Wang Junping, Zhengl Yuling, Pian Yaya, Guo Jie, Hao Huaijie, Jiang Yongqiang
Wei Sheng Wu Xue Bao. 2015 May 4;55(5):643-9.
In order to study the role of SS2 Type IV Secretion System VirD4 in evasion of the host innate immune killing, we constructed a knockout mutant AVirD4. Then we studied its biological activity and virulence.
The two VirD4 flanking DNA sequences were amplified using genome of 05ZYH33 as template. We also amplified the Cm sequence of shuttle vector pSET1, and through overlap extension PCR we connected the three fragments together. Using suicide vector pSET4s, we constructed the recombinant gene knockout vector pSET4s::VirD4. The mutant AVirD4 was successfully constructed by allelic replacement. Virulence of mutant strain was compared with wild type strain 05ZYH33 through in vitro bactericidal assays, competitive infection and challenge experiment of CD1 mice.
Mutant strain AVirD4 was constructed successfully, its virulence attenuated compared to the wild type strain.
These findings indicated that Type IV Secretion System component VirD4 contributed to the virulence of S. suis with important functions in evading innate immunocyte killing.
为研究猪链球菌2型IV型分泌系统VirD4在逃避宿主固有免疫杀伤中的作用，构建了敲除突变株AVirD4。随后对其生物学活性和毒力进行了研究。
以05ZYH33基因组为模板扩增VirD4两侧的两段DNA序列。同时扩增穿梭载体pSET1的Cm序列，通过重叠延伸PCR将三个片段连接在一起。利用自杀载体pSET4s构建重组基因敲除载体pSET4s::VirD4。通过等位基因替换成功构建突变株AVirD4。通过体外杀菌试验、竞争感染和对CD1小鼠的攻毒实验，比较突变株与野生型菌株05ZYH33的毒力。
成功构建突变株AVirD4，其毒力较野生型菌株减弱。
这些结果表明，IV型分泌系统组分VirD4对猪链球菌的毒力有贡献，在逃避固有免疫细胞杀伤中具有重要作用。
