人源性鼻咽癌抗独特型单链抗体基因G22真核表达载体的构建及表达鉴定

Construction of eukaryotic expression vector encoding human nasopharyngeal carcinoma anti-idiotype antibody single chain fragment gene G22 and its expression

目的:构建含人源性鼻咽癌抗独特型单链抗体基因G22的真核表达载体,并观察其在直肠癌细胞(rectalcancercells,CMT-93)中的表达。方法:将目的基因G22克隆入真核质粒pcDNA3.1(+)中构建真核表达载体pcDNA3.1(+)-G22,并进行酶切鉴定和DNA序列测定。用脂质体法将重组质粒转染入CMT-93细胞,以Western免疫印迹、流式细胞术和免疫荧光检测G22基因的表达。结果:酶切鉴定和DNA序列分析证实,重组质粒pcDNA3.1(+)-G22含有人源性鼻咽癌抗独特型单链抗体基因G22的全长序列,转染实验表明G22基因能在真核细胞CMT-93中正确表达。结论:人源性鼻咽癌抗独特型抗体基因G22真核表达载体构建及其在CMT-93细胞中的表达均获成功,为基因疫苗的进一步研究奠定了基础。

Objective To construct a eukaryotic expression vector encoding human nasopharyngeal carcinoma anti-idiotype antibody single chain fragment （ScFv） gene G22, and to identify its expression in rectal cancer cells （ CMT-93 ）. Methods The G22 gene was ligated into the sites of EcoRI and NotⅠ of eukaryotic expression vector pcDNA3. 1 （ ＋ ）. After the identification and DNA sequencing, the recombinant plasmid pc DNA3. 1 （ ＋ ）-G22 was stably transfected into CMT-93 cells, and the expression of G22 was detected by Western blot, flow cytometry and immunofluorescence staining. Results Restriction enzyme digestion and DNA sequencing showed that the recombinant plasmid contained the human nasopharyngeal carcinoma anti-idiotype antibody ScFv gene G22. Transfection experiment verified that G22 gene could be expressed in CMT-93 cells in the right way. Conclusion The eukaryotic expression vector containing the human nasopharyngeal carcinoma antiidiotype antibody ScFv gene G22 is successfully constructed and expressed, which is the basis for further study of its DNA vaccine.