兔抗人乳酸脱氢酶C4(LDHC4)多克隆抗体制备与应用

Preparation and application of rabbit polyclonal antibody against human lactate dehydrogenase C4(LDHC4)

目的 制备兔抗人乳酸脱氢酶C4(LDHC4)的多克隆抗体。方法 采用PCR点突变LDHC基因,并将该基因构建到pET-28a载体上,形成pET-28a-LDHC重组表达载体、将重组载体转化至大肠杆菌表达宿主BL21(DE3),诱导表达获得重组人LDHC4蛋白,将重组蛋白作为抗原免疫新西兰兔,3次加强免疫后得到抗血清,采用ELISA检测抗血清效价,采用Western blot法检测抗血清的特异性,采用免疫组织化学法检测人三阴性乳腺癌组织中LDHC4的表达。结果 获得了特异性的兔抗人LDHC4多克隆抗体,抗体效价达1∶51 200,该抗体可用于Western blot法和免疫组织化学法。结论 成功制备了特异性兔抗人LDHC4多克隆抗体。

Objective To prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4(LDHC4).Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene,and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector.The recombinant vector was introduced into E.coli BL21(DE3),and LDHC4 protein was obtained by induced expression.The recombinant protein was used as an antigen to immunize New Zealand rabbits,and the antiserum was obtained after three boosted immunizations.The titer of the antiserum against LDHC4 were detected by ELISA.Western blot was used to detect the specificity of the antiserum,and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue.Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51200.The antibody can be used for Western blot and immunohistochemistry.Conclusion Thespecificrabbitanti-human LDHC4 polyclonal antibody is successfully prepared.