羊种布鲁氏菌WadC基因缺失株的构建与鉴定

doi:10.16431/j.cnki.1671-7236.2020.12.007

摘要/Abstract

摘要： 试验旨在分析糖基转移酶编码基因WadC影响布鲁氏菌胞内存活的作用。以羊种布鲁氏菌Rev.1基因组为模板，通过同源重组方法获得WadC基因上、下游同源臂融合片段，并与载体pUC19-SacB连接，构建pUC19-SacB-ΔwadC重组载体，电转至羊种布鲁氏菌Rev.1，构建ΔwadC缺失株（Rev.1ΔwadC），检测菌株Rev.1ΔwadC的遗传稳定性，比较分析亲本株Rev.1和缺失株Rev.1ΔwadC的生长特性及其在BMDC和RAW264.7细胞中的生存能力。结果显示，试验成功构建基因缺失株，连续传代30次未发现基因回复突变；在体外相同培养条件下，缺失株Rev.1ΔwadC与亲本株Rev.1生长趋势相似，均在20 h到达对数生长期，44 h进入平台期；侵染BMDC细胞48和72 h时，其胞内存活率显著低于亲本株（P<0.05）；而侵染小鼠RAW264.7巨噬细胞试验显示，亲本菌株和基因缺失株无显著性差异（P>0.05）。综上所述，本试验成功构建并获得了具有良好遗传稳定性的布鲁氏菌WadC基因缺失株，该缺失株在体外培养条件下与亲本株生长趋势相似；但该缺失株在BMDC细胞内的存活能力显著变弱，为深入研究布鲁氏菌WadC基因功能奠定基础。

关键词: 布鲁氏菌; 糖基转移酶; WadC基因缺失株; 疫苗

Abstract: The purpose of the test was to analyze the role of the glycosyltransferase-encoding gene WadC in affecting the intracellular survival of Brucella.Using the Brucella sheep Rev.1 genome as template,the fusion fragments of the homologous arms of the upper and lower arms of WadC gene were obtained by homologous recombination,and ligated to the vector pUC19-SacB to construct the pUC19-SacB-ΔwadC recombinant vector,which was transferred to sheep species Brucella Rev.1,constructing a ΔwadC deletion strain (Rev.1ΔwadC),testing the genetic stability of the strain Rev.1ΔwadC,comparing and analyzing the growth characteristics of the parental strain Rev.1 and the deletion strain Rev.1ΔwadC and the BMDC and RAW264.7 viability of cells.The results showed that the gene-deficient strain was successfully constructed in the experiment,and no genetic back mutation was found in 30 consecutive passages.Under the same culture conditions in vitro,the growth trend of the deleted strain Rev.1ΔwadC was similar to that of the parental strain Rev.1,and both reached logarithmic growth period at 20 h and reached plateau period at 44 h.When the BMDC cells were infected at 48 and 72 h,the intracellular survival rate was significantly lower than that of the parent strain (P<0.05).The RAW264.7 macrophage test of infected mice showed that the parent strain had no significant difference with the gene deletion strain (P>0.05).To sum up,this experiment successfully constructed and obtained a strain of Brucella WadC gene with good genetic stability.The deletion strain had similar growth trend with the parent strain under in vitro culture conditions;However,the survival ability of the deletion strain in BMDC cells was significantly weakened.This study laid a foundation for further study on the function of WadC gene of Brucella.

Key words: Brucella; glycosyltransferase; WadC gene deletion strain; vaccine

中图分类号:

S852.61

孙浩杰, 徐磊, 孙佳丽, 丁家波, 毛开荣, 蔡亚南, 王楠. 羊种布鲁氏菌WadC基因缺失株的构建与鉴定[J]. 中国畜牧兽医, 2020, 47(12): 3852-3860.

SUN Haojie, XU Lei, SUN Jiali, DING Jiabo, MAO Kairong, CAI Yanan, WANG Nan. Construction and Identification of Brucella WadC Gene Deletion Strain[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 3852-3860.

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