利用免疫共沉淀验证HT036与P311间的相互作用

Identification of interaction between HT036 and P311 by co-immunoprecipitation

目的利用免疫共沉淀技术验证未知蛋白HT036(hypotheticalprotein,HT036)和P311间的相互作用。方法构建能在哺乳动物细胞中表达带HA标签的HT036融合蛋白(HA-HT036)的重组载体pCMV-HA-HT036,经酶切鉴定正确后,和表达带Myc标签的P311融合蛋白(Myc-P311)的重组真核表达载体pCMV-Myc-p311,单独或者共转染人293细胞,利用免疫共沉淀技术验证HT036与P311间的相互作用。结果构建的重组表达载体pCMV-HA-HT036经双酶切鉴定正确,转染293细胞,抗Myc单克隆抗体沉淀Myc-P311相互作用蛋白复合物后,用抗HA单克隆抗体进行Westernblot检测,可以检测到HA-HT036的表达。结论成功构建了HA-HT036融合蛋白真核表达重组载体,在哺乳动物细胞中表达后利用免疫共沉淀技术证实HT036与P311之间存在着相互作用。

Objective To explore the interaction between HT036 （hypothetical protein HT036） and P311 by co-immunoprecipitation. Methods HA-tagged fusion protein （HA-HT036） expression vector was constructed, identified and transfected into human embryo kidney 293 （HEK293）cells alone or with Myc-tagged fusion protein （Myc-P311 ） expression vector pCMV-Myc-p311. The interaction between P311 and HT036 was detected by co-immunoprecipitation. Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly. When Myc-P311 was immunoprecipitated by anti-Myc antibody, HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex. Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully. The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311. The result provides an important basis for further study of the intracellular signal transduction of P311.