一种有效敲低原代B细胞中目的基因表达以快速鉴定基因功能方法的建立

B细胞介导的抗病毒体液免疫应答过程涉及大量基因的上调表达，为快速鉴定这些基因的功能，需在体外建立一种有效敲低B细胞中目的基因表达以研究基因功能的方法，但目前已有的方法转导效率较低且很少将B细胞转移到小鼠体内以观察其增殖和分化。本研究首先将Drosha酶特异性小分子干扰RNA(small interfering RNA，siRNA)与反转录病毒包装质粒共转染至病毒包装细胞中，大大提高了病毒的滴度；其次，在培养基中加入抗CD180抗体，构建了原代B细胞的体外培养体系，使B细胞在保持自身特性的同时具有较强的增殖能力；再次，增加离心感染(spin infection)次数，进一步提高了B细胞的转导效率；另外，通过小鼠预先感染，可收获更多增殖、分化的B细胞以供表型分析。通过上述改进措施，成功敲除了B细胞功能基因Bcl6的表达，验证了其抗凋亡功能。该方法的建立为研究病毒急、慢性感染中B细胞的增殖、分化与缺陷机制奠定了良好基础。

Establishment of a method to effectively knock down gene expression in primary B cells to quickly identify functional gene

The process of B cell-mediated antiviral humoral immune response involves the up-regulation of genes. In order to identify the function of these genes quickly, we need a method to knock down target gene expression in B cells effectively in vitro and in vivo. In this study, four procedures were adopted to improve the assay. Firstly, we co-transfected the Drosha enzyme-specific siRNA with the retroviral packaging plasmid into virus packaging cells. Secondly, we constructed an in vitro culture system for primary B cell by adding anti-CD180 antibody to the culture medium, in which B cells can proliferate robustly. Then, by increasing the times of spin infection, the transduction efficiency of B cells was further improved. Besides, through pre-infection of mice, more proliferated and differentiated B cells after adoptive transfer can be harvested for phenotypic analysis. By adopting the above-mentioned improvement measures, the expression of B cell functional gene Bcl6 was successfully knocked down, and its anti-apoptotic function was verified. Establishment of this method would lay a good foundation for studying the mechanism of B cell proliferation, differentiation and defection in acute and chronic viral infections in the future.