CD163基因敲除大白猪的抗蓝耳病性能和主要生产性能研究

Resistance to blue ear disease and production performance assessment of CD163 gene-edited Large White pigs

[目的]运用CRISPR/Cas9基因编辑技术和体细胞核移植技术获得CD163基因全敲除(CD163 gene knockout,CD163-KO)的克隆猪,验证该基因敲除大白猪的抗蓝耳病特性,并研究其与野生型大白猪在生理、生产和繁殖性能等方面的差别,评估CD163-KO大白猪的主要生产性能。[方法]用猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)流行毒株NADC30-like对本研究所获得的11头CD163-KO大白猪和5头年龄、体质量相当的野生型大白猪进行攻毒试验。连续14 d观察猪的临床症状,记录直肠温度变化,检测血清中PRRSV病毒含量和抗体水平。14 d后取肺部组织进行切片,通过PRRSV免疫荧光试验观察肺脏感染情况。采集CD163-KO和野生型大白猪肺泡巨噬细胞进行免疫染色,观察肺泡巨噬细胞表面CD163蛋白的表达情况;采集猪外周血单核细胞进行诱导分化,观察CD163-KO与野生型大白猪诱导分化的巨噬细胞对血红蛋白-结合珠蛋白复合物的摄取情况。最后,统计分析CD163-KO与野生型大白猪的生产性能及公猪的繁殖性能。[结果]本研究获得的CD163-KO大白猪对蓝耳病NADC30-like毒株具有完全抗性,敲除CD163基因不影响巨噬细胞的生理功能,CD163-KO与野生型大白猪的生长性能和繁殖性能无明显差异。[结论]本研究是对CD163-KO猪抵抗蓝耳病的又一佐证和补充,证明CD163基因敲除操作对生产性能没有潜在的负面影响,为抗蓝耳病猪的生物安全提供了支撑。

【Objective】The purpose of this study was to generate CD163 gene knockout(CD163-KO)Large White pigs by CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies,investigate the resistance to blue ear disease and the biosafety effect including physiology,productive and reproductive performances of the gene knockout pigs,and assess the main production performances of CD163-KO Large White pigs.【Method】In this study,the 11 CD163-KO pigs and five age-and body weight-matched wild type Large White pigs were challenged with NADC30-like strain of porcine reproductive and respiratory syndrome virus(PRRSV).The rectal temperature,PRRSV antibody and virus variation were monitored continuously for 14 days.The lung tissues were examined by immunofluorescence of PRRSV antigen.Expression of CD163 protein on the surface of pulmonary alveolar macrophages in wild type and CD163-KO Large White pigs were examined through immunofluorescence staining.We compared the differentiation potential of monocytes into macrophages between CD163-KO and wild type pigs,and observed their uptake capacities to hemoglobin-haptoglobin complex.In addition,we analyzed the growth and reproductive production of the boars between CD163-KO pigs and wild type control to assess their biosafety and breeding value.【Result】CD163-KO pigs were completely resistant to NADC30-like strain without impairing the biological function associated with the modified gene,as well as productive and reproductive performances.【Conclusion】This study is an evidence and supplement of CD163-KO pigs resistance to blue ear disease,and demonstrates that CD163 gene knockout has no potentially negative effects on production performance,which provides evidences for the biosecurity of CD163-KO pigs.