分别含有HIS,GST-Tag),并进行SDS-PAGE及Western blotting鉴定。结果:经RT-PCR、PCR扩增后成功获得一条450 bp的DNA片段,经测序鉴定与预期序列一致;并成功在大肠杆菌中实现了高表达,经Western blotting鉴定为SCCA融合蛋白。结论:成功获得SCCA目的基因并获得高纯度的SCCA融合蛋白,为进一步开发针对肝癌的SCCA诊断试剂打下基础,开辟诊断肝癌新途径。
Objective: Recombinant human squamous cell carcinoma antigen (SCCA) gene and expression of fusion protein, which will lay the foundation for the next step to establish a new diagnostic method of liver cancer. Methods: The total RNA of cervical cancer cell line (hela) was extracted. The SCCA gene was amplified by RT-PCR and PCR. The gene was ligated with PET-32a and PGEX-4T-1 vector, In the cloning, and sequencing identification. The SCCA fusion protein (containing HIS and GST-Tag respectively) was expressed in BL21 strain induced by IPTG and identified by SDS-PAGE and Western blotting. Results: A 450 bp DNA fragment was successfully obtained by RT-PCR and PCR amplification. The DNA fragment was identified by sequencing. The DNA sequence was successfully expressed in E. coli and identified as SCCA fusion protein by Western blotting. CONCLUSION: The SCCA fusion protein obtained from SCCA gene was successfully obtained, which laid the foundation for the further development of SCCA diagnostic reagent for liver cancer and opened up a new way to diagnose liver cancer.