摘要
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 Using recombinant adeno associated virus (AAV) as a mediator, to transduce a sense or an antisense DNA fragment to 6A8 α mannosidase gene into EB virus transformed B lymphocytes and to establish cell lines with long term expression of the transduced DNA. Methods The recombinant AAV vector pAGX(+) containing a sense or an antisense 6A8 DNA fragment was constructed. Recombinant AAV (rAAV) particles were transduced into EB virus transformed B cell line SKW6 and B lymphoid leukemia cell line BJAB. mRNA expression was detected by Northern blot and RT PCR assay. The change of 6A8 α mannosidase activity was assayed with Con A binding. Results rAAV particles containing a sense or an antisense 6A8 DNA fragment were successfully developed. After successful transduction of the sense or the antisense 6A8 DNA, SKW 6 and BJAB cells were cloned by limiting dilution method. Northern blot demonstrated the mRNA expression of the transduced DNA. RT PCR indicated that the transduced sense or antisense 6A8 DNA was significantly expressed in the cells cultured in vitro for more than one year. The mRNA expression of neomycin resistant gene (neo R) was also detected in the transduced cells, but not in the wild type cells. Con A binding to the cells transduced with the antisense 6A8 DNA was increased suggesting a change of protein glycosylation resulted from the effect on 6A8 α mannosidase expression caused by the transcripts of the antisense 6A8 DNA. Conclusion A sense or an antisense 6A8 DNA has been successfully transduced into EB virus transformed B cell line SKW6 and B lymphoid leukemia cell line BJAB by using a rAAV as a mediator. The transduced sense or antisense 6A8 DNA has been expressed in the cells for more than one year. [
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第6期594-599,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金资助项目 ( 396 30 30 0 )
中国医学科学院创新项目资助
关键词
重组腺相关病病毒
EB病毒转化
B淋巴细胞
外源基因
转导
Adeno associated virus vector
Sense and antisense 6A8 DNA
Transduction
EB virus transformed B cell
