猪XCL1蛋白纯化、多克隆抗体的制备及其生物活性鉴定  

Biological activity of purificated swine XCL1 protein and preparation of its polyclonal antibody

作　　者：吴耀丽[1] 温俊歌[1] 孙岩[1] 张德礼[1] 

机构地区：[1]西北农林科技大学动物医学院兽医免疫学研究所农业部动物生物技术重点开放实验室预防兽医学系兽医微生物学与病毒学课程组病毒免疫与生物信息研究室生物信息学研究中心分子病毒免疫与肿瘤系统生物学研究组,陕西杨凌712100

出　　处：《细胞与分子免疫学杂志》2011年第12期1335-1338,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(31072115;30270342);生物大分子国家重点实验室开放基金(O5SY021107);遗传资源与进化国家重点实验室开放基金资助(GREKF09-05);"十二五"国家科技计划农村领域基础研究类首批预备项目(NC2010CD0178)

摘　　要：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。AIM： To obtain purificated his-XCLl recombinant protein of porcine in vitro and prepare the associated polyclonal antibody; To study how the recombinant protein affects on lymphocytes proliferation. METHODS： Purify the recombinant protein using HiTrapTM Chelating HP chromatographic column; Check the purified product using SDS- PAGE; Detect the expression of the recombinant protein using Western blot; Immunize the experimental animals with the purified fusion protein to prepare the serum containing the associated polyclonal antibody. The serum will then undergo double immnodiffusion test and indirect ELISA test to determine the polyclonal antibody titer. Then, test the condition of lymphocytes proliferation by MTT. RESULTS： Single target strip could be seen under conducting the SDS- PAGE electrophoresis when the concentration of the binding buffer is 40 mmol/L and the concentration of the elution buffer is 500 mmol/L; Western blot test showed that the recombinant protein could be successfully expressed; Double immunodiffusion test showed the antigen-antibody binding ratio to be 1：8, and the titre of antibodies was 1：12 800 detected by indirect ELISA. The result of MTT showed that both the native XCLI and the recombinant protein could stimulate lymphocytes proliferation, and this stimulating effect could be effectively blocked by the polyclonal antibody we prepared. CONCLUSION： To conclude, this recombinant protein has biological activity and this research can provide basic material for further investigation of the function of XCL1 in swine.

关 键 词：XCL1 纯化蛋白 多克隆抗体 间接ELISA MTT 

分 类 号：Q75[生物学—分子生物学]