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目的 采用重组腺相关病毒 (AAV)载体pAGX(+) ,把 6A8α 甘露糖苷酶基因的正义或反义DNA片段转导入EB病毒转化的B淋巴细胞 ,以获得正义或反义 6A8DNA长期表达的B淋巴细胞株。方法 构建含正义或反义 6A8DNA片段的重组pAGX(+)质粒。经包装后转导淋巴细胞 ,经G418筛选 ,用有限稀释法克隆转导成功的淋巴细胞。Northern杂交和RT PCR检测转导基因的mRNA表达水平。ConA结合试验检测细胞在转导基因后 6A8α 甘露糖苷酶活性的改变。结果 pAGX 反义6A8DNA及pAGX 正义 6A8DNA经包装获得了高复制滴度的重组AAV(rAAV)颗粒 ,藉助rAAV成功地把 6A8基因转导入EB病毒转化的B细胞株SKW6和B淋巴样白血病细胞株BJAB。Northern杂交结果显示 ,转导的正义或反义 6A8DNA获得表达。经 1年余传代培养 ,RT PCR检测见BJAB和SKW6细胞中转导的正义或反义 6A8DNA的mRNA表达增加 ,新霉素抗性基因 (neoR)也获得表达 ,表明转导基因获得了长期表达。ConA结合强度在转导反义 6A8DNA的细胞升高 ,提示转导反义 6A8DNA可影响 6A8α 甘露糖苷酶的表达。结论 用AAV载体成功地把 6A8α 甘露糖苷酶基因的正义或反义DNA片段转导入EB病毒转化的B淋巴细胞SKW6和B淋巴样白血病细胞株BJAB ,转导的基因获得长期表达 ,并干扰 6A8α 甘露糖苷酶的表达。
OBJECTIVE: To transduce sense or antisense DNA fragment of 6A8α mannosidase gene into Epstein-Barr virus transformed B lymphocytes by recombinant adeno-associated virus (AAV) vector pAGX (+) to obtain long-term expressed B lymphoid cells of sense or antisense 6A8 DNA Cell lines. Methods Recombinant pAGX (+) plasmids containing sense or antisense 6A8 DNA fragments were constructed. After packaging, the transduced lymphocytes were screened by G418 and the transduced lymphocytes were cloned by limiting dilution. Northern blot and RT PCR were used to detect the mRNA expression levels of transduction genes. ConA binding assay was used to detect the change of 6A8α-mannosidase activity after transduction of cells. Results pAGX antisense 6A8 DNA and pAGX sense 6A8 DNA were packaged to obtain recombinant replication-competent AAV (rAAV) particles with high replication titer. 6A8 gene was successfully transduced into EBV-transformed B cell line SKW6 and B lymphoblastic leukemia cell line BJAB by rAAV . Northern blot results showed that transduced sense or antisense 6A8 DNA was expressed. After 1 year of subculturing, the mRNA expression of sense or antisense 6A8 DNA transduced in BJAB and SKW6 cells was increased by RT PCR assay, and the neoR gene was also expressed, indicating that the transgene had been expressed for a long time . ConA binding intensity in the transduction of antisense 6A8DNA cells increased, suggesting that the transfection of antisense 6A8DNA 6A8α mannosidase expression can be affected. CONCLUSIONS: The sense or antisense DNA fragment of 6A8α-mannosidase gene was successfully transduced into EBV-transformed B lymphocyte cell line SKW6 and B lymphoblastic leukemia cell line BJAB using AAV vector. The transduced genes were obtained for long-term expression and interfere with 6A8α mannan Glycosidase expression.