CRISPR/Cas9介导TLR5敲除猪肺泡巨噬细胞系建立及对革兰阴性菌黏附能力的影响

Establishment of TLR5 gene knockout porcine alveolar macrophage cell line mediated by CRISPR/Cas9 and its effect on Gram-negative bacterial adhesion

Toll样受体5 (TLR5)是Toll-like受体家族中的重要成员,在革兰阴性菌引起的炎症反应中发挥着重要的调控作用。通过CRISPR/Cas9基因敲除技术对猪肺泡巨噬细胞(PAMS)的TLR5基因进行敲除,然后经菌落计数,检测TLR5基因敲除后对大肠埃希菌F18ab和F18ac以及沙门菌黏附能力的影响。结果表明:设计的2条sgRNAs成功插入CRISPR/Cas9载体中,序列正确,转染猪肺泡巨噬细胞后均能稳定表达,且PCR测序峰图出现套峰,表明敲除载体具有切割活性。利用有限稀释法,最终获得1株缺失15个碱基的单克隆细胞系TLR5-sg1。对TLR5敲除细胞进行qPCR和Western blot验证,结果显示TLR5基因的转录水平极显著下调,其蛋白质水平也明显降低。细菌黏附试验结果显示,TLR5敲除细胞中黏附的大肠埃希菌F18ab、F18ac和沙门菌(ATCC13312)的数量均极显著低于对照组。这一研究成功建立了猪TLR5基因敲除的3D4/21细胞系,且TLR5基因的敲除会降低大肠埃希菌和沙门菌对3D4/21细胞的黏附能力,为进一步探究TLR5基因的生物学功能建立了细胞模型,为深入探讨TLR5基因在炎症反应中的免疫调控作用提供了试验素材。

As an important member of the toll like receptor family, toll like receptor 5(TLR5) plays an important regulatory role in inflammatory response induced by Gram-negative bacteria. In this study, porcine alveolar macrophages with TLR5 gene knocked out was established by CRISPR/Cas9 genome editing technique, and the effect of TLR5 gene knockout on E.coli F18 ab, F18 ac and Salmonella adhesion ability was detected by colony counting. The results showed that sgRNAs were successfully inserted into the CRISPR/Cas9 vector with correct sequence and stably expressed after transfection into porcine alveolar macrophages, and nested peaks appeared in the PCR sequencing peak maps indicating that the vector had cleavage activity. Limiting dilution method was used to obtain a monoclonal cell line TLR5 with 15 base deletions. The qPCR and Western blot were used to detect the expression of TLR5 in TLR5 knockdown cells, which showed that the mRNA expression level of TLR5 gene was significantly down-regulated(P<0.01), and its protein level was also significantly down-regulated.The results of Gram-negative bacterial adhesion test showed that the number of colonies of E.coli F18 ab,F18 ac and Salmonella adhered to TLR5 knockdown cells was significantly less than that of the control group(P<0.01).In summary,we obtained 3 D4/21 cell lines with porcine TLR5 knockout and verified that knockout of TLR5 gene would reduce the adhesion ability of E.coli and Salmonella to 3 D4/21 cells.This study established a cell model for further exploring the biological function of TLR5 gene,and provided experimental materials and theoretical basis for further exploring the immunoregulatory role of TLR5 gene in inflammatory response.