论文部分内容阅读
目的:纯化获得体外表达猪his-XCL1的重组蛋白,制备多克隆抗体并研究其对淋巴细胞增殖的影响。方法:首先,采用HiTrapTM Chelating HP纯化方法,SDS-PAGE检测重组蛋白纯化情况,Western blot检测重组蛋白表达情况。再免疫动物制备多抗血清,双向琼脂扩散试验、间接ELISA检测多克隆抗体效价,MTT法检测猪XCL1对淋巴细胞增殖的影响。结果:当结合缓冲液的浓度为40 mmol/L,洗脱缓冲液500 mmol/L时,SDS-PAGE电泳可观察到有单一目的条带,Western blot显示重组蛋白成功表达,双向琼脂扩散试验证实抗原抗体的结合比为1∶8,间接ELISA检测到抗体水平达到1∶12 800。重组蛋白XCL1能促进淋巴细胞的增殖,而此实验制备的多克隆抗体可以阻断这种效应。结论:体外表达的重组蛋白具有生物活性,制备的多克隆抗体为研究XCL1在猪体中的功能提供科技资料。
OBJECTIVE: To purify porcine his-XCL1 recombinant protein and to prepare polyclonal antibody to study its effect on lymphocyte proliferation. Methods: Firstly, HiTrapTM Chelating HP purification method was used to detect the purification of recombinant protein by SDS-PAGE and the expression of recombinant protein by Western blot. The animals were re-immunized with multi-antiserum, two-dimensional agar diffusion test, polyclonal antibody titer by indirect ELISA, and the effect of XCL1 on lymphocyte proliferation by MTT assay. Results: When the binding buffer concentration was 40 mmol / L and the elution buffer was 500 mmol / L, a single band was observed by SDS-PAGE electrophoresis. Western blot showed that the recombinant protein was successfully expressed. The two-dimensional agar diffusion test confirmed The antigen-antibody binding ratio was 1: 8, and the indirect ELISA detected the antibody level reaching 1:12 800. Recombinant protein XCL1 can promote the proliferation of lymphocytes, while the polyclonal antibody prepared in this experiment can block this effect. CONCLUSION: The recombinant protein expressed in vitro has biological activity. The prepared polyclonal antibody provides scientific material for studying the function of XCL1 in pigs.