边缘革蜱AQPs重组蛋白的表达及抗蜱效果
Conditional screening of recombinant proteins of Dermacentor marginatus AQPs and anti-tick effect
伍军 1何文文 1普浩 1金敏 1石文玉 1马爱军 1罗亭祥 1杨德鹏 1巴音查汗 1呼尔查2
作者信息
- 1. 新疆农业大学动物医学学院,新疆乌鲁木齐 830052
- 2. 新疆农业大学动物医学学院,新疆乌鲁木齐 830052;新疆农业大学兽医学博士后流动站,新疆乌鲁木齐 830052
- 折叠
摘要
对边缘革蜱(Dermacentor marginatus)水通道蛋白(aquaporin,AQP)rDmAQP1、rDmAQP2 和 rDmAQP3 纯化后免疫家兔,采血,进行 Western blot和ELISA试验,第3次免疫2周后进行抗蜱试验.利用SDS-PAGE凝胶电泳筛选出rDmAQP1、rDmAQP2和rDmAQP3的最优表达条件,经HisSep Ni-NTA 6FF纯化柱将3种重组蛋白纯化,家兔分4组,每组3只家兔,对照组1组,免疫组3组.纯化的3种重组蛋白分别单独免疫3组家兔,在0、14、21 d分别进行1次免疫,每隔7 d采血1次,制备多克隆抗体,Western blot检测其反应原性,用ELISA检测其抗体效价,待抗体效价升高后开展攻蜱试验.结果显示,rDmAQP1的最优表达条件是IPTG浓度为1.0 mmol/L、37 ℃诱导8 h;rDmAQP2的最优表达条件是IPTG浓度为1.0 mmol/L、37 ℃诱导7 h;rDmAQP3的最优表达条件是IPTG浓度为1.0 mmol/L、37 ℃条件下诱导5 h.Western blot结果显示rDmAQP1、rDmAQP2和rDmAQP3都具有一定的反应原性.ELISA结果显示rDmAQP1、rDmAQP2和rDmAQP3免疫家兔的抗体效价分别为:1:409 600、1:512 00和1:409 600,rDmAQP1蛋白免疫后总的抗蜱效果为79.74％,对边缘革蜱的平均饱血蜱质量、平均卵质量及平均卵孵化率的抑制率分别为9.43％、25.17％和63.81％;rDmAQP2蛋白免疫后总的抗蜱效果为78.78％,对边缘革蜱的平均饱血蜱质量、平均卵质量及平均卵孵化率的抑制率分别为8.30％、20.14％和68.26％;rDmAQP3蛋白免疫后总的抗蜱效果为87.91％,对边缘革蜱的平均饱血蜱质量、平均卵质量及平均卵孵化率的抑制率分别为3.23％、22.47％和80.50％.本研究通过血清学试验及抗蜱试验,发现rDmAQP1、rDmAQP2和rDmAQP3都具有抗蜱疫苗候选抗原潜力,其中rDmAQP3的免疫效果最佳,为rDmAQP1、rDmAQP2和rDmAQP3功能研究奠定了基础.
Abstract
In order to mine candidate vaccine antigens against ticks and to control ticks safely and effectively,the aim of this study was to immunize rabbits with purified aquaporins(AQPs)rD-mAQP1,rDmAQP2 and rDmAQP3 of Dermacentor marginatus.Blood collections for Western blot and ELISA tests were performed.The anti-tick challenge was conducted.The optimal expression conditions of rDmAQP1,rDmAQP2 and rDmAQP3 were screened by SDS-PAGE gel electrophore-sis.The three recombinant proteins were purified by HisSepNi-NTA6FF purification column.Rab-bits were divided into four groups of three rabbits each,including a control group and three immu-nized groups.The three purified recombinant proteins were separately immunized to three groups of rabbits,and the rabbits were immunized once on the 0th,14th and 21st day.Blood samples were collected every 7 days to prepare polyclonal antibodies.The reactivity was detected by Western blot and the antibody titer was detected by ELISA.Tick challenge test was carried out after the anti-body titer increased.The results showed that the optimal expression conditions for rDmAQP1 were induced for 8 h at IPTG concentration of 1.0 mmol/L and 37 ℃;the optimal expression conditions for rDmAQP2 were induced for 7 h at IPTG concentration of 1.0 mmol/L and 37 ℃;and the opti-mal expression conditions for rDmAQP3 were induced for 5 h at IPTG concentration of 1.0 mmol/L and 37 ℃.Western blot results showed that rDmAQP1,rDmAQP2 and rDmAQP3 all had certain reactivity.The ELISA results showed that the antibody titers of rabbits immunized with rD-mAQP1,rDmAQP2 and rDmAQP3 were as follows:the total anti-tick effect of rDmAQP1 protein was 79.74％,and the inhibition rates on average full-blooded tick weight,average egg weight and average egg hatching rate were 9.43％,25.17％and 63.81％,respectively.The total anti-tick effect of rDmAQP2 protein was 78.78％,and the inhibition rates of average full-blooded tick weight,av-erage egg weight and average egg hatching rate of Dermacentor marginatus were 8.30％,20.14％and 68.26％,respectively.The total anti-tick effect of rDmAQP3 protein was 87.91％,and the inhi-bition rates of average full-blooded tick weight,average egg weight and average egg hatching rate were 3.23％,22.47％and 80.5％,respectively.Through serological test and anti-tick test,it has been found that rDmAQP1,rDmAQP2 and rDmAQP3 all have the potential of candidate antigens against ticks,among which rDmAQP3 has the best immune effect,which lays a foundation for the study of the function of rDmAQP1,rDmAQP2 and rDmAQP3.
关键词
边缘革蜱/水通道蛋白/多克隆抗体的制备/抗蜱效果评价Key words
Dermacentor marginatus/aquaporin/preparation of polyclonal antibodies/evaluation of anti-tick effect引用本文复制引用
基金项目
上海合作组织科技合作伙伴计划(2021E01001)
出版年
2024
NETL
NSTL
万方数据