VEGF111b的表达载体构建及抗体制备

Construction of VEGF111b expression vector and preparation of VEGF111b antibody

目的克隆VEGF111b基因,构建真核表达载体pc DNA3.1-VEGF111b,并进行测序,制备VEGF111b多克隆抗体。方法丝裂霉素C诱导处理卵巢癌SKOV3细胞24 h,设计VEGF111b酶切引物,以SKOV3细胞的c DNA为模板,通过PCR得到VEGF111b特异性基因产物,克隆入真核表达载体pc DNA3.1中,获得重组真核表达载体pc DNA3.1-VEGF111b,测序验证。用KLH耦联VEGF111b特异性短肽,制备多克隆抗体,Western blot检测其特异性。结果成功扩增了VEGF111b基因,双酶切、测序鉴定证实目的基因成功克隆到真核表达载体pc DNA3.1中,测序结果与预测完全一致,成功制备了VEGF111b多克隆抗体,使用其多克隆抗体,Western blot能够检测到VEGF111b对应分子量的蛋白条带。结论本研究鉴定了pc DNA3.1-VEGF111b真核表达载体,并验证了VEGF111b多克隆抗体的特异性,为进一步研究VEGF111b蛋白的功能奠定了基础。

Objective To clone the VEGF111 b gene, construct eukaryotic expression system pc DNA3.1-VEGF111 b, sequence VEGF111 b gene, and prepare VEGF111 b polyclonal antibody. Methods With VEGF111b-specific primers and RT-PCR, VEGF111 b gene was amplified from ovarian cancer SKOV3 cells treated with mitomycin C for 24 h. PCR product was cloned into eukaryotic expression vector pc DNA3.1to construct pc DNA3.1-VEGF111 b, and was then sequenced. VEGF111b-specific peptide was connected KLH to prepare the polyclonal antibody. Western blot was used to detect its specificity. Results VEGF111 b gene was successfully amplified by RT-PCR and it was proved to be cloned into pc DNA3.1vector by double digestion with restriction endonuclease and sequencing analysis. We have prepared VEGF111 b polyclonal antibody successfully, and detected protein band of the corresponding molecular weight using the polyclonal antibody by Western blotting. Conclusion This study not only identified eukaryotic expression vector pc DNA3.1-VEGF111 b, but also verified the specificity of VEGF111 b polyclonal antibody, which provides a basis for further research on VEGF111 b function.