稳定表达小鼠CD40L的NIH3T3细胞系的建立及其在B细胞培养和激活中的应用

该研究构建小鼠CD40L真核表达重组质粒pcDNA3.1-mCD40L,通过电转法将重组质粒转至NIH3T3细胞中。利用G418对转染后细胞进行压力筛选,获得稳定转染细胞株。提取稳定转染细胞株RNA,通过RT-PCR法检测Neo基因的mRNA表达情况。分离稳定转染细胞上清,利用ELISA法检测小鼠CD40L蛋白水平的表达情况。RT-PCR结果显示,Neo基因能够在稳定转染细胞中表达,ELISA结果显示,获得的稳定转染细胞株NIH3T3-mCD40L细胞上清中CD40L的表达量高达1.286 ng/mL。进一步活性研究表明,该细胞系能够在体外与IL-2和IL-21共同作用培养B细胞至14天,并刺激B细胞产生特异性抗体。该细胞系的成功构建,为利用体外B细胞分离培养和活化法分离特异性单克隆抗体奠定了良好的基础。

Establishment of Mouse CD40L Stably Expressed NIH3T3 Cell Lines and Its Application in the Culture and Activation of B Lymphocytes

Recombinant plasmid pcDNA3.1-mCD40L was constructed and transfected into NIH3T3 cells to express mouse CD40L.The transfected cells were selected with G418 to obtain stably transfected cell lines.The RNA of stable transfected cells was isolated and the mRNA expression level of Neo gene was detected by RT-PCR.The expression level of CD40L protein in the culture supernatant of transfected cells was detected by ELISA analysis.RT-PCR revealed that Neo gene could be expressed in the transfected cells.ELISA results showed that the concentration of CD40L in the supernatant of the stable transfected cell line NIH3T3-mCD40L was 1.286 ng/mL.Further activity studies showed that the addition of NIH3T3-mCD40L cells,IL-2 and IL-21 could successfully culture and stimulate B cells to produce specific antibodies.NIH3T3-mCD40L stable cell lines was successfully obtained.This study lays a good foundation for generation of monoclonal antibody from B lymphocytes.