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、21d分别进行1次免疫,每隔7d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1.0mmol/L、37℃诱导8h;rDmAQP2的最优表达条件是IPTG浓度为1.0mmol/L、37℃诱导7h;rDmAQP3的最优表达条件是IPTG浓度为1.0mmol/L、37℃条件下诱导5h。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∶409600、1∶51200和1∶409600,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。
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)rDmAQP1,rDmAQP2and rDmAQP3of Dermacentor marginatus.Blood collections for Western blot and ELISA tests were performed.The anti-tick challenge was conducted.The optimal expression conditions of rDmAQP1,rDmAQP2and rDmAQP3were screened by SDS-PAGE gel electrophoresis.The three recombinant proteins were purified by HisSepNi-NTA6FF purification column.Rabbits were divided into four groups of three rabbits each,including a control group and three immunized 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 7days 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 antibody titer increased.The results showed that the optimal expression conditions for rDmAQP1were induced for 8hat IPTG concentration of 1.0mmol/L and 37℃;the optimal expression conditions for rDmAQP2were induced for 7hat IPTG concentration of 1.0mmol/L and 37℃;and the optimal expression conditions for rDmAQP3were induced for 5hat IPTG concentration of 1.0mmol/L and 37℃.Western blot results showed that rDmAQP1,rDmAQP2and rDmAQP3all had certain reactivity.The ELISA results showed that the antibody titers of rabbits immunized with rDmAQP1,rDmAQP2and rDmAQP3were as follows:the total anti-tick effect of rDmAQP1protein 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 rDmAQP2protein was 78.78%,and the inhibition rates of average full-blooded tick weight,average 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 rDmAQP3protein was 87.91%,and the inhibition 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,rDmAQP2and rDmAQP3all have the potential of candidate antigens against ticks,among which rDmAQP3has the best immune effect,which lays a foundation for the study of the function of rDmAQP1,rDmAQP2and rDmAQP3.
作者
伍军
何文文
普浩
金敏
石文玉
马爱军
罗亭祥
杨德鹏
巴音查汗
呼尔查
WU Jun;HE Wenwen;PU Hao;JIN Min;SHI Wenyu;MA Aijun;LUO Tingxiang;YANG Depeng;Bayinchahan;HU Ercha(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Postdoctoral Research Station of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2024年第7期1466-1472,1506,共8页
Chinese Journal of Veterinary Science
基金
上海合作组织科技合作伙伴计划资助项目(2021E01001)。
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
Dermacentor marginatus
aquaporin
preparation of polyclonal antibodies
evaluation of anti-tick effect
