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目的:制备Cyclin D1b蛋白C端多肽的多克隆抗体,为后续Cyclin D1b的研究奠定基础。方法:用生物信息学方法分析Cyclin D1b蛋白的同源性、亲水性和抗原性,设计并合成Cyclin D1b C端的氨基酸序列,将合成后的多肽与钥孔血蓝蛋白(Keyhole-limpet hemocyanin,KLH)偶联并免疫新西兰大白兔,制备多克隆抗体。ELISA法检测多抗效价,Western blotting检测Cyclin D1b的表达,免疫组织化学法检测该抗体的特异性与适用范围。结果:成功制备兔抗人Cyclin D1b蛋白C端多肽的多克隆抗体,ELISA法效价为1∶18 000。纯化后的抗体用于Western blotting可特异识别MCF-7细胞中的Cyclin D1b,并可用于免疫组织化学检测。结论:制备成功Cyclin D1b蛋白C端多肽的多克隆抗体,此多抗可用于Western blotting和免疫组织化学等分析。
OBJECTIVE: To prepare polyclonal antibodies against C-terminal polypeptide of Cyclin D1b protein, which will lay the foundation for the further study of Cyclin D1b. METHODS: The homology, hydrophilicity and antigenicity of Cyclin D1b protein were analyzed by bioinformatics methods. The amino acid sequence of the C-terminal of Cyclin D1b was designed and synthesized. The synthesized peptide was fused with Keyhole-limpet hemocyanin KLH) were conjugated and immunized New Zealand white rabbits to prepare polyclonal antibodies. The multi-antibody titer was detected by ELISA, the expression of Cyclin D1b was detected by Western blotting, and the specificity and the applicable range of the antibody were detected by immunohistochemistry. Results: The polyclonal antibody against C-terminal domain of rabbit anti-human Cyclin D1b protein was successfully prepared. The ELISA titer was 1:18 000. The purified antibody can be used to detect Cyclin D1b in MCF-7 cells by Western blotting and can be used for immunohistochemistry. Conclusion: The polyclonal antibody of C-terminal polypeptide of Cyclin D1b protein was successfully prepared. This polyclonal antibody can be used for Western blotting and immunohistochemistry analysis.