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目的构建表皮生长因子受体-3(v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3,ErbB3)真核表达质粒,并检测其免疫小鼠血清抗体滴度。方法以乳腺cDNA文库为模板,PCR扩增ErbB3全长基因,插入pcDNA3.1-myc/His载体,构建重组表达质粒pcDNA3.1-myc/His-ErbB3。同时以质粒pcDNA3.1-myc/His-ErbB3为模板,PCR扩增ErbB3-ECD和ErbB3-RLD基因,插入pET-28a(+)和pGEX-KG载体中,构建重组表达质粒pET-28a(+)-ECD和pGEX-KG-RLD;将质粒pET-28a(+)-ECD和pGEX-KG-RLD转化感受态大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物经Western blot鉴定后,经GST柱或镍亲和柱纯化,BCA法定量纯化后蛋白。以60μg质粒pcDNA3.1-myc/His-ErbB3免疫BALB/c小鼠,4次免疫后,经小鼠尾静脉采血,分离血清,以纯化后的ErbB3-ECD和ErbB3-RLD蛋白为包被抗原,采用间接ELISA法检测小鼠血清抗体滴度。结果 3种质粒经双酶切及测序鉴定,证明构建正确。表达的重组蛋白ErbB3-ECD和ErbB3-RLD在相对分子质量约76 000和46 000处可见特异蛋白条带,浓度分别为2和1 mg/ml,免疫后的小鼠血清抗体滴度分别为1︰400和1︰10 000。结论 ErbB3全长质粒免疫BALB/c小鼠后,经ErbB3-ECD和ErbB3-RLD蛋白初步筛选,已获得ErbB3的多克隆抗体,为后期ErbB3单抗的制备以及以ErbB3为靶点的核酸免疫治疗奠定了基础。
Objective To construct the eukaryotic expression plasmid of v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3 (ErbB3), and to detect the serum antibody titers of the immunized mice. Methods The full-length ErbB3 gene was amplified by PCR from breast cDNA library and inserted into pcDNA3.1-myc / His vector to construct recombinant plasmid pcDNA3.1-myc / His-ErbB3. The ErbB3-ECD and ErbB3-RLD genes were amplified by PCR using plasmid pcDNA3.1-myc / His-ErbB3 as a template and inserted into pET-28a (+) and pGEX-KG vectors to construct recombinant expression plasmid pET-28a ) -ECD and pGEX-KG-RLD. The plasmid pET-28a (+) - ECD and pGEX-KG-RLD were transformed into competent E. coli BL21 (DE3), induced by IPTG. After purification by GST column or nickel affinity column, the purified protein was quantified by BCA method. BALB / c mice were immunized with 60μg plasmid pcDNA3.1-myc / His-ErbB3. After four immunizations, blood was collected from the tail vein of mice and the serum was separated. The purified ErbB3-ECD and ErbB3-RLD proteins were coated antigen , The indirect ELISA method was used to detect the serum antibody titers in mice. Results The three plasmids were identified by double enzyme digestion and sequencing. The expressed recombinant proteins ErbB3-ECD and ErbB3-RLD showed specific protein bands at about 76 000 and 46 000 in relative molecular mass at concentrations of 2 and 1 mg / ml, respectively, and the serum antibody titers of the immunized mice were 1 : 400 and 1: 10,000. Conclusion ErbB3 polyclonal antibody was obtained after preliminary screening of ErbB3-ECD and ErbB3-RLD protein by BALB / c mice with ErbB3 full-length plasmid. The preparation of ErbB3 monoclonal antibody and ErbB3-targeted nucleic acid immunotherapy Foundation.