CAJ | 학술논문

目的：构建人CD63真核表达质粒，观察其在人舌鳞癌细胞TCA8113中的表达与定位。方法提取TCA8113细胞总RNA，RT-PCR方法扩增CD63全长基因序列，亚克隆至pcDNA3.1真核表达质粒中，构建pcDNA3.1-CD63表达质粒，转化E.coli DH5α，筛选阳性克隆，酶切及测序鉴定正确后，将重组质粒转染TCA8113细胞，Western blot检测蛋白表达，间接免疫荧光方法观察CD63蛋白在TCA8113细胞中的表达与定位。结果成功构建了pcDNA3.1-CD63真核表达质粒， Western bolt结果检测到外源CD63高表达，间接免疫荧光结果表明CD63融合蛋白与内源CD63蛋白定位相似，均表达于细胞膜。结论外源CD63融合蛋白能在TCA8113中高表达并定位于细胞膜。
목적：구건인CD63진핵표체질립，관찰기재인설린암세포TCA8113중적표체여정위。방법제취TCA8113세포총RNA，RT-PCR방법확증CD63전장기인서렬，아극륭지pcDNA3.1진핵표체질립중，구건pcDNA3.1-CD63표체질립，전화E.coli DH5α，사선양성극륭，매절급측서감정정학후，장중조질립전염TCA8113세포，Western blot검측단백표체，간접면역형광방법관찰CD63단백재TCA8113세포중적표체여정위。결과성공구건료pcDNA3.1-CD63진핵표체질립， Western bolt결과검측도외원CD63고표체，간접면역형광결과표명CD63융합단백여내원CD63단백정위상사，균표체우세포막。결론외원CD63융합단백능재TCA8113중고표체병정위우세포막。
Objective To construct an eukaryotic expressing plasmid of human CD63 gene and observe its expression and location in TCA8113 tongue squamous cell carcinoma cell line. MethodsTotal RNA was extracted from TCA8113 cells. The CD63 gene was amplified by PCR method and subcloned to pcDNA3.1 (+) plasmid. The recombinant plasmids were transformed into E.coli DH5α and screened by restriction enzyme digestion and DNA sequencing. Then, pcDNA3.1-CD63 plasmids were transfected into TCA8113 cells. Western blot was used to detect the expression of protein, the subcellular location between endogenous CD63 and pcDNA3.1-CD63 in TCA8113 cells was detected and compared by indirect immunofluorescence staining. Results The recombinant eukaryotic expressing plasmid, pcDNA3.1-CD63, was successfully constructed. The expression of recombinant plasmid in TCA8113 cells was proved by Western blot. And the indirect immunofluorescence staining showed that the location of pcDNA3.1-CD63 protein was similar with endogenous CD63 protein, and they both located in the outer membrane of TCA8113 cells.ConclusionThe fusion protein pcDNA3.1-CD63 can be successfully expressed in TCA8113 cells and located in the outer membrane.