猪树突状细胞表面分子CD103的表达及其抗血清的制备

Expression of and polyclonal antiserum preparation for the porcine surface molecule CD103 protein of the dendritic cell

为制备抗猪树突状细胞表面分子CD103蛋白的多克隆抗体,首先从NCBI基因库中获取查找猪源CD103的基因序列,并设计引物;其次,采集猪呼吸道淋巴结样品,提取RNA反转录为cDNA,通过PCR的方法扩增得到CD103基因片段;然后将所获扩增的CD103基因克隆入原核表达载体pET-30a(+)并构建表达重组质粒pET-30a-CD103,将构建的重组质粒转化入BL21(DE3)感受态细胞,利用IPTG诱导蛋白表达,用NI-NTA亲和层析的方法纯化目的蛋白;最后,将纯化的目的蛋白与ISA206佐剂混合乳化后通过背部皮下免疫途径免疫BALB/c小鼠,经3次加强免疫后收集血清获得多克隆抗体。利用ELISA、Western blot和间接免疫荧光(IFA)试验评价制备的多克隆抗体的效价和反应性。结果表明,本研究成功扩增得到猪CD103基因并成功构建重组表达质粒;利用原核表达系统成功表达了猪CD103目的蛋白。Western blot以及IFA结果显示,本研究制备的多克隆抗体特异性高,能与CD103蛋白发生特异性结合;经ELISA滴定结果显示,制备的多克隆抗体在稀释至1∶25 600倍时依旧具有良好的反应性。综上表明,本研究成功制备抗猪CD103蛋白多克隆抗体,为后期制备抗CD103单克隆抗体及研究黏膜免疫过程中CD103阳性树突状细胞的相关研究奠定了基础。

For preparation of polyclonal antibodies against the surface molecule CD103 protein of porcine dendritic cells, primers were designed based on porcine CD103 sequence in gene bank. Initially, total RNA was extracted from porcine respiratory lymph nodes and reversely transcribed into cDNA, which was then used as template to amplify CD103 gene by PCR. Moreover, CD103 gene was cloned into vector pET-30 a(+) to construct recombinant plasmid pET-30 a-CD103, which was subsequently transformed into BL21(DE3) competent cells to express CD103 protein induced by IPTG. Furthermore, the target protein was purified by NI-NTA affinity chromatography and inoculated to BALB/c mice via subcutaneous injection three times with ISA206 adjuvant to prepare polyclonal antibodies. The prepared polyclonal antibodies were evaluated using ELISA, Western blot and indirect immunofluorescence assay(IFA). Our results not only showed that recombinant plasmid was constructed successfully and CD103 protein was expressed efficiently, but aslo display that prepared CD103 antibodies could react with CD103 protein in Western blot, IFA and ELISA. In conclusion, the polyclonal antibodies against CD103 protein was prepared successfully that providing the basis for the preparation of monoclonal antibodies against CD103 protein, and related immunologic study of CD103;porcine dendritic cells.