猪链球菌2型Δ0948互补株构建及对溶血素分泌和毒力的影响

Construction ofΔ0948 complementary strain of Streptococcus suis 2 and its effect on hemolysin secretion and virulence

目的:构建猪链球菌2型Δ0948互补株,验证对溶血素分泌和毒力的影响。方法:PCR扩增带启动子的SSU05_0948基因序列并连接至pAT18载体构建互补株,Western blot验证表达情况;绘制生长曲线比较互补株和野生株、突变株在不同时期的生长情况;CD1小鼠攻毒模型验证互补株能否恢复突变株毒力;溶血素(SLY)溶血活性及Western blot比较互补株和野生株、突变株不同时间点对SLY蛋白分泌的影响。结果:互补株构建成功但SSU05_0948基因的表达低于野生株;互补株在对数生长期的生长速度明显慢于野生株和突变株,但在平台期一致;CD1小鼠毒力试验证明互补株可以基本恢复突变株毒力;SLY蛋白溶血活性及Western blot实验证明SSU05_0948在对数期早中期可以抑制SLY蛋白的分泌,而在对数期晚期及平台期则不影响SLY蛋白的分泌,互补株可以恢复SLY蛋白的分泌。结论:猪链球菌互补株CΔ0948能够恢复突变株Δ0948的毒力,SSU05_0948影响SLY蛋白的分泌,为猪链球菌防治提供新思路。

Objective To construct Streptococcus suis type 2Δ0948 complementary strain and verify its effect on suilysin(SLY)secretion and virulence.Methods The SSU05_0948 gene sequence with promoter was amplified by PCR and ligated to pAT18 vector to construct complementary strain and verify its expression through Western blot.Growth curve was drawn to compare the growth of complementary strain against the wild-type strain and mutant strain in different periods.CD1 mice challenge model was used to verify whether complementary strain could restore the virulence of mutant.SLY hemolytic activity and Western blot were compared the effect of complementary strain and wild-type strain and mutant strain on SLY protein secretion at different time points.Results The complementary strain was successfully constructed,but the expression of SSU05_0948 was lower than the wild-type strain.The growth rate of the complementary strain was significantly slower than the wild-type strain and mutant strain in the logarithmic growth phase,but the same in the platform phase.The CD1 mice challenge model showed the complementary strain could basically restore the virulence of the mutant strain.The hemolytic activity of SLY and Western blot showed that SSU05_0948 could inhibit the secretion of SLY protein in the early and middle logarithmic phase,but did not affect the secretion of SLY in the late logarithmic and platform phase,while the complementary strain could restore the secretion of SLY protein.Conclusions The complementary strain CΔ0948 of Streptococcus suis can restore the virulence of mutant strainΔ0948,and SSU05_0948 affects the virulence ofΔ0948,which provides a new idea for the prevention and treatment of Streptococcus suis.