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

Abstract

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

CLC Number:

S852.61

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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Construction and Identification of Brucella WadC Gene Deletion Strain

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