分泌抗PRRSV抗体的猪源单克隆B细胞永生化方法的建立

A method for immortalizing swine monoclonal B cells secreting anti-PRRSV antibodies

摘要:

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)引起的高度接触性传染疾病，对社会经济造成重大损失。目前并未获得有效的中和抗体应用于科研及治疗中，所以建立一种筛选具有中和活性的单克隆抗体的方法，对PRRSV防治及抗原位点筛选具有重要意义。单克隆抗体在人类和动物的众多疾病治疗及诊断中得到广泛应用，而针对不同病原如何筛选出有效的中和抗体是目前急需解决的问题。在单克隆抗体筛选的众多方法中，B细胞永生化方法是一种有效的且大概率能获得单克隆中和抗体的方法。通过将bcl-6和bcl-xl两个基因中间通过f2a序列连接后构建到一个载体上，进行逆转录病毒包装。包装成熟的逆转录病毒感染免疫PRRSV的猪源淋巴细胞，并使用加入CD40L和IL21细胞因子的完全培养基培养，然后使用CD21作为筛选标记通过磁珠法进行B细胞筛选，最后对筛选得到的B细胞单克隆化并进行检测，验证是否有抗体分泌。结果显示，构建的质粒不论单独转染还是与病毒包装时的两种质粒共同转染，均可以成功表达，且包装病毒可成功感染细胞。感染后的淋巴细胞可以检测到抗体分泌，进一步筛选得到的B细胞也可以检测到抗体分泌。因此，本研究成功建立了一种针对PRRSV的单克隆抗体筛选方法。

Abstract:

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which causes great economic losses. At the moment, no effective neutralizing antibody is available for scientific research and treatment. Therefore, developing a method for screening the neutralizing monoclonal antibodies is of great significance for the prevention and treatment of PRRSV and the screening of antigen sites. Monoclonal antibodies have been widely used in the treatment and diagnosis of many human and animal diseases. Therefore, screening effective neutralizing antibodies for different pathogens is an urgent task. Among the methods for monoclonal antibody screening, B cell immortalization is an effective method to obtain neutralizing monoclonal antibody. Specifically, in this study, the bcl-6 and bcl-xl genes were connected by f2a and then the yielded product was ligated to a vector for retrovirus packaging. The swine lymphocytes immunized with PRRSV were infected the yielded mature viruses and cultured in the complete medium containing CD40L and IL21 cytokines. Then, CD21 was used as the marker to screen B cells with the magnetic bead method. Finally, monoclonal B cells were obtained and the secretion of antibodies was tested. The results showed that the plasmid, either being transfected alone or with the packaged plasmids, could be expressed, and that the packaged retrovirus could infect the cells. Moreover, the infected lymphocytes secreted antibodies, so did the screened B cells. Therefore, the method for screening monoclonal antibody against PRRSV was successfully established.