两种分离纯化介质对虾肝肠胞虫孢子发芽率的影响

The Effect of Two Purification Media on the Germination Rate of Enterocytozoon hepatopenaei Spores

【目的】比较蔗糖和Percoll两种不同介质的密度梯度离心分离纯化虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)孢子的效果及对EHP孢子发芽活力的影响。【方法】取感染EHP的凡纳滨对虾(Litopenaeus vannamei)肝胰腺充分研磨,经3次差速离心后,设置介质质量分数为30%,60%,90%的梯度,分别进行蔗糖和Percoll密度梯度离心,比较两种介质不同分层中EHP孢子的数量和纯化效果。纯化样品分别用0.1mol·L^-1的KOH溶液、0.15mol·L^-1的HCl溶液、0.15mol·L^-1的NaOH溶液、0.025mol·L^-1 pH为11.5的磷酸盐缓冲液、0.05mol·L^-1 pH为8.8的TrisHCl溶液、0.01mol·L^-1 pH为4.8的柠檬酸盐缓冲液等6种溶液于28℃孵育30min,比较EHP孢子的发芽率。【结果】蔗糖密度梯度离心后EHP孢子主要分布在蔗糖质量分数为90%层的底层,数量为1.53×108个。Percoll密度梯度离心后EHP孢子主要分布在Percoll质量分数为90%层的下层,数量为2.64×10^7个。经Percoll或蔗糖密度梯度离心分离纯化的EHP孢子再经上述6种不同溶液处理后,孢子发芽率存在差异;经0.1mol·L^-1的KOH溶液或0.05mol·L^-1pH为8.8的Tris-HCl溶液诱导时,两种介质分离纯化所得的EHP孢子发芽率之间差异具有统计学意义(p<0.05)。【结论】以介质质量分数为30%,60%,90%的梯度对EHP孢子进行密度梯度离心,蔗糖的分离纯化效果比Percoll更好,对EHP孢子的发芽率影响更小;经蔗糖或Percoll分离纯化的EHP孢子再经0.05mol·L^-1 pH为8.8的Tris-HCl溶液或0.025mol·L^-1 pH为11.5的磷酸盐缓冲液处理,可获得较高的发芽率。

[Purposes]To investigate the effect of sucrose or Percoll density gradient centrifugation on the purification and germination of Enterocytozoon hepatopenaei(EHP)spores under different conditions.[Methods]EHP-infected hepatopancreas of Penaeus vannamei was grind absolutely and spores were isolated by 3 times of differential centrifugation.30%,60%,and 90%sucrose or Percoll density gradient centrifugation were performed and spores in different layers were counted and observed by microscopy.Sucrose and Percoll purified samples were treated with 0.1 mol·L^-1 KOH,0.15 mol·L^-1 HCl,0.15 mol·L^-1 NaOH,0.025 mol·L^-1 PBS(pH 11.5),0.05 mol·L^-1 Tris-HCl(pH 8.8)and 0.01 mol·L^-1 Citrate buffer(pH 4.8)at 28℃for 30 min,respectively and then calculate the germination rates.[Findings]The spores were mainly distributed in the bottom of the 90%layer after sucrose density gradient centrifugation,the content was 1.53×108 spores while 2.64×10^7 spores were distributed in the middle of the 90%layer.The germination rates of EHP spores purified by Percoll or sucrose had a certain difference after being treated with different solutions.The effect of the two media used for purification on the germination rate of EHP spores was statistically significant(p<0.05)when induced by 0.1 mol·L^-1 KOH or 0.05 mol·L^-1 Tris-HCl(pH 8.8).[Conclusions]Density gradients of 30%,60%,and 90%were used to separate and purify the EHP spores using sucrose and Percoll,respectively.The purification of EHP spores by sucrose was better than Percoll as the medium for the above mentioned density gradients centrifugation.And sucrose was less effected on spore germination.Sucrose and Percoll-purified EHP spores had higher germination rates while treated with 0.05 mol·L^-1 Tris-HCl(pH 8.8)and 0.025 mol·L^-1 PBS(pH 11.5),respectively.