新疆褐牛CD46基因的原核表达及其多克隆抗体的制备

发布时间：2018-06-24 04:07

本文选题：新疆褐牛 + CD　；参考：《动物医学进展》2017年08期

【摘要】：旨在克隆并表达新疆褐牛CD46基因,制备其多克隆抗体,为进一步研究牛CD46分子生物学功能奠定基础。采用RT-PCR方法从新疆褐牛脾脏中扩增CD46基因全长,进行测序,并对测序结果进行生物信息学分析,将CD46部分序列亚克隆于pET-28a(+)和pVAX1载体中。将阳性重组质粒pET-28a-△CD46转化于E.coli Rosetta-gamiB(DE3)感受态细胞,诱导表达截短CD46蛋白。用切胶纯化的His-△CD46融合蛋白免疫新西兰大白兔,制备多克隆抗体;采用ELISA测定多克隆抗体的效价;取无内毒素重组质粒pVAX1-△CD46转入BHK-21(仓鼠肾细胞)细胞,表达产物以制备的多克隆抗体为一抗,采用Western blot法检测该多克隆抗体的特异性。CD46基因的测序结果表明,部分基因蛋白表达、纯化条带大小与预期一致;制备的抗牛CD46多克隆抗体效价高于1∶128 000,并具有良好的特异性。
[Abstract]:The aim of this study was to clone and express the CD46 gene of Xinjiang Brown cattle and to prepare its polyclonal antibody, which laid a foundation for further study on the molecular biological function of bovine CD46. The full-length CD46 gene was amplified from the spleen of Xinjiang brown cattle by RT-PCR and sequenced. The results were analyzed by bioinformatics. The CD46 partial sequence was subcloned into pET-28a () and pVAX1 vectors. The positive recombinant plasmid pET-28a- CD46 was transformed into E. coli Rosetta-gamiB (DE3) competent cells, and the truncated CD46 protein was induced to express in E. coli Rosetta-gamiB (DE3) cells. New Zealand white rabbits were immunized with His-CD46 fusion protein purified by gelatin, polyclonal antibody was prepared, the titer of polyclonal antibody was determined by Elisa, and recombinant plasmid pVAX1-CD46 without endotoxin was transferred into BHK-21 (hamster kidney cell) cells. The specific. CD46 gene of the polyclonal antibody was detected by Western blot assay. The results showed that some of the protein expressed and purified were the same size as expected. The titer of anti-bovine CD46 polyclonal antibody was higher than 1: 128 000 and had good specificity.
【作者单位】：新疆农业大学动物医学学院;
【基金】：新疆维吾尔自治区自然科学基金项目(2013211A030)
【分类号】：S823

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