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目的构建含人的肿瘤侵袭和转移的特异性抗原CD44v6编码基因的真核表达的重组载体,进行核苷酸序列分析,并在肿瘤细胞中表达。方法①以pBud作为真核表达载体,选择CD44v6编码基因进行RT-PCR扩增,构建重组表达质粒,进行酶切鉴定和测序分析。②在Lipofectamine 2000的介导下,将构建成功的pBud-CD44v6重组表达质粒转染到肿瘤细胞中,通过免疫组织化学方法检测CD44v6的表达。结果经酶切鉴定和测序分析表明,插入的目的基因片段为CD44v6的编码基因,长为129bp,与GenBank中登录的cDNA相比较,同源性高达100%,且方向正确;重组质粒pBud-CD44v6转染对数生长期胃癌细胞株SGC-7901,进行免疫组织化学检测结果显示,与空质粒转染组对比,重组质粒组肿瘤细胞的细胞质中可见大量CD44v6基因编码的表达,其蛋白呈棕黄色,而对照组呈阴性。结论成功构建了pBud-CD44v6真核表达的重组载体,并在肿瘤细胞中表达。
Objective To construct an eukaryotic expression recombinant vector containing the specific antigen CD44v6 encoding human tumor invasion and metastasis, perform nucleotide sequence analysis and express it in tumor cells. Methods 1 pBud was used as eukaryotic expression vector, CD44v6 encoding gene was selected for RT-PCR amplification, recombinant expression plasmid was constructed, and enzyme digestion and sequencing analysis were performed. 2 Under the guidance of Lipofectamine 2000, the recombinant plasmid pBud-CD44v6 was successfully transfected into tumor cells, and the expression of CD44v6 was detected by immunohistochemistry. RESULTS: Enzyme digestion analysis and sequencing analysis showed that the inserted gene fragment was a gene encoding CD44v6 with a length of 129 bp. Compared with the cDNA registered in GenBank, the homology was 100% and the orientation was correct. Recombinant plasmid pBud-CD44v6 The immunohistochemistry results of the transfected logarithmic growth gastric cancer cell line SGC-7901 showed that compared with the empty plasmid transfection group, a large number of CD44v6 gene-encoded expressions were observed in the cytoplasm of the recombinant plasmid group tumor cells, and the protein was brownish yellow. While the control group was negative. Conclusion The pBud-CD44v6 eukaryotic expression recombinant vector was successfully constructed and expressed in tumor cells.