布鲁氏菌vceA基因缺失株的构建及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2019.04.004

Abstract

Abstract:

This study was aimed to construct Brucella 2308 (S2308) vceA gene mutants then analyse its growth character and survival ability in human embryonic trophoblast cells (HPT-8).The upstream,downstream homologous arms of vceA gene and kan gene were amplified by PCR method with wild S2308 strain as template.The fusion PCR technology was used to fuse the three genes fragments and connected to pMD19-T vector and then electric transported to E. coli DH5α competent cells to construct S2308ΔvceA strain.The genetic stability of the mutant strain was tested by screening continuously with kanar resistance,and the growth rates between the S2308ΔvceA and S2308 strains were compared.The HPT-8 cells were infected with S2308ΔvceA and S2308 strains at a multiplicity of infection of 100:1 and the intracellular survival ability was determined by CFU counting.The results showed that the target gene fragments of upstream homologous arms (372 bp),downstream homologous arms (510 bp) of vceA gene and kan gene (1 093 bp) were amplified respectively.The mutants was successfully constructed and the virulence not recover after culture at least 10 generations.The growth curves of S2308ΔvceA and S2308 strains were similar under the same condition.These two strains reached the logarithm growth phase at 12 h,entered in platform period after 30 h.However,the survival rates of the S2308ΔvceA strain was significantly lower than its parent strain S2308 after the infection of HPT-8 cells at 12 h (P<0.05).In conclusion,This experiment successfully constructed and obtained a vceA gene deletion strain of Brucella with good genetic stability,and the growth trend of this deleted strain was similar to parent strain under the condition of in vitro culture.However,the viability of the deleted strain in HPT-8 cells was significantly weakened.

Key words: Brucella; vceA gene mutant strain; growth curve; biological characteristics

CLC Number:

S852.61

WANG Xiaofeng, LI Mingqi, GUO Jia, ZHAO Tianyi, LIU Hang, ZHANG Fan, SUN Xiaolu, HE Jiaqi, WANG Zhen, ZHANG Hui. Construction and Biological Characteristics of Brucella vceA Gene Deletion Strain[J]. , 2019, 46(4): 986-993.

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Construction and Biological Characteristics of Brucella vceA Gene Deletion Strain

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