| 摘要: |
| 【目的】克隆草原短尾羊Brachyury基因（即T基因）编码区（CDS），并制备Brachyury蛋白特异性多克隆抗体，为草原短尾羊短尾表型机制研究提供重要的试验材料。【方法】提取草原短尾羊16 d胚胎的组织RNA，反转成cDNA，以cDNA为模板，采用PCR方法克隆草原短尾羊Brachyury基因CDS全长序列。将Brachyury基因CDS全长序列连接至pEASY-Blunt E1载体，构建原核表达载体pEASY-Blunt E1-Brachyury，并进行Nhe Ⅰ和EcoR Ⅰ双酶切鉴定。将原核表达载体导入RosettagamiB（DE3）大肠杆菌，并进行IPTG诱导表达，对表达产物进行回收纯化。利用纯化后的表达产物免疫6周龄日本大耳白兔，用间接ELISA（iELISA）法测定血清多克隆抗体效价。以制备的多克隆抗体为一抗，采用Western blot法检测草原短尾羊16，20，25和30 d胚胎中Brachyury蛋白的表达水平。【结果】克隆了1 335 bp的草原短尾羊Brachyury基因CDS全长序列，成功构建了原核表达载体pEASY-Blunt E1-Brachyury，表达获得了49.16 ku的重组Brachyury蛋白。成功制备了重组Brachyury蛋白多克隆抗体，抗体效价达1∶1 500，该多克隆抗体可以特异性识别草原短尾羊胚胎中的Brachyury蛋白。草原短尾羊16 d胚胎中Brachyury蛋白的表达量最高。【结论】成功制备草原短尾羊Brachyury蛋白兔血清多克隆抗体，抗体效价为1∶1 500,该多克隆抗体可以特异性识别草原短尾羊胚胎中的Brachyury蛋白。 |
| 关键词: 草原短尾羊 胚胎 Brachyury基因 多克隆抗体制备 |
| DOI： |
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| 基金项目:国家自然科学基金项目（3186130045）；内蒙古自治区科技计划项目（2019GG241） |
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| Preparation of polyclonal antibody against Brachyury protein of prairie short-tail sheep |
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YANG Guang,HUO Yujia,SU Hong,et al
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| Abstract: |
| 【Objective】This study cloned the Brachyury (also known as T) protein coding region and prepared Brachyury protein-specific polyclonal antibody to provide experimental materials for the study of short-tail phenotype mechanism of prairie bobtail sheep.【Method】Tissue RNA from 16-day embryos of prairie short tail sheep was extracted and reversely transcribed into cDNA.Using cDNA as a template,PCR was used to clone the full-length sequence of Brachyury CDS.The Brachyury CDS full-length sequence was ligated to the pEASY-Blunt E1 vector to construct the prokaryotic expression vector pEASY-Blunt E1-Brachyury,and the Nhe Ⅰ and EcoR Ⅰ double restriction enzyme digestion was performed.The prokaryotic expression vector was transformed into RosettagamiB (DE3) Escherichia coli for IPTG induced expression,and the expression product was recovered and purified.The purified expression product was used to immunize 6-week-old Japanese big-eared white rabbits,and the serum polyclonal antibody titer was determined by indirect ELISA.Using the prepared polyclonal antibody as primary antibody,Western blot was used to detect the expression levels of Brachyury in 16,20,25 and 30 d embryos of prairie bobtail sheep.【Result】The 1 335 bp prairie short-tail sheep Brachyury CDS full-length sequence was cloned,the prokaryotic expression vector pEASY-Blunt E1-Brachyury was successfully constructed,and the recombinant Brachyury protein of 49.16 ku was expressed.The recombinant Brachyury protein polyclonal antibody was successfully prepared with a titer of 1∶1 500.The polyclonal antibody can specifically recognize the Brachyury protein in the embryos of prairie bobtail sheep.The highest expression of Brachyury protein was in 16 d embryos of prairie bobtail sheep.【Conclusion】The rabbit serum polyclonal antibody against Brachyury protein of prairie bobtail sheep with a titer of 1∶1 500 was successfully prepared.The polyclonal antibody can specifically recognize the Brachyary protein in embryos of prairie bobtail sheep. |
| Key words: prairie short-tail sheep embryo Brachyury gene polyclonal antibody preparation |
