弓形虫钙依赖蛋白激酶CDPK3多克隆抗体的制备与初步应用  

Preparation and preliminary application of polyclonal antibodies against Toxoplasma gondii calcium dependent protein kinase CDPK3

作　　者：李丹 韩萌 都建 安然 LI Dan;HAN Meng;DU Jian;AN Ran(Department of Biochemistry,School of Basic Medicine,Anhui Medical University,Anhui Key Laboratory of Pathogen Biology,Anhui Provincial Key Laboratory of Zoonoses,Hefei 230032,China)

机构地区：[1]安徽医科大学基础医学院生物化学教研室,病原生物学安徽省重点实验室,人畜共患病安徽高校省级重点实验室,安徽合肥230032

出　　处：《中国病原生物学杂志》2023年第8期922-926,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81902084)。

摘　　要：目的制备抗弓形虫钙依赖蛋白激酶(TgCDPK3)多克隆抗体,为进一步探究TgCDPK3在弓形虫中的作用奠定基础。方法将酶切鉴定正确的原核重组表达载体pET28a-TgCDPK3转化BL21,使用IPTG诱导表达重组TgCDPK3蛋白,利用His-Ni Beads亲和层析法纯化重组pET-28a-TgCDPK3蛋白。取纯化蛋白用弗氏佐剂乳化后通过皮下多点注射免疫新西兰大白兔,一周后加强免疫,共2次。末次免疫后1周取血,分离血清,通过ELISA测定抗体效价,Western blot检测抗血清与内源天然TgCDPK3蛋白以及重组表达蛋白TgCDPK3的反应性。同时以该抗血清为一抗采用免疫荧光法进行TgCDPK3蛋白的定位。结果pET-28a-TgCDPK3转化BL21后表达相对分子质量为59×103的His-TgCDPK3蛋白,纯化后免疫新西兰大白兔,制备的抗血清抗体效价为1∶64000。Western blot检测该抗血清能识别来源于弓形虫ME-49株的TgCDPK3蛋白及外源重组TgCDPK3蛋白。免疫荧光检测显示TgCDPK3定位于弓形虫速殖子膜表面。结论成功制备TgCDPK3多克隆抗体,该抗体效价高且特异性强,可用于相关基础实验,为进一步探究TgCDPK3在弓形虫中的作用奠定了基础。Objective to lay a foundation for further investigation of the role of TgCDPK3 in Toxoplasma gondii,prepare polyclonal antibodies against calcium dependent protein kinase(TgCDPK3).Methods The enzymatically identified prokaryotic recombinant expression vector pET28a-TgCDPK3 was transformed into BL21,and the recombinant TgCDPK3 protein was induced to be expressed in vitro using IPTG.After purification of the recombinant pET-28a-TgCDPK3 protein using His-Ni Beads affinity chromatography,and combined with Freund's adjuvant,New Zealand rabbits were immunized by subcutaneous injection of multiple sites Blood was collected afterwards.The prepared polyclonal antibodies were tested by ELISA to determine their potency and Western Blot to detect their ability to bind endogenous natural TgCDPK3 protein as well as recombinantly expressed TgCDPK3 protein,so as to evaluate the specificity of the polyclonal antibodies,and also applied to observe the localization of TgCDPK3 protein by immunofluorescence technique.Results the pET-28a-TgCDPK3 was transformed into BL21 and expressed His-TgCDPK3 protein with a relative molecular mass of 59×10~3.pET-28a-TgCDPK3 was purified and immunized in New Zealand rabbits,and the antiserum was prepared with an antibody potency of 1∶64000.pET-28a-TgCDPK3 recognized TgCDPK3 protein from Toxoplasma gondii strain ME-49 and exogenous recombinant TgCDPK3 protein by Western blot.TgCDPK3 protein and exogenous recombinant TgCDPK3 protein.Immunofluorescence showed that TgCDPK3 was localized on the surface of T.gondii tachyzoites.Conclusion The TgCDPK3 polyclonal antibody was successfully prepared with high potency and specificity,which can be used in relevant basic experiments,laying the foundation for further investigation of the role of TgCDPK3 in T.gondii.

关 键 词：弓形虫 TgCDPK3 多克隆抗体 免疫荧光 

分 类 号：R382.5[医药卫生—医学寄生虫学]